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Background: The existing frameworks for the evaluation of genetic and genomic applications clearly address the technical and clinical value of a test, but are less concerned with the way genetic services are delivered and organized. We therefore aimed to develop a comprehensive new framework that includes an assessment of service delivery. Methods: A new framework was built on the evaluation dimensions identified through a systematic review of the existing frameworks and a Delphi survey of Italian experts in public health genomics. Results: Our framework has four sections. The first two sections, respectively, guide the evidence collection process for the genetic test (analytic validity; clinical validity; clinical utility; personal utility) and its delivery models (organizational aspects; economic evaluation; ethical, legal and social implications; patient perspective). The third section guides the formulation of the research priorities to be addressed in future research. Finally, the fourth section suggests three criteria to summarize the collected evidence (net benefit, cost-effectiveness, feasibility). Conclusion: We have successfully developed an evaluation framework for the evaluation of genetic tests that includes an assessment of service delivery. It also introduces some neglected evaluation dimensions such as personal utility and patient perspective. [ABSTRACT FROM AUTHOR]

Local neocortical circuits play critical roles in information processing, including synaptic plasticity, circuit physiology, and learning, and GABAergic inhibitory interneurons have key roles in these circuits. Moreover, specific neurological disorders, including schizophrenia and autism, are associated with deficits in GABAergic transmission in these circuits. GABAergic synapses represent a small fraction of neocortical synapses, and are embedded in complex local circuits that contain many neuron and synapse types. Thus, it is challenging to study the physiological roles of GABAergic inhibitory interneurons and their synapses, and to develop treatments for the specific disorders caused by dysfunction at these GABAergic synapses. To these ends, we report a novel technology that can deliver different genes into pre- and post-synaptic neocortical interneurons connected by a GABAergic synapse: First, standard gene transfer into the presynaptic neurons delivers a synthetic peptide neurotransmitter, containing three domains, a dense core vesicle sorting domain, a GABAA receptor-binding domain, a single-chain variable fragment anti-GABAA ß2 or ß3, and the His tag. Second, upon release, this synthetic peptide neurotransmitter binds to GABAA receptors on the postsynaptic neurons. Third, as the synthetic peptide neurotransmitter contains the His tag, antibody-mediated, targeted gene transfer using anti-His tag antibodies is selective for these neurons. We established this technology by expressing the synthetic peptide neurotransmitter in GABAergic neurons in the middle layers of postrhinal cortex, and the delivering the postsynaptic vector into connected GABAergic neurons in the upper neocortical layers. Targeted gene transfer was 61% specific for the connected neurons, but untargeted gene transfer was only 21% specific for these neurons. This technology may support studies on the roles of GABAergic inhibitory interneurons in circuit physiology and learning, and support gene therapy treatments for specific disorders associated with deficits at GABAergic synapses. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is the most common type of malignant gliomas, characterized by genetic instability, intratumoral histopathological variability and unpredictable clinical behavior. Disappointing results in the treatment of gliomas with surgery, radiation and chemotherapy have fueled a search for new therapeutic targets and treatment modalities. Here we report new approach towards RNA interference therapy of glioblastoma multiforme based on the magnetic nanoparticles delivery of the double-stranded RNA (dsRNA) with homological sequences to mRNA of tenascin-C (TN-C), named ATN-RNA. The obtained nanocomposite consisted of polyethyleneimine (PEI) coated magnetic nanoparticles conjugated to the dsRNA show high efficiency in ATN-RNA delivery, resulting not only in significant TN-C expression level suppressesion, but also impairing the tumor cells migration. Moreover, synthesized nanomaterials show high contrast properties in magnetic resonance imaging (MRI) and low cytotoxicity combining with lack of induction of interferon response. We believe that the present work is a successful combination of effective, functional, non-immunostimulatory dsRNA delivery system based on magnetic nanoparticles with high potential for further application in GBM therapy. [ABSTRACT FROM AUTHOR]

Papillomavirus capsids are known to have the ability to package DNA plasmids and deliver them both in vitro and in vivo. Of all known papillomavirus types, human papillomaviruses (HPVs) are by far the most intensely studied. Although HPVs work well as gene transfer vectors, their use is limited as most individuals are exposed to this virus either through a HPV vaccination or natural infection. To circumvent these constraints, we produced pseudovirions (PsVs) of ten non-human papillomavirus types and tested their transduction efficiencies in vitro. PsVs based on Macaca fascicularis papillomavirus-11 and Puma concolor papillomavirus-1 were further tested in vivo. Intramuscular transduction by PsVs led to months-long expression of a reporter plasmid, indicating that PsVs have potential as gene delivery vectors. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) have enabled numerous basic research discoveries and therapeutic approaches for many protein classes. However, there still exist a number of target classes, such as multi-pass membrane proteins, for which antibody discovery is difficult, due in part to lack of high quality, recombinant protein. Here we describe the impact of several parameters on antigen expression and the development of mAbs against human claudin 4 (CLDN4), a potential multi-indication cancer target. Using gene gun-based DNA delivery and bioluminescence imaging, we optimize promoter type by comparing expression profiles of four robust in vivo promoters. In addition, we observe that most vectors rapidly lose expression, ultimately reaching almost background levels by three days post-delivery. Recognizing this limitation, we next explored skin pretreatment strategies as an orthogonal method to further boost the efficiency of mAb generation. We show that SDS pretreatment can boost antigen expression, but fails to significantly increase mAb discovery efficiency. In contrast, we find that sandpaper pretreatment yields 5-fold more FACS+ anti-CLDN4 hybridomas, without impacting antigen expression. Our findings coupled with other strategies to improve DNA immunizations should improve the success of mAb discovery against other challenging targets and enable the generation of critical research tools and therapeutic candidates. [ABSTRACT FROM AUTHOR]

Since development of plasmid gene therapy for therapeutic angiogenesis by J. Isner this approach was an attractive option for ischemic diseases affecting large cohorts of patients. However, first placebo-controlled clinical trials showed its limited efficacy questioning further advance to practice. Thus, combined methods using delivery of several angiogenic factors got into spotlight as a way to improve outcomes. This study provides experimental proof of concept for a combined approach using simultaneous delivery of VEGF165 and HGF genes to alleviate consequences of myocardial infarction (MI). However, recent studies suggested that angiogenic growth factors have pleiotropic effects that may contribute to outcome so we expanded focus of our work to investigate potential mechanisms underlying action of VEGF165, HGF and their combination in MI. Briefly, Wistar rats underwent coronary artery ligation followed by injection of plasmid bearing VEGF165 or HGF or mixture of these. Histological assessment showed decreased size of post-MI fibrosis in both—VEGF165- or HGF-treated animals yet most prominent reduction of collagen deposition was observed in VEGF165+HGF group. Combined delivery group rats were the only to show significant increase of left ventricle (LV) wall thickness. We also found dilatation index improved in HGF or VEGF165+HGF treated animals. These effects were partially supported by our findings of c-kit+ cardiac stem cell number increase in all treated animals compared to negative control. Sporadic Ki-67+ mature cardiomyocytes were found in peri-infarct area throughout study groups with comparable effects of VEGF165, HGF and their combination. Assessment of vascular density in peri-infarct area showed efficacy of both–VEGF165 and HGF while combination of growth factors showed maximum increase of CD31+ capillary density. To our surprise arteriogenic response was limited in HGF-treated animals while VEGF165 showed potent positive influence on a-SMA+ blood vessel density. The latter hinted to evaluate infiltration of monocytes as they are known to modulate arteriogenic response in myocardium. We found that monocyte infiltration was driven by VEGF165 and reduced by HGF resulting in alleviation of VEGF-stimulated monocyte taxis after combined delivery of these 2 factors. Changes of monocyte infiltration were concordant with a-SMA+ arteriole density so we tested influence of VEGF165 or HGF on endothelial cells (EC) that mediate angiogenesis and inflammatory response. In a series of in vitro experiments we found that VEGF165 and HGF regulate production of inflammatory chemokines by human EC. In particular MCP-1 levels changed after treatment by recombinant VEGF, HGF or their combination and were concordant with NF-κB activation and monocyte infiltration in corresponding groups in vivo. We also found that both–VEGF165 and HGF upregulated IL-8 production by EC while their combination showed additive type of response reaching peak values. These changes were HIF-2 dependent and siRNA-mediated knockdown of HIF-2α abolished effects of VEGF165 and HGF on IL-8 production. To conclude, our study supports combined gene therapy by VEGF165 and HGF to treat MI and highlights neglected role of pleiotropic effects of angiogenic growth factors that may define efficacy via regulation of inflammatory response and endothelial function. [ABSTRACT FROM AUTHOR]

The vehicle routing problem (VRP) has a wide range of applications in the field of logistics distribution. In order to reduce the cost of logistics distribution, the distance-constrained and capacitated VRP with split deliveries by order (DCVRPSDO) was studied. We show that the customer demand, which can’t be split in the classical VRP model, can only be discrete split deliveries by order. A model of double objective programming is constructed by taking the minimum number of vehicles used and minimum vehicle traveling cost as the first and the second objective, respectively. This approach contains a series of constraints, such as single depot, single vehicle type, distance-constrained and load capacity limit, split delivery by order, etc. DCVRPSDO is a new type of VRP. A new tabu search algorithm is designed to solve the problem and the examples testing show the efficiency of the proposed algorithm. This paper focuses on constructing a double objective mathematical programming model for DCVRPSDO and designing an adaptive tabu search algorithm (ATSA) with good performance to solving the problem. The performance of the ATSA is improved by adding some strategies into the search process, including: (a) a strategy of discrete split deliveries by order is used to split the customer demand; (b) a multi-neighborhood structure is designed to enhance the ability of global optimization; (c) two levels of evaluation objectives are set to select the current solution and the best solution; (d) a discriminating strategy of that the best solution must be feasible and the current solution can accept some infeasible solution, helps to balance the performance of the solution and the diversity of the neighborhood solution; (e) an adaptive penalty mechanism will help the candidate solution be closer to the neighborhood of feasible solution; (f) a strategy of tabu releasing is used to transfer the current solution into a new neighborhood of the better solution. [ABSTRACT FROM AUTHOR]

Delivery of recombinant proteins to therapeutic cells is limited by a lack of efficient methods. This hinders the use of transcription factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleoproteins to develop cell therapies. Here, we report a soluble peptide designed for the direct delivery of proteins to mammalian cells including human stem cells, hard-to-modify primary natural killer (NK) cells, and cancer cell models. This peptide is composed of a 6x histidine-rich domain fused to the endosomolytic peptide CM18 and the cell penetrating peptide PTD4. A less than two-minute co-incubation of 6His-CM18-PTD4 peptide with spCas9 and/or asCpf1 CRISPR ribonucleoproteins achieves robust gene editing. The same procedure, co-incubating with the transcription factor HoxB4, achieves transcriptional regulation. The broad applicability and flexibility of this DNA- and chemical-free method across different cell types, particularly hard-to-transfect cells, opens the way for a direct use of proteins for biomedical research and cell therapy manufacturing. [ABSTRACT FROM AUTHOR]

Virus-mediated expression of defined transcription factor (TF) genes can effectively induce cellular reprogramming. However, sustained expression of the TFs often hinders pluripotent stem cell (PSC) differentiation into specific cell types, as each TF exerts its effect on PSCs for a defined period of time during differentiation. Here, we applied a bacterial type III secretion system (T3SS)-based protein delivery tool to directly translocate TFs in the form of protein into human PSCs. This transient protein delivery technique showed high delivery efficiency for hPSCs, and it avoids potential genetic alterations caused by the introduction of transgenes. In an established cardiomyocyte de novo differentiation procedure, five transcriptional factors, namely GATA4, MEF2C, TBX5, ESRRG and MESP1 (abbreviated as GMTEM), were translocated at various time points. By detecting the expression of cardiac marker genes (Nkx2.5 and cTnT), we found that GMTEM proteins delivered on mesoderm stage of the cardiomyocytes lineage differentiation significantly enhanced both the human ESC and iPSC differentiation into cardiomyocytes, while earlier or later delivery diminished the enhancing effect. Furthermore, all of the five factors were required to enhance the cardiac differentiation. This work provides a virus-free strategy of transient transcription factors delivery for directing human stem cell fate without jeopardizing genome integrity, thus safe for biomedical applications. [ABSTRACT FROM AUTHOR]

Nanoparticles represent promising gene delivery systems in biomedicine to facilitate prolonged gene expression with low toxicity compared to viral vectors. Specifically, nanoparticles of calcium phosphate (nCaP), the main inorganic component of human bone, exhibit high biocompatibility and good biodegradability and have been reported to have high affinity for protein or DNA, having thus been used as gene transfer vectors. On the other hand, Octa-arginine (R8), which has a high permeability to cell membrane, has been reported to improve intracellular delivery systems. Here, we present an optimized method for nCaP-mediated gene delivery using an octa-arginine (R8)-functionalized nCaP vector containing a marker or functional gene construct. nCaP particle size was between 220–580 nm in diameter and all R8-functionalized nCaPs carried a positive charge. R8 concentration significantly improved nCaP transfection efficiency with high cell compatibility in human mesenchymal stem cells (hMSC) and human osteoblasts (hOB) in particular, suggesting nCaPs as a good option for non-viral vector gene delivery. Furthermore, pre-treatment with different endocytosis inhibitors identified that the endocytic pathway differed among cell lines and functionalized nanoparticles, with amiloride increasing transfection efficiency of R8-functionalized nCaPs in hMSC and hOB. [ABSTRACT FROM AUTHOR]

Different viral and non-viral vectors have been designed to allow the delivery of nucleic acids in gene therapy. In general, non-viral vectors have been associated with increased safety for in vivo use; however, issues regarding their efficacy, toxicity and stability continue to drive further research. Thus, the aim of this study was to evaluate the potential use of the polymerizable diacetylenic lipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) as a strategy to formulate stable cationic lipopolymers in the delivery and protection of plasmid DNA. Cationic lipopolymers were prepared following two different methodologies by using DC8,9PC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the cationic lipids (CL) 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), stearylamine (SA), and myristoylcholine chloride (MCL), in a molar ratio of 1:1:0.2 (DMPC:DC8,9PC:CL). The copolymerization methodology allowed obtaining cationic lipopolymers which were smaller in size than those obtained by the cationic addition methodology although both techniques presented high size stability over a 166-day incubation period at 4°C. Cationic lipopolymers containing DOTAP or MCL were more efficient in complexing DNA than those containing SA. Moreover, lipopolymers containing DOTAP were found to form highly stable complexes with DNA, able to resist serum DNAses degradation. Furthermore, neither of the cationic lipopolymers (with or without DNA) induced red blood cell hemolysis, although metabolic activity determined on the L-929 and Vero cell lines was found to be dependent on the cell line, the formulation and the presence of DNA. The high stability and DNA protection capacity as well as the reduced toxicity determined for the cationic lipopolymer containing DOTAP highlight the potential advantage of using lipopolymers when designing novel non-viral carrier systems for use in in vivo gene therapy. Thus, this work represents the first steps toward developing a cationic lipopolymer-based gene delivery system using polymerizable and cationic lipids. [ABSTRACT FROM AUTHOR]

Background: Development of an effective non-viral vaccine against hepatitis C virus infection is of a great importance. Gelatin nanoparticles (Gel.NPs) have an attention and promising approach as a viable carrier for delivery of vaccine, gene, drug and other biomolecules in the body. Aim of work: The present study aimed to develop stable Gel.NPs conjugated with nonstructural protein 2 (NS2) gene of Hepatitis C Virus genotype 4a (HCV4a) as a safe and an efficient vaccine delivery system. Methods and results: Gel.NPs were synthesized and characterized (size: 150±2 nm and zeta potential +17.6 mv). NS2 gene was successfully cloned and expressed into E. coli M15 using pQE-30 vector. Antigenicity of the recombinant NS2 protein was confirmed by Western blotting to verify the efficiency of NS2 as a possible vaccine. Then NS2 gene was conjugated to gelatin nanoparticles and a successful conjugation was confirmed by labeling and imaging using Confocal Laser Scanning Microscope (CLSM). Interestingly, the transformation of the conjugated NS2/Gel.NPs complex into E. coli DH5-α was 50% more efficient than transformation with the gene alone. In addition, conjugated NS2/Gel.NPs with ratio 1:100 (w/w) showed higher transformation efficiency into E. coli DH5-α than the other ratios (1:50 and 2:50). Conclusion: Gel.NPs effectively enhanced the gene delivery in bacterial cells without affecting the structure of NS2 gene and could be used as a safe, easy, rapid, cost-effective and non-viral vaccine delivery system for HCV. [ABSTRACT FROM AUTHOR]

Glycolipids might become a new type of promising non-viral gene delivery systems because of their low cytotoxicity, structural diversity, controllable aqua- and lipo-solubility, appropriate density and distribution of positive charges, high transfer efficiency and potential targeting function. In this study, four kinds of L-arabinose-based cationic glycolipids (Ara-DiC12MA, Ara-DiC14MA, Ara-DiC16MA and Ara-DiC18MA) containing quaternary ammonium as hydrophilic headgroup and two alkane chains as hydrophobic domain were synthesized and characterized. They were observed to have strong affinities for plasmid DNA (pDNA) and siRNA, the pDNA can be completely condensed at N/P ratio less than 2, and the siRNA can be completely retarded at N/P ratio less than 3. The dynamic light scattering (DLS) experiment and atomic force microscopy (AFM) experiment demonstrated that cationic lipids and their lipoplexes possessed suitable particle sizes with near-spherical shape and proper ζ-potentials for cell transfection. The Ara-DiC16MA liposome was found to have good transfection efficacy in HEK293, PC-3 and Mat cells compared with other three kinds of liposomes, and also maintain low cytotoxicity and better uptake capability in vitro. Furthermore, the gene silencing assay showed that Ara-DiC14MA and Ara-DiC16MA liposomes have demonstrated effective delivery and higher gene knockdown activity (>80%) in the above mentioned cells than Lipofectamine 2000. These results indicated Ara-DiC16MA can be developed for efficient and low toxic gene delivery. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B. [ABSTRACT FROM AUTHOR]

Recent advances in gene delivery into cells allow improved therapeutic effects in gene therapy trials. To increase the bioavailability of applied cells, it is of great interest that transfected cells remain at the application site and systemic spread is minimized. In this study, we tested clinically used biodegradable poly(lactic acid-co-glycolic acid) (PLGA) scaffolds (Vicryl & Ethisorb) as transient carriers for genetically modified cells. To this aim, we used human fibroblasts and examined attachment and proliferation of untransfected cells on the scaffolds in vitro, as well as the mechanical properties of the scaffolds at four time points (1, 3, 6 and 9 days) of cultivation. Furthermore, the adherence of cells transfected with green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF165) and also VEGF165 protein secretion were investigated. Our results show that human fibroblasts adhere on both types of PLGA scaffolds. However, proliferation and transgene expression capacity were higher on Ethisorb scaffolds most probably due to a different architecture of the scaffold. Additionally, cultivation of the cells on the scaffolds did not alter their biomechanical properties. The results of this investigation could be potentially exploited in therapeutic regiments with areal delivery of transiently transfected cells and may open the way for a variety of applications of cell-based gene therapy, tissue engineering and regenerative medicine. [ABSTRACT FROM AUTHOR]

Corneal scarring is due to aberrant activity of the transforming growth factor β (TGFβ) signaling pathway following traumatic, mechanical, infectious, or surgical injury. Altered TGFβ signaling cascade leads to downstream Smad (Suppressor of mothers against decapentaplegic) protein-mediated signaling events that regulate expression of extracellular matrix and myogenic proteins. These events lead to transdifferentiation of keratocytes into myofibroblasts through fibroblasts and often results in permanent corneal scarring. Hence, therapeutic targets that reduce transdifferentiation of fibroblasts into myofibroblasts may provide a clinically relevant approach to treat corneal fibrosis and improve long-term visual outcomes. Smad7 protein regulates the functional effects of TGFβ signaling during corneal wound healing. We tested that targeted delivery of Smad7 using recombinant adeno-associated virus serotype 5 (AAV5-Smad7) delivered to the corneal stroma can inhibit corneal haze post photorefractive keratectomy (PRK) in vivo in a rabbit corneal injury model. We demonstrate that a single topical application of AAV5-Smad7 in rabbit cornea post-PRK led to a significant decrease in corneal haze and corneal fibrosis. Further, histopathology revealed lack of immune cell infiltration following AAV5-Smad7 gene transfer into the corneal stroma. Our data demonstrates that AAV5-Smad7 gene therapy is relatively safe with significant potential for the treatment of corneal disease currently resulting in fibrosis and impaired vision. [ABSTRACT FROM AUTHOR]

PIM1, a pro-survival gene encoding a serine/ threonine kinase, influences cell proliferation and survival. Modification of cardiac progenitor cells (CPCs) or cardiomyocytes with PIM1 using a lentivirus-based delivery method showed long-term improved cardiac function after myocardial infarction (MI). However, lentivirus based delivery methods have stringent FDA regulation with respect to clinical trials. To provide an alternative and low risk PIM1 delivery method, this study examined the use of a non-viral modified plasmid-minicircle (MC) as a vehicle to deliver PIM1 into mouse CPCs (mCPCs) in vitro and the myocardium in vivo. MC containing a turbo gfp reporter gene (gfp-MC) was used as a transfection and injection control. PIM1 was subcloned into gfp-MC (PIM1-MC) and then transfected into mCPCs at an efficiency of 29.4±3.7%. PIM1-MC engineered mCPCs (PIM1-mCPCs) exhibit significantly (P<0.05) better survival rate under oxidative treatment. PIM1-mCPCs also exhibit 1.9±0.1 and 2.2±0.2 fold higher cell proliferation at 3 and 5 days post plating, respectively, as compared to gfp-MC transfected mCPCs control. PIM1-MC was injected directly into ten-week old adult FVB female mice hearts in the border zone immediately after MI. Delivery of PIM1 into myocardium was confirmed by GFP+ cardiomyocytes. Mice with PIM1-MC injection showed increased protection compared to gfp-MC injection groups measured by ejection fraction at 3 and 7 days post injury (P = 0.0379 and P = 0.0262 by t-test, respectively). Success of PIM1 delivery and integration into mCPCs in vitro and cardiomyocytes in vivo by MC highlights the possibility of a non-cell based therapeutic approach for treatment of ischemic heart disease and MI. [ABSTRACT FROM AUTHOR]

Double-stranded RNA (dsRNA)-mediated gene silencing, also known as RNA interference (RNAi), has been a breakthrough technology for functional genomic studies and represents a potential tool for the management of insect pests. Since the inception of RNAi numerous studies documented successful introduction of exogenously synthesized dsRNA or siRNA into an organism triggering highly efficient gene silencing through the degradation of endogenous RNA homologous to the presented siRNA. Managing hemipteran insect pests, especially Halyomorpha halys (Stål) (Heteroptera: Pentatomidae), the brown marmorated stink bug (BMSB), is critical to food productivity. BMSB was recently introduced into North America where it is both an invasive agricultural pest of high value specialty, row, and staple crops, as well as an indoor nuisance pest. RNAi technology may serve as a viable tool to manage this voracious pest, but delivery of dsRNA to piercing-sucking insects has posed a tremendous challenge. Effective and practical use of RNAi as molecular biopesticides for biocontrol of insects like BMSB in the environment requires that dsRNAs be delivered in vivo through ingestion. Therefore, the key challenge for molecular biologists in developing insect-specific molecular biopesticides is to find effective and reliable methods for practical delivery of stable dsRNAs such as through oral ingestion. Here demonstrated is a reliable delivery system of effective insect-specific dsRNAs through oral feeding through a new delivery system to induce a significant decrease in expression of targeted genes such as JHAMT and Vg. This state-of-the-art delivery method overcomes environmental delivery challenges so that RNAi is induced through insect-specific dsRNAs orally delivered to hemipteran and other insect pests. [ABSTRACT FROM AUTHOR]

A recent theory suggests that endocytosis is involved in uptake and intracellular transport of electrotransfected plasmid DNA (pDNA). The goal of the current study was to understand if approaches used previously to improve endocytosis of gene delivery vectors could be applied to enhancing electrotransfection efficiency (eTE). Results from the study showed that photochemically induced endosomal escape, which could increase poly-L-lysine (PLL)-mediated gene delivery, decreased eTE. The decrease could not be blocked by treatment of cells with endonuclease inhibitors (aurintricarboxylic acid and zinc ion) or antioxidants (L-glutamine and ascorbic acid). Chemical treatment of cells with an endosomal trafficking inhibitor that blocks endosome progression, bafilomycin A1, resulted in a significant decrease in eTE. However, treatment of cells with lysosomotropic agents (chloroquine and ammonium chloride) had little effects on eTE. These data suggested that endosomes played important roles in protecting and intracellular trafficking of electrotransfected pDNA. [ABSTRACT FROM AUTHOR]

Lack of capacity to cross the nucleus membrane seems to be one of the main reasons for the lower transfection efficiency of gene vectors observed in vivo study than in vitro. To solve this problem, a new non-viral gene vector was designed. First, a degradable polyethylenimine (PEI) derivate was synthesized by crosslinking low-molecular-weight (LMW) PEI with N-octyl-N-quaternary chitosan (OTMCS), and then adopting a designed trifunctional peptide (RGDC-TAT-NLS) with good tumor targeting, cell uptake and nucleus transport capabilities to modify OTMCS-PEI. The new gene vector was termed as OTMCS-PEI-R18 and characterized in terms of its chemical structure and biophysical parameters. Gene transfection efficiency and nucleus transport mechanism of this vector were also evaluated. The polymer showed controlled degradation and remarkable buffer capabilities with the particle size around 100–300 nm and the zeta potential ranged from 5 mV to 40 mV. Agraose gel electrophoresis showed that OTMCS-PEI-R18 could effectively condensed plasmid DNA at a ratio of 1.0. Besides, the polymer was stable in the presence of sodium heparin and could resist digestion by DNase I at a concentration of 63U DNase I/DNA. OTMCS-PEI-R18 also showed much lower cytotoxicity and better transfection rates compared to polymers OTMCS-PEI-R13, OTMCS-PEI and PEI 25 KDa in vitro and in vivo. Furthermore, OTMCS-PEI-R18/DNA complexes could accumulate in the nucleus well soon and not rely on mitosis absolutely due to the newly incorporated ligand peptide NLS with the specific nuclear delivery pathway indicating that the gene delivery system OTMCS-PEI-R18 could reinforce gene transfection efficiency in vivo. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is the most common and aggressive primary intracranial brain tumor in adults with a mean survival of 14 to 15 months. Aberrant activation of the epidermal growth factor receptor (EGFR) plays a significant role in GBM progression, with amplification or overexpression of EGFR in 60% of GBM tumors. To target EGFR expressed by GBM, we have developed a strategy to deliver the coding sequence for cetuximab, an anti-EGFR antibody, directly to the CNS using an adeno-associated virus serotype rh.10 gene transfer vector. The data demonstrates that single, local delivery of an anti-EGFR antibody by an AAVrh.10 vector coding for cetuximab (AAVrh.10Cetmab) reduces GBM tumor growth and increases survival in xenograft mouse models of a human GBM EGFR-expressing cell line and patient-derived GBM. AAVrh10.CetMab-treated mice displayed a reduction in cachexia, a significant decrease in tumor volume and a prolonged survival following therapy. Adeno-associated-directed delivery of a gene encoding a therapeutic anti-EGFR monoclonal antibody may be an effective strategy to treat GBM. [ABSTRACT FROM AUTHOR]

Introduction: Expressing exogenous genes after naked DNA delivery into hepatocytes might achieve sustained and high expression of human proteins. Tail vein DNA injection is an efficient procedure for gene transfer in murine liver. Hydrodynamic procedures in large animals require organ targeting, and improve with liver vascular exclusion. In the present study, two closed liver hydrofection models employing the human alpha-1-antitrypsin (hAAT) gene are compared to reference standards in order to evaluate their potential clinical interest. Material and Methods: A solution of naked DNA bearing the hAAT gene was retrogradely injected in 7 pig livers using two different closed perfusion procedures: an endovascular catheterization-mediated procedure (n = 3) with infrahepatic inferior vena cava and portal vein blockage; and a surgery-mediated procedure (n = 4) with completely sealed liver. Gene transfer was performed through the suprahepatic inferior cava vein in the endovascular procedure and through the infrahepatic inferior vena cava in the surgical procedure. The efficiency of the procedures was evaluated 14 days after hydrofection by quantifying the hAAT protein copies per cell in tissue and in plasma. For comparison, samples from mice (n = 7) successfully hydrofected with hAAT and healthy human liver segments (n = 4) were evaluated. Results: Gene decoding occurs efficiently using both procedures, with liver vascular arrest improving its efficiency. The surgically closed procedure (sealed organ) reached higher tissue protein levels (4x10^5- copies/cell) than the endovascular procedure, though the levels were lower than in human liver (5x10^6- copies/cell) and hydrofected mouse liver (10^6- copies/cell). However, protein levels in plasma were lower (p<0.001) than the reference standards in all cases. Conclusion: Hydrofection of hAAT DNA to “in vivo” isolated pig liver mediates highly efficient gene delivery and protein expression in tissue. Both endovascular and surgically closed models mediate high tissue protein expression. Impairment of protein secretion to plasma is observed and might be species-related. This study reinforces the potential application of closed liver hydrofection for therapeutic purposes, provided protein secretion improves. [ABSTRACT FROM AUTHOR]

Background: Five-to-eighteen percent of pregnancies worldwide end in preterm birth, which is the major cause of neonatal death and morbidity. Approximately 30% of the variation in gestational age at birth can be attributed to genetic factors. Genome-wide association studies (GWAS) have not shown robust evidence of association with genomic loci yet. Methods: We separately investigated 1921 Norwegian mothers and 1199 children from pregnancies with spontaneous onset of delivery. Individuals were further divided based on the onset of delivery: initiated by labor or prelabor rupture of membranes. Genetic association with ultrasound-dated gestational age was evaluated using three genetic models and adaptive permutations. The top-ranked loci were tested for enrichment in 12 candidate gene-sets generated by text-mining PubMed abstracts containing pregnancy-related keywords. Results: The six GWAS did not reveal significant associations, with the most extreme empirical p = 5.1 × 10−7. The top loci from maternal GWAS with deliveries initiated by labor showed significant enrichment in 10 PubMed gene-sets, e.g., p = 0.001 and 0.005 for keywords "uterus" and "preterm" respectively. Enrichment signals were mainly caused by infection/inflammation-related genes TLR4, NFKB1, ABCA1, MMP9. Literature-informed analysis of top loci revealed further immunity genes: IL1A, IL1B, CAMP, TREM1, TFRC, NFKBIA, MEFV, IRF8, WNT5A. Conclusion: Our analyses support the role of inflammatory pathways in determining pregnancy duration and provide a list of 32 candidate genes for a follow-up work. We observed that the top regions from GWAS in mothers with labor-initiated deliveries significantly more often overlap with pregnancy-related genes than would be expected by chance, suggesting that increased sample size would benefit similar studies. [ABSTRACT FROM AUTHOR]

RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease. [ABSTRACT FROM AUTHOR]

Metal complexes might become a new type of promising gene delivery systems because of their low cytotoxicity, structural diversity, controllable aqua- and lipo-solubility, and appropriate density and distribution of positive charges. In this study, Zn2+ complexes (1–10) formed with a series of ligands contained benzimidazole(bzim)were prepared and characterized. They were observed to have different affinities for DNA, dependent on their numbers of positive charges, bzim groups, and coordination structures around Zn2+. The binding induced DNA to condensate into spherical nanoparticles with ~ 50 nm in diameter. The cell transfection efficiency of the DNA nanoparticles was poor, although they were low toxic. The sequential addition of the cell-penetrating peptide (CPP) TAT(48–60) and polyethylene glycol (PEG) resulted in the large DNA condensates (~ 100 nm in diameter) and the increased cellular uptake. The clathrin-mediated endocytosis was found to be a key cellular uptake pathway of the nanoparticles formed with or without TAT(48–60) or/and PEG. The DNA nanoparticles with TAT(48–60) and PEG was found to have the cell transfection efficiency up to 20% of the commercial carrier Lipofect. These results indicated that a simple Zn2+-bzim complex-based composite system can be developed for efficient and low toxic gene delivery through the combination with PEG and CPPs such as TAT. [ABSTRACT FROM AUTHOR]

Objective: Hepatocellular carcinoma (HCC), mostly derived from hepatitis or cirrhosisis, is one of the most common types of liver cancer. T-cell mediated immune response elicited by CD/TK double suicide gene has shown a substantial antitumor effect in HCC. Integrin αVβ3 over expresssion has been suggested to regulate the biology behavior of HCC. In this study, we investigated the strategy of incorporating CD/TK double suicide gene and anti-αVβ3 integrin monoclonal antibodies into cationic microbubbles (CMBsαvβ3), and evaluated its killing effect in HCC cells. Methods: To improve the transfection efficiency of targeted CD/TK double suicide gene, we adopted cationic microbubbles (CMBs), a cationic delivery agent with enhanced DNA-carrying capacity. The ultrasound and high speed shearing method was used to prepare the non-targeting cationic microbubbles (CMBs). Using the biotin-avidin bridge method, αVβ3 integrin antibody was conjugated to CMBs, and CMBsαvβ3 was generated to specifically target to HepG2 cells. The morphology and physicochemical properties of the CMBsαvβ3 was detected by optical microscope and zeta detector. The conjugation of plasmid and the antibody in CMBsαvβ3 were examined by immunofluorescent microscopy and flow cytometry. The binding capacities of CMBsαvβ3 and CMBs to HCC HepG2 and normal L-02 cells were compared using rosette formation assay. To detect EGFP fluorescence and examine the transfection efficiencies of CMBsαvβ3 and CMBs in HCC cells, fluorescence microscope and contrast-enhanced sonography were adopted. mRNA and protein level of CD/TK gene were detected by RT-PCR and Western blot, respectively. To evaluate the anti-tumor effect of CMBsαvβ3, HCC cells with CMBsαvβ3 were exposed to 5-flurocytosine / ganciclovir (5-FC/GCV). Then, cell cycle distribution after treatment were detected by PI staining and flow cytometry. Apoptotic cells death were detected by optical microscope and assessed by MTT assay and TUNEL-staining assay. Results: CMBsαvβ3 had a regular shape and good dispersion. Compared to CMBs, CMBsαvβ3 had more stable concentrations of αVβ3 ligand and pEGFP-KDRP-CD/TK, and CMBsαvβ3 was much sticker to HepG2 HCC cells than normal liver L-02cells. Moreover, after exposed to anti-αVβ3 monoclonal antibody, the adhesion of CMBsαvβ3 to HepG2 cells and L-02 cells were significantly reduced. Also, CMBsαvβ3 demonstrated a substantially higher efficiency in pEGFP-KDRP-CD/TK plasmid transfection in HepG2 cells than CMBs. In addition, CMBsαvβ3 could significantly facilitate 5-FC/GCV-induced cell cycle arrest in S phase. Moreover, treatment of 5-FC/GCV combined with CMBsαvβ3 resulted in a marked apoptotic cell death in HepG2 and SK-Herp-1 HCC cells. In vitro, treatment of 5-FC/GCV combined with CMBsαvβ3 suppresed cell proliferation. In nude mice model, 5-FU + GCV combined with plasmid + CMBsαvβ3were able to significantly suppress tumor volumes. Conclusion: Through biotin-avidin mediation system, CMBsαvβ3 were successfully generated to specifically target HCC HepG2 cells. More importantly, CMBsαvβ3 could significantly facilitate 5-FC/GCV-induced cell cycle arrest and apoptotic cell death in HepG2 cells. Our study demonstrated a potential strategy that could be translated clinically to improve liver tumor gene delivery. [ABSTRACT FROM AUTHOR]

Recent clinical trials have shown that in vivo and ex vivo gene therapy strategies can be an option for the treatment of several neurological disorders. Both strategies require efficient and safe vectors to 1) deliver the therapeutic gene directly into the CNS or 2) to genetically modify stem cells that will be used as Trojan horses for the systemic delivery of the therapeutic protein. A group of target diseases for these therapeutic strategies are mitochondrial encephalopathies due to mutations in nuclear DNA genes. In this study, we have developed a lentiviral vector (CCoq9WP) able to overexpress Coq9 mRNA and COQ9 protein in mouse embryonic fibroblasts (MEFs) and hematopoietic progenitor cells (HPCs) from Coq9R239X mice, an animal model of mitochondrial encephalopathy due to primary Coenzyme Q (CoQ) deficiency. Ectopic over-expression of Coq9 in both cell types restored the CoQ biosynthetic pathway and mitochondrial function, improving the fitness of the transduced cells. These results show the potential of the CCoq9WP lentiviral vector as a tool for gene therapy to treat mitochondrial encephalopathies. [ABSTRACT FROM AUTHOR]

The heterogeneity of spontaneous preterm birth (SPTB) requires an interdisciplinary approach to determine potential predictive risk factors of early delivery. The aim of this study was to investigate maternal whole blood gene expression profiles associated with spontaneous preterm birth (SPTB, <37 weeks) in asymptomatic pregnant women. The study population was a matched subgroup of women (51 SPTBs, 114 term delivery controls) who participated in the All Our Babies community based cohort in Calgary (n = 1878). Maternal blood at 17–23 (sampling time point 1, T1) and 27–33 weeks of gestation (T2) were collected. Total RNA was extracted and microarray was performed on 326 samples (165 women). Univariate analyses determined significant clinical factors and differential gene expression associated with SPTB. Thirteen genes were validated using qRT-PCR. Three multivariate logistic models were constructed to identify gene expression at T1 (Model A), T2 (Model B), and gene expression fold change from T1 to T2 (Model C) associated with SPTB. All models were adjusted for clinical factors. Model C can predict SPTB with 65% sensitivity and 88% specificity in asymptomatic women after adjusting for history of abortion and anaemia (occurring before T2). Clinical data enhanced the sensitivity of the Models to predict SPTB. In conclusion, clinical factors and whole blood gene expression are associated with SPTB in asymptomatic women. An effective screening tool for SPTB during pregnancy would enable targeted preventive approaches and personalised antenatal care. [ABSTRACT FROM AUTHOR]

Purpose: To test the efficacy of systemic gene delivery of a mutant form of erythropoietin (EPO-R76E) that has attenuated erythropoietic activity, in a mouse model of autosomal dominant retinitis pigmentosa. Methods: Ten-day old mice carrying one copy of human rhodopsin with the P23H mutation and both copies of wild-type mouse rhodopsin (hP23H RHO+/-,mRHO+/+) were injected into the quadriceps with recombinant adeno-associated virus (rAAV) carrying either enhanced green fluorescent protein (eGFP) or EpoR76E. Visual function (electroretinogram) and retina structure (optical coherence tomography, histology, and immunohistochemistry) were assessed at 7 and 12 months of age. Results: The outer nuclear layer thickness decreased over time at a slower rate in rAAV.EpoR76E treated as compared to the rAAV.eGFP injected mice. There was a statistically significant preservation of the electroretinogram at 7, but not 12 months of age. Conclusions: Systemic EPO-R76E slows death of the photoreceptors and vision loss in hP23H RHO+/-,mRHO+/+ mice. Treatment with EPO-R76E may widen the therapeutic window for retinal degeneration patients by increasing the number of viable cells. Future studies might investigate if co-treatment with EPO-R76E and gene replacement therapy is more effective than gene replacement therapy alone. [ABSTRACT FROM AUTHOR]

The ability of monocytes and monocyte-derived macrophages (MDM) to travel towards chemotactic gradient, traverse tissue barriers, and accumulate precisely at diseased sites makes them attractive candidates as drug carriers and therapeutic gene delivery vehicles targeting the brain, where treatments are often hampered by the blockade of the blood brain barrier (BBB). This study was designed to fully establish an optimized cell-based delivery system using monocytes and MDM, by evaluating their homing efficiency, engraftment potential, as well as carriage and delivery ability to transport nano-scaled particles and exogenous genes into the brain, following the non-invasive intravenous (IV) cell adoptive transfer in an acute neuroinflammation mouse model induced by intracranial injection of Escherichia coli lipopolysaccharides. We demonstrated that freshly isolated monocytes had superior inflamed-brain homing ability over MDM cultured in the presence of macrophage colony stimulating factor. In addition, brain trafficking of IV infused monocytes was positively correlated with the number of adoptive transferred cells, and could be further enhanced by transient disruption of the BBB with IV administration of Mannitol, Bradykinin or Serotonin right before cell infusion. A small portion of transmigrated cells was detected to differentiate into IBA-1 positive cells with microglia morphology in the brain. Finally, with the use of superparamagnetic iron oxide nanoparticles SHP30, the ability of nanoscale agent-carriage monocytes to enter the inflamed brain region was validated. In addition, lentiviral vector DHIV-101 was used to introduce green fluorescent protein (GFP) gene into monocytes, and the exogenous GFP gene was detected in the brain at 48 hours following IV infusion of the transduced monocytes. All together, our study has set up the optimized conditions for the more-in-depth tests and development of monocyte-mediated delivery, and our data supported the notion to use monocytes as a non-invasive cell-based delivery system for the brain. [ABSTRACT FROM AUTHOR]

Diffuse large B-cell lymphoma (DLBCL) is characterized by great genetic and clinical heterogeneity which complicates prognostic prediction and influences treatment efficacy. The most common regimen, R-CHOP, consists of a combination of anthracycline- and immuno-based drugs including Rituximab. It remains elusive how and to which extent genetic variability impacts the response and potential tolerance to R-CHOP. Hence, an improved understanding of mechanisms leading to drug tolerance in B-cells is crucial, and modelling by genetic intervention directly in B-cells is fundamental in such investigations. Lentivirus-based gene vectors are widely used gene vehicles, which in B-cells are an attractive alternative to potentially toxic transfection-based methodologies. Here, we investigate the use of VSV-G-pseudotyped lentiviral vectors in B-cells for exploring the impact of microRNAs on tolerance to Rituximab. Notably, we find that robust lentiviral transduction of cancerous B-cell lines markedly and specifically enhances the resistance of transduced germinal center B-cells (GCBs) to Rituximab. Although Rituximab works partially through complement-mediated cell lysis, increased tolerance is not achieved through effects of lentiviral transduction on cell death mediated by complement. Rather, reduced levels of PARP1 and persistent high levels of CD43 in Rituximab-treated GCBs demonstrate anti-apoptotic effects of lentiviral transduction that may interfere with the outcome and interpretation of Rituximab tolerance studies. Our findings stress that caution should be exercised exploiting lentiviral vectors in studies of tolerance to therapeutics in DLBCL. Importantly, however, we demonstrate the feasibility of using the lentiviral gene delivery platform in studies addressing the impact of specific microRNAs on Rituximab responsiveness. [ABSTRACT FROM AUTHOR]

Objective and Methods: This study investigated the potential for protective effects of human umbilical cord blood mononuclear cells (UCB-MCs) genetically modified with the VEGF and GNDF genes on contusion spinal cord injury (SCI) in rats. An adenoviral vector was constructed for targeted delivery of VEGF and GDNF to UCB-MCs. Using a rat contusion SCI model we examined the efficacy of the construct on tissue sparing, glial scar severity, the extent of axonal regeneration, recovery of motor function, and analyzed the expression of the recombinant genes VEGF and GNDF in vitro and in vivo. Results: Transplantation of UCB-MCs transduced with adenoviral vectors expressing VEGF and GDNF at the site of SCI induced tissue sparing, behavioral recovery and axonal regeneration comparing to the other constructs tested. The adenovirus encoding VEGF and GDNF for transduction of UCB-MCs was shown to be an effective and stable vehicle for these cells in vivo following the transplantation into the contused spinal cord. Conclusion: Our results show that a gene delivery using UCB-MCs-expressing VEGF and GNDF genes improved both structural and functional parameters after SCI. Further histological and behavioral studies, especially at later time points, in animals with SCI after transplantation of genetically modified UCB-MCs (overexpressing VEGF and GDNF genes) will provide additional insight into therapeutic potential of such cells. [ABSTRACT FROM AUTHOR]

Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy. [ABSTRACT FROM AUTHOR]

Gene delivery holds therapeutic promise for the treatment of neurological diseases and spinal cord injury. Although several studies have investigated the use of non-viral vectors, such as polyethylenimine (PEI), their clinical value is limited by their cytotoxicity. Recently, biodegradable poly (lactide-co-glycolide) (PLGA) nanospheres have been explored as non-viral vectors. Here, we show that modification of PLGA nanospheres with 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) enhances gene transfection efficiency. PLGA/DC-Chol nanospheres encapsulating DNA were prepared using a double emulsion-solvent evaporation method. PLGA/DC-Chol nanospheres were less cytotoxic than PEI both in vitro and in vivo. DC-Chol modification improved the uptake of nanospheres, thereby increasing their transfection efficiency in mouse neural stem cells in vitro and rat spinal cord in vivo. Also, transgene expression induced by PLGA nanospheres was higher and longer-lasting than that induced by PEI. In a rat model of spinal cord injury, PLGA/DC-Chol nanospheres loaded with vascular endothelial growth factor gene increased angiogenesis at the injury site, improved tissue regeneration, and resulted in better recovery of locomotor function. These results suggest that DC-Chol-modified PLGA nanospheres could serve as therapeutic gene delivery vehicles for spinal cord injury. [ABSTRACT FROM AUTHOR]

The nuclear receptor FXR acts as an intracellular bile salt sensor that regulates synthesis and transport of bile salts within their enterohepatic circulation. In addition, FXR is involved in control of a variety of crucial metabolic pathways. Four FXR splice variants are known, i.e. FXRα1-4. Although these isoforms show differences in spatial and temporal expression patterns as well as in transcriptional activity, the physiological relevance hereof has remained elusive. We have evaluated specific roles of hepatic FXRα2 and FXRα4 by stably expressing these isoforms using liver-specific self-complementary adeno-associated viral vectors in total body FXR knock-out mice. The hepatic gene expression profile of the FXR knock-out mice was largely normalized by both isoforms. Yet, differential effects were also apparent; FXRα2 was more effective in reducing elevated HDL levels and transrepressed hepatic expression of Cyp8b1, the regulator of cholate synthesis. The latter coincided with a switch in hydrophobicity of the bile salt pool. Furthermore, FXRα2-transduction caused an increased neutral sterol excretion compared to FXRα4 without affecting intestinal cholesterol absorption. Our data show, for the first time, that hepatic FXRα2 and FXRα4 differentially modulate bile salt and lipoprotein metabolism in mice. [ABSTRACT FROM AUTHOR]

Gene therapy with human adenovirus type 5 (Ad5) has been extensively explored for the treatment of diseases resistant to traditional therapies. Intravenous administration leads to rapid clearance from blood circulation and high liver accumulation, which restrict the use of Ad-based vectors in clinical gene therapy protocols that involve systemic administration. We have previously proposed that such limitations can be improved by engineering artificial lipid envelopes around Ad and designed a variety of artificial lipid bilayer envelopes around the viral capsid. In this study, we sought to explore further opportunities that the artificially enveloped virus constructs could offer, by designing a previously unreported gene therapy vector by simultaneous envelopment of Ad and siRNA within the same lipid bilayer. Such a dual-activity vector can offer efficacious therapy for different genetic disorders where both turning on and switching off genes would be needed. Dynamic light scattering, transmission electron microscopy and atomic force microscopy were used to characterize these vectors. Agarose gel electrophoresis, Ribo green and dot blot assays showed that siRNA and Ad virions can be enveloped together within lipid bilayers at high envelopment efficiency. Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors. [ABSTRACT FROM AUTHOR]