Polymers for DNA delivery

Nucleic acid delivery has many applications in basic science, biotechnology, agriculture, and medicine. One of the main applications is DNA or RNA delivery for gene therapy purposes. Gene therapy, an approach for treatment or prevention of diseases associated with defective gene expression, involves the insertion of a therapeutic gene into cells, followed by expression and production of the required proteins. This approach enables replacement of damaged genes or expression inhibition of undesired genes. Following two decades of research, there are two major methods for delivery of genes. The first method, considered the dominant approach, utilizes viral vectors and is generally an efficient tool of transfection. Attempts, however, to resolve drawbacks related with viral vectors (e.g., high risk of mutagenicity, immunogenicity, low production yield, limited gene size, etc.), led to the development of an alternative method, which makes use of non-viral vectors. This review describes non-viral gene delivery vectors, termed "self-assembled" systems, and are based on cationic molecules, which form spontaneous complexes with negatively charged nucleic acids. It introduces the most important cationic polymers used for gene delivery. A transition from in vitro to in vivo gene delivery is also presented, with an emphasis on the obstacles to achieve successful transfection in vivo.     

Library selection approaches to engineering enhanced retroviral and lentiviral vectors

Retroviral and lentiviral based gene delivery vectors have been used in numerous pre-clinical studies and clinical trials due to their advantages, including stable and prolonged expression of therapeutic transgenes and minimal immune responses against the vector. Despite such advantages, however, retroviral vectors also have several limitations for gene therapy applications. For example, they can suffer from a lack of efficient or targeted gene delivery to key cell types. In addition, retroviral vector stability can be compromised by their envelope proteins. This review briefly describes how such limitations have been overcome by recently developed library selection approaches that borrow a lesson from nature: the ability of evolution to generate biomolecules with novel function. These library selection approaches are based on the construction of retroviral libraries where the sequences encoding natural viral components are partially randomized using a variety of methods in order to generate diverse libraries that can be selected to create improved or novel functions. These high throughput, library-based approaches provide a strong complement to rational engineering of viral components for the rapid development of efficient and safe retroviral and lentiviral vector systems for gene therapy.     

Polymers in gene therapy technology

There are several barriers to gene delivery. One of the biggest challenges is the design of appropriate vectors. Currently, nonviral vectors have received significant attention because of low toxicity, potential for tissue specificity, stability during storage, lack of immunogenicity, and relatively low production cost. Despite the high efficiency of viral vectors, they show limited clinical applications because of potentially fatal adverse effects and because of the likelihood of the immune response shutting down the transgene expression system. Nonviral technologies comprise plasmid‐based expression systems harboring a gene that encodes a therapeutic protein along with a synthetic gene delivery system. This review provides a broad perspective on recent improvements in the development of four kinds of nonviral vectors that are based on polymers, peptides, lipids, and DNA and discusses the cytotoxicity associated with gene therapy. Copyright © 2014 John Wiley & Sons, Ltd.     

Transcriptional targeting of gene expression in breast cancer by the promoters of protein regulator of cytokinesis 1 and ribonuclease reductase 2

For cancer gene therapy, cancer-specific over- expression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoter's cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.     

Monitoring transfected cells without selection agents by using the dual-cassette expression EGFP vectors

Conventional methods of selecting gene transfected cells by toxic agents may yield ambiguous results. It is difficult to determine whether cell death is due to selection agents or gene transfection, owing to the substantial overlap of the time-courses for both effects. Therefore, to determine transfection-induced cell toxicity, the mammalian expression vector pEGFP-N1 (CLONTECH Lab., Palo Alto, CA, USA) has been modified to the dual-cassette expression vectors named pEGFP-Ks by the relocation of its EGFP expression cassette. We have precisely monitored the cells transfected with this vector on our custom culture dishes, thereby bypassing the need for selection agent or fluorescent cell sorting. This is a useful method to screen genes encoding potential toxic or useful proteins without performing undesirable selection agent and also can be used to monitor the transfected cells for various purposes, either the inhibition or proliferation of mammalian cells for applications in biotechnology.     

Inverted Quasi‐Spherical Droplets on Polydopamine–TiO2 Substrates for Enhancing Gene Delivery

Devising efficient gene delivery systems is crucial to enhancing the therapeutic efficacy of gene–cell therapy approaches. Herein, inverted quasi‐spherical (iQS) droplet systems, which enhance gene delivery efficiencies by reducing the path lengths of gene vectors, mediating motions of vectors at early stages, and raising the contact frequencies of vectors with cells, are developed by adopting the principle of 3D hanging‐drop cell culture. Micrometer‐sized polydopamine (pDA) holes are created on superhydrophobic titanium isopropoxide (TiO₂)‐coated substrates by physical scraping; droplets are loaded on the pDA holes, and inversion of the substrate generates iQS droplets with large contact angles. Both human neural stem cells (hNSCs) and adeno‐associated viral vectors are simultaneously incorporated into the iQS droplets to assess gene delivery efficiencies. The steep angles of iQS droplets and enhanced cell/vector contact frequencies facilitate the viral association with hNSCs and enhancing cell–cell interactions, thereby significantly promoting gene delivery efficiencies. Even with reduced viral quantities/exposure times and cell numbers, the iQS droplet systems elicit sufficient gene expression (i.e., interleukin‐10). The ability of the iQS droplet systems to maximize beneficial gene delivery effects with minimal materials (e.g., medium, cells, and vectors) should enable their extensive use as a platform for preparing genetically stimulated cellular therapeutics.     

高聚物基因载体的功能化研究

寻找安全、高效的基因载体是基因治疗的关键问题之一，聚合物基因载体具有低毒、低成本、可设计性强等优良的性质，是未来基因载体发展的方向。传统的高聚物作为基因载体时，功能单一，不能完全满足基因治疗的需要，因此对载体进行功能化修饰是十分必要的。本文在分析基因治疗过程的基础上，详述了基因载体应具有的四种基本功能——在细胞外保持稳定的功能、进入细胞的功能、逃离溶酶体的功能、进入细胞核的功能，并详细介绍了有关基因载体功能化修饰的方法和目前国际上的进展情况，最后本文总结了高聚物载体目前存在的主要问题及未来的发展方向。     

Low-dose radiation response of the p21WAF1/CIP1 gene promoter transduced by adeno-associated virus vector

In cancer gene therapy, restriction of antitumor transgene expression in a radiation field by use of ionizing radiation-inducible promoters is one of the promising approaches for tumor-specific gene delivery. Although tumor suppressor protein p53 is induced by low doses (< 1 Gy) of radiation, there have been only a few reports indicating potential utilization of a p53-target gene promoter, such as that of the p21 gene. This is mainly because the transiently transfected promoter of p53-target genes is not much sensitive to radiation. We examined the response of the p21 gene promoter to low-dose radiation when transduced into a human breast cancer cell line MCF-7 by use of recombinant adeno-associated virus (rAAV) vectors. It was shown that the p21 gene promoter transduced by rAAV vectors was more highly radiation-responsive than that transiently transfected by electroporation. A significant induction of the p21 gene promoter by radiation of low doses down to 0.2 Gy was observed. When cells were transduced with the p21 gene promoter-driven HSVtk gene by rAAV vector, they were significantly sensitized to repetitive treatment with low dose radiation (1 Gy) in the presence of the prodrug ganciclovir. It was therefore considered that the p21 gene promoter in combination with a rAAV vector is potentially usable for the development of a low-dose radiation-inducible vector for cancer gene therapy.     

Nanoparticles as an emerging tool to alter the gene expression: Preparation and conjugation methods

Many human diseases occur due to the over or under-expression of genes which can be corrected either by silencing or over-expression, respectively by transforming with specific nucleic acid (NA). NA transformation for medical purposes to alter the cellular gene expression is challenging because NA cannot cross efficiently the cellular biomembrane. One option, the viral vectors, is risky for patients and, the non-viral vectors have lower transformation efficiency. From the past few years, nanoparticles (NPs) are being studied extensively for their use as a vector to deliver NA. They are of a sub-micron size, have a large surface area, rapid absorption ability and can reach inside of the cells. These properties make them a suitable gene carrier. NPs types - organic, inorganic, organic/inorganic hybrid and polymeric NPs, having different properties that can be used to deliver the NA. They possess various properties like biocompatibility, targeted delivery of gene, controlled release of NA which makes them suitable for different uses. In this review, we are describing and comparing various methods to synthesize various kinds of NPs and how they can be conjugated with NA. A series of modifications in NPs to form the polyplex are also discussed along with the varying outcomes in terms of changes in the gene expression and its cytotoxicity towards different cell lines. This review is helpful for nano-scientists to decide which method to be followed for a specific need via controlling gene expression.     

Construction of the ie1-Bacmid Expression System and Its Use to Express EGFP and BmAGO2 in BmN Cells

The presently available expression tools and vectors (e.g., eukaryotic expression vectors and the adenovirus expression system) for studying the functional genes in Bombyx mori are insufficient. The baculovirus expression system is only used as a protein production tool; therefore, recombinant proteins expressed by B. mori using the baculovirus expression system equipped with a polyhedrin promoter cannot be used for in vivo research applications. In this work, we constructed and screened a eukaryotic expression vector for silkworm cells The EGFP and B. mori Argonaute2 proteins were found to be efficiently expressed using the screened pIEx-1 vector with the FuGENE 6 transfection reagent. Additionally, we constructed a novel nucleopolyhedrovirus ie1-Bacmid expression system for the production of recombinant protein; we then used the system to highly express the EGFP and B. mori Argonaute2 proteins. In this system, the protein of interest can be efficiently expressed 13 h after infection by controlling the B. mori nucleopolyhedrovirus immediate early ie1 promoter. The ie1-Bacmid system provides a powerful “adenovirus-like” expression tool; not only can the tool be used to study baculovirus molecular biology for the silkworm but it is also useful in other research applications as well, such as the study of gene functions involved in cellular physiological processes.     

非病毒性基因载体的合成研究

基因治疗是一种有效的治疗先天性遗传性疾病以及后天获得性疾病的手段。它通过激发细胞的生物活性或者抑制细胞非正常的功能来治疗或者预防疾病的发生,例如细胞的基因紊乱,细胞的无序增殖。目前基因治疗所面临的问题是缺乏有效的基因递送载体。基因载体主要分为病毒性基因载体和非病毒性基因载体。与病毒性基因载体相比,非病毒性基因载体具有毒性小、安全性高、易于制备、能够荷载分子量大的DNA等优点。本文综述了非病毒性基因载体的合成研究进展。     

Dendrimers as Non-Viral Vectors in Gene-Directed Enzyme Prodrug Therapy

Gene-directed enzyme prodrug therapy (GDEPT) has been intensively studied as a promising new strategy of prodrug delivery, with its main advantages being represented by an enhanced efficacy and a reduced off-target toxicity of the active drug. In recent years, numerous therapeutic systems based on GDEPT strategy have entered clinical trials. In order to deliver the desired gene at a specific site of action, this therapeutic approach uses vectors divided in two major categories, viral vectors and non-viral vectors, with the latter being represented by chemical delivery agents. There is considerable interest in the development of non-viral vectors due to their decreased immunogenicity, higher specificity, ease of synthesis and greater flexibility for subsequent modulations. Dendrimers used as delivery vehicles offer many advantages, such as: nanoscale size, precise molecular weight, increased solubility, high load capacity, high bioavailability and low immunogenicity. The aim of the present work was to provide a comprehensive overview of the recent advances regarding the use of dendrimers as non-viral carriers in the GDEPT therapy.     

Retrovirus-mediated transduction of a cytosine deaminase gene preserves the stemness of mesenchymal stem cells

Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation.     

On-chip fabrication of mutifunctional envelope-type nanodevices for gene delivery

Microfluidic devices may be highly beneficial to the rapid fabrication of small quantities of various nonviral vectors with different functionalities, which is indispensable for effective order-made gene therapy. We adapted a microfluidic chip-based approach for fabricating small quantities of nonviral vectors in a short time in preparation for order-made gene therapy applications. This approach permitted us to fabricate multifunctional envelope-type nanodevices (MENDs), composed of a compacted (or condensed) DNA core and a lipid bilayer membrane shell, which are considered as promising nonviral vectors for gene therapy applications. The on-chip fabrication of the MEND was very simple, rapid, convenient, and cost-effective compared with conventional methods. The size of the MEND showed strong dependence on the concentration and flow rate of the reaction precursors and could be controlled to be much smaller than that achievable by conventional methods. This, together with abovementioned merits, makes our microfluidic chip-based approach very attractive for the fabrication of MENDs for effective application to order-made gene therapy.     

Bi-functional gold-coated magnetite composites with improved biocompatibility

The effect of gold attachment on the physical characteristics, cellular uptake, gene expression efficiency, and biocompatibility of magnetic iron oxide (MNP) vector was investigated in vitro in BHK21 cells. The surface modification of magnetite with gold was shown to alter the morphology and surface charge of the vector. Nonetheless, despite the differences in the surface charge with and without gold attachment, the surface charge of all vectors were positive when conjugated with PEI/DNA complex, and switched from positive to negative when suspended in cell media containing serum, indicating the adsorption of serum components onto the composite. The cellular uptake of all MNP vectors under the influence of a magnetic field increased when the composite loadings increased, and was higher for the MNP vector that was modified with gold. Both bare magnetite and gold-coated magnetite vectors gave similar optimal gene expression efficiency, however, the gold-coated magnetite vector required a 25-fold higher overall loading to achieve a comparable efficiency as the attachment of gold increased the particle size, thus reducing the surface area for PEI/DNA complex conjugation. The MNP vector without gold showed optimal gene expression efficiency at a specific magnetite loading, however further increases beyond the optimum loading decreased the efficiency of gene expression. The drop in efficiency at high magnetite loadings was attributed to the significant reduction in cellular viability, indicating the bare magnetite became toxic at high intracellular levels. The gene expression efficiency of the gold-modified vector, on the other hand, did not diminish with increasing magnetite loadings. Intracellular examination of both bare magnetite and gold-coated magnetite vectors at 48h post-magnetofection using transmission electron microscopy provided evidence of the localization of both vectors in the cell nucleus for gene expression and elucidated the nuclear uptake mechanism of both vectors. The results of this work demonstrate the efficacy of gold-modified vectors to be used in cellular therapy research that can function both as a magnetically-driven gene delivery vehicle and an intracellular imaging agent with negligible impact on cell viability.     

The baculovirus display technology--an evolving instrument for molecular screening and drug delivery

High throughput screening is a core technology in drug discovery. During the past decade, several strategies have been developed to screen (poly)peptide libraries for diverse applications including disease diagnosis and profiling, imaging, as well as therapy. The recently established baculovirus display vector system (BDVS) represents a eukaryotic screening platform that combines the positive attributes of both cell and virus-based display approaches, allowing presentation of complex polypeptides on cellular and viral surfaces. Compared to microbial display systems, the BDVS has the advantage of correct protein folding and post-translational modifications similar to those in mammals, facilitating expression and analysis of proteins with therapeutic interest. The applicability of the system is further expanded by the availability of genetically engineered insect cell lines capable of performing e.g. mammalianized glycosylation in combination with high level of expression. In addition to insect cells, baculovirus can mediate delivery and expression of heterologous genes in a broad spectrum of primary and established mammalian cells. Currently, a variety of baculovirus-based assays aiming at routine high throughput identification of agents targeting cell surface receptors or studies on ligand-receptor interactions are under construction. Here, the advancements and future prospects of the baculovirus display technologies with emphasis on molecular screening and drug delivery applications using insect cell display, mammalian cell display, and virion display are described.     

Development of a ZHER3‐Affibody‐Targeted Nano‐Vector for Gene Delivery to HER3‐Overexpressed Breast Cancer Cells

Despite the initial successes of gene delivery applications, they faced on several intrinsic drawbacks including toxicity and immunogenicity. Therefore, alternative gene‐delivery systems derived from recombinant peptides have emerged and is rapidly developing. Human epidermal growth factor receptor‐3 (HER3) shows high activity in tumor resistance to anti‐human epidermal growth factor receptor 2 (HER2) therapies. In this study, an affibody molecule against HER3 is conjugated to a biomimetic peptide RALA (an amphipathic and cationic peptide enriched with arginine) and the ability of the fusion vector for targeting HER3 and afterward delivering specific genes in breast cancer cells is evaluated. The results demonstrate that the biopolymeric platform, which contains an affibody‐conjugated RALA peptide, can effectively condense DNA into nanoparticles and target the overexpressed HER3 receptors in breast cancer cells and transfer specific genes. The use of such a recombinant biopolymer may pave the way for the development of sensitive and effective diagnostic and treatment tool for breast cancer.     

Tangential flow filtration for virus purification

Purification of virus particles and viral vectors for viral vaccines and gene therapy applications is a major large-scale separations challenge. Purification of parvovirus particles such as adeno-associated virus, the leading candidate for gene therapy applications, is particularly challenging given their small size, typically 18–26 nm. We have investigated the use of ultrafiltration for purification of Aedes aegypti densonucleosisvirus, a mosquito parvovirus.