Gene Delivery Techniques Research Articles

Surgical methods for cardiac gene transfer.

Over the last few decades, progress in the molecular biology of cardiomyocytes and the decoding of their genome through DNA sequencing has improved our insight into the pathophysiological mechanisms underlying the development of many heart diseases. The further evolution of genetic methods to investigate signal transduction, microarray analysis, and the development of transgenic animal models of various diseases including myocardial hypertrophy and others has contributed to the emergence of cardiovascular gene therapy as an important new paradigm in clinical and experimental research. These factors have resulted in the translation of cardiovascular therapy from animal studies to clinical applications more rapidly than expected. Successful plasmid DNA transfection after direct injection into beating rat hearts was demonstrated by expression of a recombinant β-galactosidase gene after 3–4 weeks. These “The further evolution of genetic methods ... has contributed to the emergence of cardiovascular gene therapy as an important new paradigm in clinical and experimental research.” results showed for the first time that cardiac muscle has a unique ability to take up and express injected recombinant DNA without recipient cell division [2]. The next historical step was the proof that viral-mediated gene transfer after intramyocardial injection is thousands of times more efficient than plasmid DNA; however, the percentage of cardiomyocytes expressing marker genes was largely limited to regions adjacent to the injection site [3,4]. Subsequent development involved intracoronary infusion of adenovirus. Viral DNA was detected in the rabbit myocardium for 2 weeks after delivery, although significant gene expression in other main organs was also recorded [5]. These and other data obtained two decades ago showed that: the route of gene delivery is no less important than the choice of vector system; and the basic methods of cardiac gene transfer were primarily intramyocardial injection and intracoronary infusion. Over time, although, it became clear that both of these methods had severe limitations and did not adequately reflect all the potentially more novel technologies of gene delivery. A classification of existing cardiac gene delivery techniques

Near-infrared-light-based nano-platform boosts endosomal escape and controls gene knockdown in vivo.

Current nanoparticle-based gene delivery techniques face two major limitations, namely, endosomal degradation and poor cytosolic release of the nanoparticles and nonspecificity of treatment. These limitations can be overcome with certain light-based techniques, such as photochemical internalization to enable endosomal escape of the delivered nanoparticles and light-controlled gene expression to overcome the nonspecific effects. However, these techniques require UV/visible light, which is either phototoxic and/or has low tissue penetration capabilities, thus preventing their use in deep tissues in a clinical setting. In an effort to overcome these barriers, we have successfully demonstrated a light-based gene delivery system that significantly boosts cytosolic gene delivery, with precise control over gene expression and the potential for use in nonsuperficial tissues. Core-shell fluorescent upconversion nanoparticles excited by highly penetrating near-infrared radiation and emitting simultaneously in the ultraviolet and visible ranges were synthesized and used as remote nanotransducers to simultaneously activate endosomal escape and gene knockdown. Gene knockdown using photomorpholinos was enhanced as much as 30% in vitro compared to the control without endosomal escape facilitation. A similar trend was seen in vivo in a murine melanoma model, demonstrating the enormous clinical potential of this system.

GSK3β, But Not GSK3α, Inhibits the Neuronal Differentiation of Neural Progenitor Cells As a Downstream Target of Mammalian Target of Rapamycin Complex1

Glycogen synthase kinase 3 (GSK3) acts as an important regulator during the proliferation and differentiation of neural progenitor cells (NPCs), but the roles of the isoforms of this molecule (GSK3α and GSK3β) have not been clearly defined. In this study, we investigated the functions of GSK3α and GSK3β in the context of neuronal differentiation of murine NPCs. Treatment of primary NPCs with a GSK3 inhibitor (SB216763) resulted in an increase in the percentage of TuJ1-positive immature neurons, suggesting an inhibitory role of GSK3 in embryonic neurogenesis. Downregulation of GSK3β expression increased the percentage of TuJ1-positive cells, while knock-down of GSK3α seemed to have no effect. When primary NPCs were engineered to stably express either isoform of GSK3 using retroviral vectors, GSK3β, but not GSK3α, inhibited neuronal differentiation and helped the cells to maintain the characteristics of NPCs. Mutant GSK3β (Y216F) failed to suppress neuronal differentiation, indicating that the kinase activity of GSK3β is important for this regulatory function. Similar results were obtained in vivo when a retroviral vector expressing GSK3β was delivered to E9.5 mouse brains using the ultrasound image-guided gene delivery technique. In addition, SB216763 was found to block the rapamycin-mediated inhibition of neuronal differentiation of NPCs. Taken together, our results demonstrate that GSK3β, but not GSK3α, negatively controls the neuronal differentiation of progenitor cells and that GSK3β may act downstream of the mammalian target of rapamycin complex1 signaling pathway.

Transient transformation of date palm via Agrobacterium-mediated and particle bombardment

Abstract A study was conducted to develop an effective technique for gene delivery into date palm tissues. Two steps were established to implement the process; first, embryogenic callus was transformed by particle bombardment and Agrobacterium with uidA gene (pCAMBIA3301 plasmid); second, different parameters were optimized for the delivering of uidA gene into somatic embryos. The results indicated that bombardment of embryogenic callus shows a higher level of GUS activity (22 blue spots) as compared to transformation by integrated particle bombardment with Agrobacterium , Agl1 and LBA4404strains with 12 and 11 blue spots, respectively. A higher level of uidA gene expression was observed in somatic embryos bombarded with 1350 psi helium pressure at 28 inHg chamber vacuum, 6 cm target distance, 0.4 M mannitol and pAct1-D plasmid (Act1 promoter). PCR analysis using GUS specific primers confirmed integration of the uidA gene in the genomic DNA of transformed tissues. Key words

PLGA-Carbon Nanotube Conjugates for Intercellular Delivery of Caspase-3 into Osteosarcoma Cells

Cancer has arisen to be of the most prominent health care issues across the world in recent years. Doctors have used physiological intervention as well as chemical and radioactive therapeutics to treat cancer thus far. As an alternative to current methods, gene delivery systems with high efficiency, specificity, and safety that can reduce side effects such as necrosis of tissue are under development. Although viral vectors are highly efficient, concerns have arisen from the fact that viral vectors are sourced from lethal diseases. With this in mind, rod shaped nano-materials such as carbon nanotubes (CNTs) have become an attractive option for drug delivery due to the enhanced permeability and retention effect in tumors as well as the ability to penetrate the cell membrane. Here, we successfully engineered poly (lactic-co-glycolic) (PLGA) functionalized CNTs to reduce toxicity concerns, provide attachment sites for pro-apoptotic protein caspase-3 (CP3), and tune the temporal release profile of CP3 within bone cancer cells. Our results showed that CP3 was able to attach to functionalized CNTs, forming CNT-PLGA-CP3 conjugates. We show this conjugate can efficiently transduce cells at dosages as low as 0.05 μg/ml and suppress cell proliferation up to a week with no further treatments. These results are essential to showing the capabilities of PLGA functionalized CNTs as a non-viral vector gene delivery technique to tune cell fate.

Gene Therapy Delivery Systems for Enhancing Viral and Nonviral Vectors for Cardiac Diseases: Current Concepts and Future Applications

Gene therapy is one of the most promising fields for developing new treatments for the advanced stages of ischemic and monogenetic, particularly autosomal or X-linked recessive, cardiomyopathies. The remarkable ongoing efforts in advancing various targets have largely been inspired by the results that have been achieved in several notable gene therapy trials, such as the hemophilia B and Leber's congenital amaurosis. Rate-limiting problems preventing successful clinical application in the cardiac disease area, however, are primarily attributable to inefficient gene transfer, host responses, and the lack of sustainable therapeutic transgene expression. It is arguable that these problems are directly correlated with the choice of vector, dose level, and associated cardiac delivery approach as a whole treatment system. Essentially, a delicate balance exists in maximizing gene transfer required for efficacy while remaining within safety limits. Therefore, the development of safe, effective, and clinically applicable gene delivery techniques for selected nonviral and viral vectors will certainly be invaluable in obtaining future regulatory approvals. The choice of gene transfer vector, dose level, and the delivery system are likely to be critical determinants of therapeutic efficacy. It is here that the interactions between vector uptake and trafficking, delivery route means, and the host's physical limits must be considered synergistically for a successful treatment course.

Gene Delivery Techniques for Adult Stem Cell-Based Regenerative Therapy

Over the past decade, stem cells have been considered to be a promising resource to cure and regenerate damaged or diseased tissues with research extending from basic studies to clinical application. Furthermore, genetically modified stem cells have the potential to reduce tumorigenic risks and achieve safe tissue formation. Recent advances in genetic modification of stem cells have rendered these cells more accessible and stable. The successful genetic modification of stem cells relies heavily on designing vector systems, either viral or nonviral vectors, which can efficiently deliver therapeutic genes to the cells with minimum toxicity. Currently, viral vectors showing high transfection efficiencies still raise safety issues, whereas safer nonviral vectors exhibit extremely poor transfection in stem cells. Here, we attempt to review and discuss the main factors raising concern in previous reports, and devise strategies to solve the issues in gene delivery systems for successful stem cell-targeting regenerative therapy.

RETRACTED ARTICLE: Surface-functionalized gold nanoparticles mediate bacterial transformation: a nanobiotechnological approach

Transformation of bacteria is an important step in molecular biology. Viral and non-virus-based gene delivery techniques, including chemical/biological and physical approaches, have been applied to bacterial, mammalian and plant cells. E. coli is not competent to take up DNA; hence, different methods are used to incorporate plasmid DNA. A novel method has been developed using glutathione-functionalized gold nanoparticles to mediate transformation of plasmid DNA (pUC19) into E. coli DH5α that does not require the preparation of competent cells. The glutathione-functionalized gold nanoparticles acted as a vector and facilitated the entry of DNA into the host cell. The method also gave a higher transformation efficiency (4.2×10(7)/μg DNA) compared to 2.3×10(5)/μg DNA using the conventional CaCl2-mediated method. It was also non-toxic to the bacterium making it suitable for biotechnological applications.

Suppression of thymosin β10 increases cell migration and metastasis of cholangiocarcinoma

BackgroundThymosin β10 (Tβ10) expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of Tβ10 in liver fluke-associated cholangiocarcinoma (CCA) are not fully understood. In this study, we investigated the expression of Tβ10 in CCA tumor tissues and cell lines as well as molecular mechanisms of Tβ10 in tumor metastasis of CCA cell lines.MethodsTβ10 expression was determined by real time RT-PCR or immunocytochemistry. Tβ10 silence or overexpression in CCA cells was achieved using gene delivery techniques. Cell migration was assessed using modified Boyden chamber and wound healing assay. The effect of silencing Tβ10 on CCA tumor metastasis was determined in nude mice. Phosphorylation of ERK1/2 and the expression of EGR1, Snail and matrix metalloproteinases (MMPs) were studied.ResultsTen pairs of CCA tissues (primary and metastatic tumors) and 5 CCA cell lines were studied. With real time RT-PCR and immunostaining analysis, Tβ10 was highly expressed in primary tumors of CCA; while it was relatively low in the metastatic tumors. Five CCA cell lines showed differential expression levels of Tβ10. Silence of Tβ10 significantly increased cell migration, invasion and wound healing of CCA cells in vitro; reversely, overexpression of Tβ10 reduced cell migration compared with control cells (P<0.05). In addition, silence of Tβ10 in CCA cells increased liver metastasis in a nude mouse model of CCA implantation into the spleen. Furthermore, silence of Tβ10 activated ERK1/2 and increased the expression of Snail and MMPs in CCA cell lines. Ras-GTPase inhibitor, FPT inhibitor III, effectively blocked Tβ10 silence-associated ERK1/2 activation, Snail expression and cell migration.ConclusionsLow expression of Tβ10 is associated with metastatic phenotype of CCA in vitro and in vivo, which may be mediated by the activation of Ras, ERK1/2 and upregulation of Snail and MMPs. This study suggests a new molecular pathway of CCA pathogenesis and a novel strategy to treat or prevent CCA metastasis.

Synergistic effects of ultrasound-targeted microbubble destruction and TAT peptide on gene transfection: An experimental study in vitro and in vivo

Cell-permeable peptides (CPPs) and ultrasound-targeted microbubble destruction (UTMD) have tremendous potential for gene delivery. However, their applications are limited due to nonspecificity of CPPs and low transfection efficiency of UTMD. Here, we developed a ‘smart’ gene delivery system by encapsulating TAT peptide (TATp) and hepatocyte growth factor (HGF) gene within lipid microbubbles, in which TATp was protected from being enzymatically cleaved and HGF gene was protected from degradation. This new strategy had synergistic effects of UTMD and TATp on gene transfection. We investigated the efficacy and safety of HGF gene transfection mediated by the combination of UTMD and TATp in vitro and in vivo. The results from MTT assay and flow cytometry analyses indicated that the combination of UTMD and TATp could enhance HGF gene expression in HUVECs without any significant side effect on cell viability. In rat myocardial infarction models, we demonstrated that the protein and mRNA expressions of HGF in myocardium caused by the combination of UTMD and TATp were the highest.Histopathological findings demonstrated that the combination of UTMD and TATp enhanced myocardial microvasculature and ameliorated myocardial fibrosis. In conclusion, the combination of UTMD and TATp might be a safe and efficient technique for gene delivery.

Diseases originate and terminate by genes: unraveling nonviral gene delivery.

The world is driving in to the era of transformation of chemical therapeutic molecules to biological genetic material therapeutics, and that is where the biological drugs especially "genes" come into existence. These genes worked as "magical bullets" to specifically silence faulty genes responsible for progression of diseases. Viral gene delivery research is far ahead of nonviral gene delivery technique. However, with more advancement in polymer science, new ways are opening for better and efficient nonviral gene delivery. But efficient delivery method is always considered as a bottleneck for gene delivery as success of which will decide the fate of gene in cells. During the past decade, it became evident that extracellular as well as intracellular barriers compromise the transfection efficiency of nonviral vectors. The challenge for gene therapy research is to pinpoint the rate-limiting steps in this complex process and implement strategies to overcome the biological physiochemical and metabolic barriers encountered during targeting. The synergy between studies that investigate the mechanism of breaking in and breaking out of nonviral gene delivery carrier through various extracellular and intracellular barriers with desired characteristics will enable the rational design of vehicles and revolutionize the treatment of various diseases.

In vitro labeling of neural stem cells with poly-L-lysine coated super paramagnetic nanoparticles for green fluorescent protein transfection.

The magnetic nanoparticle-based transfection method is a relatively new technique for delivery of functional genes to target tissues. We aimed to evaluate the transfection efficiency of rat neural stem cell (NSC) using poly-L-lysine hydrobromide (PLL)-coated super paramagnetic iron oxide nanoparticles (SPION). The SPION was prepared and coated with PLL as transfection agent and the transfection efficiency was evaluated in rat NSC using enhanced green fluorescent protein-N1 plasmid containing GFP as a reporter gene. NSC was incubated for 24 h in cell culture media containing 25 µg/ml SPION and in different concentrations of PLL (0.25, 0.50, 0.75, 1 and 2 µg/ml). Cell viability was determined before and after transfection for every concentration using Trypan blue assay. Characterization of prepared uncoated (SPION) and coated (SPION-PLL) complexes were evaluated by a transmission electron microscope and the zeta potential. PLL at 0.75 μg/ml showed optimal results with 25 μg/ml SPION concentration compared with other PLL concentrations (0.25, 0.50, 1 and 2 μg/ml). The 18% efficiency of the transfected cells showed green fluorescence. Transfection with SPION is an efficient, non-viral gene transfere method.

Identification of Rtl1, a Retrotransposon-Derived Imprinted Gene, as a Novel Driver of Hepatocarcinogenesis

We previously utilized a Sleeping Beauty (SB) transposon mutagenesis screen to discover novel drivers of HCC. This approach identified recurrent mutations within the Dlk1-Dio3 imprinted domain, indicating that alteration of one or more elements within the domain provides a selective advantage to cells during the process of hepatocarcinogenesis. For the current study, we performed transcriptome and small RNA sequencing to profile gene expression in SB–induced HCCs in an attempt to clarify the genetic element(s) contributing to tumorigenesis. We identified strong induction of Retrotransposon-like 1 (Rtl1) expression as the only consistent alteration detected in all SB–induced tumors with Dlk1-Dio3 integrations, suggesting that Rtl1 activation serves as a driver of HCC. While previous studies have identified correlations between disrupted expression of multiple Dlk1-Dio3 domain members and HCC, we show here that direct modulation of a single domain member, Rtl1, can promote hepatocarcinogenesis in vivo. Overexpression of Rtl1 in the livers of adult mice using a hydrodynamic gene delivery technique resulted in highly penetrant (86%) tumor formation. Additionally, we detected overexpression of RTL1 in 30% of analyzed human HCC samples, indicating the potential relevance of this locus as a therapeutic target for patients. The Rtl1 locus is evolutionarily derived from the domestication of a retrotransposon. In addition to identifying Rtl1 as a novel driver of HCC, our study represents one of the first direct in vivo demonstrations of a role for such a co-opted genetic element in promoting carcinogenesis.

Delivery of Short Interfering Ribonucleic Acid-Complexed Magnetic Nanoparticles in an Oscillating Field Occurs via Caveolae-Mediated Endocytosis

Gene delivery technologies to introduce foreign genes into highly differentiated mammalian cells have improved significantly over the last few decades. Relatively new techniques such as magnetic nanoparticle-based gene transfection technology are showing great promise in terms of its high transfection efficiency and wide-ranging research applications. We have developed a novel gene delivery technique, which uses magnetic nanoparticles moving under the influence of an oscillating magnetic array. Herein we successfully introduced short interfering RNA (siRNA) against green fluorescent protein (GFP) or actin into stably-transfected GFP-HeLa cells or wild-type HeLa and rat aortic smooth muscle cells, respectively. This gene silencing technique occurred in a dose- and cell density- dependent manner, as reflected using fluorescence intensity and adhesion assays. Furthermore, using endocytosis inhibitors, we established that these magnetic nanoparticle-nucleic acid complexes, moving across the cell surface under the influence of an oscillating magnet array, enters into the cells via the caveolae-mediated endocytic pathway.

Targeted gene delivery by free-tissue transfer in oncoplastic reconstruction

Surgery is the most effective curative treatment for various tumour types. Despite a current preference for conservative surgery, radical excision retains a clearly defined role in modern management of locoregional disease. Extirpative defects are reconstructed routinely using free-tissue transfer from a distant donor site. Although these free flaps currently provide no direct therapeutic benefit, advances in gene-delivery techniques offer the possibility to genetically modify flaps to produce potent targeted treatments with greater anatomical control. Several promising therapeutic strategies, including virus-directed enzyme prodrug therapy, genetic radionuclide therapy, and free-flap radioprotection, have the potential to extend the role of the free flap beyond its immediate goal of restoring form and function to patients, but challenges exist. Work to translate therapeutic free-tissue transfer from preclinical study to clinical use is in progress.

A new method of ultrasonic nonviral gene delivery to the adult myocardium

Cardiac gene transfer is a powerful molecular tool to improve our understanding of the role of new proteins and mutants in cardiac pathophysiology. There is a need for a simple efficient myocardial gene delivery technique in order to study the physiological role of proteins in their native environment. Here we tested a new method of myocardial nonviral gene delivery, by using the combination of ultrasound energy (USE), liposomes and high pressure injections to the rat heart. Wistar rats were subjected to intra-myocardial injections of liposomes-DNA or siRNA mix. The heart was exposed after an inter-costal incision, and then injections were conducted between two sets of USE heart exposure. Ultrasound application resulted in much higher transfection efficiency (2% of left ventricle) than the liposomes-DNA alone (0.12% of left ventricle) as shown by the beta-galactosidase staining. The ultrasonic based liposomes-DNA delivery resulted in low inflammatory response, as well as in low cardiac fibrosis as shown by total collagen staining. Quantitative real time polymerase chain reaction (PCR) showed that the ultrasonic delivery resulted in cardiac specific transduction. Moreover, 23,906±2197 and 71,883±4065 calcium tolerant transfected cardiac myocytes were isolated following the delivery of a GFP plasmid or tagged siRNA, respectively. This was sufficient to perform single cell physiological measurements and biochemical experiments on homogenates. We developed an interesting safe method for local gene transfer in the heart using ultrasound and liposomes gene delivery. This method is particularly useful to study the effect of gene transfer on cardiac myocytes maintained in their normal environment in animal models.

EFSUMB Guidelines 2011: Comment on Emergent Indications and Visions

The focus of this article is the emergent and potential indications of contrast-enhanced ultrasound (CEUS). Emergent applications of CEUS techniques include extravascular and intracavitary contrast-enhanced ultrasound, quantitative assessment of microvascular circulation for tumor response assessment, and tumor characterization using dynamic contrast-enhanced ultrasound (DCE-US). Potential indications for microbubble agents include novel molecular imaging and drug and gene delivery techniques, which have been successfully tested in animal models. "Comments and Illustrations of the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) Non-Liver Guidelines 2011" which focus more on established applications are published in the same supplement to Ultraschall in der Medizin (European Journal of Ultrasound).

Delivering Factors for Reprogramming a Somatic Cell to Pluripotency

An adult cell originates from stem cell. The stem cell is usually categorized into three species including an embryonic stem cell (ESc), an adult stem cell, and an induced stem cell (iPSc). iPSc features pluripotency, which is meant to be differentiated into any types of cells. Accordingly, it is much attractive to anyone who pursuit a regenerative medicine, owing to the potential almighty. They are simply produced by reprogramming a somatic cell via a transfer of transcription factors. The efficiency and productivity of iPS are considerably subject to delivering methods of exogenous genes into a variety of targeted mammalians. Conventional and well-run gene delivery techniques have been reviewed here. This details the methods and principles of delivery factors and provides an overview of the research, with an emphasis on their potential for use as clinical therapeutic platforms.

Magnetic targeting and ultrasound mediated drug delivery: Benefits, limitations and combination

This paper reviews the uses of magnetism and ultrasound in therapeutic delivery applications. Emphasis is placed upon magnetic nanoparticles and microbubble ultrasound contrast agents. The underlying physical principles, history, key developments and limitations of these techniques for drug and gene delivery in vitro and in vivo are explored. The combination of ultrasonic and magnetic techniques is also reviewed with particular focus on magnetic microbubbles as delivery agents with the potential to combine the advantages of both methods whilst addressing many of their limitations. Finally, results are presented from a study of a new magnetic microbubble formulation which shows great applicability as a therapeutic delivery vehicle.

A pH-sensitive multifunctional gene carrier assembled via layer-by-layer technique for efficient gene delivery

BackgroundThe success of gene therapy asks for the development of multifunctional vectors that could overcome various gene delivery barriers, such as the cell membrane, endosomal membrane, and nuclear membrane. Layer-by-layer technique is an efficient method with easy operation which can be used for the assembly of multifunctional gene carriers. This work describes a pH-sensitive multifunctional gene vector that offered long circulation property but avoided the inhibition of tumor cellular uptake of gene carriers associated with the use of polyethylene glycol.MethodsDeoxyribonucleic acid (DNA) was firstly condensed with protamine into a cationic core which was used as assembly template. Then, additional layers of anionic DNA, cationic liposomes, and o-carboxymethyl-chitosan (CMCS) were alternately adsorbed onto the template via layer-by-layer technique and finally the multifunctional vector called CMCS-cationic liposome-coated DNA/protamine/DNA complexes (CLDPD) was constructed. For in vitro test, the cytotoxicity and transfection investigation was carried out on HepG2 cell line. For in vivo evaluation, CMCS-CLDPD was intratumorally injected into tumor-bearing mice and the tumor cells were isolated for fluorescence determination of transfection efficiency.ResultsCMCS-CLDPD had ellipsoidal shapes and showed “core-shell” structure which showed stabilization property in serum and effective protection of DNA from nuclease degradation. In vitro and in vivo transfection results demonstrated that CMCS-CLDPD had pH-sensitivity and the outermost layer of CMCS fell off in the tumor tissue, which could not only protect CMCS- CLDPD from serum interaction but also enhance gene transfection efficiency.ConclusionThese results demonstrated that multifunctional CMCS-CLDPD had pH- sensitivity, which may provide a new approach for the antitumor gene delivery.