Adenovirus vectors for gene delivery.

Recent endeavors in the development of adenovirus as a gene vector have focused on the modification of virus tropism, the accommodation of larger genes, and the increase in stability and control of transgene expression. Whereas partial or total deletions of viral genes increase the cloning capacity and partly reduce the cellular immune response, control of the humoral response, which often precludes efficient readministration, remains a challenge.     

无机纳米粒子作为基因载体的研究进展

转染是将具生物功能的核酸转移、运送到细胞内,并使其在细胞内维持生物功能的过程。作为现代生物化学和分子生物学中的一种主要技术手段,转染对于基因治疗有重要的意义。无机纳米粒子作为基因载体受到人们日益广泛的关注,其具有易于制备,可进行多样化的表面修饰等多种优势。本文将概述无机纳米粒子作为基因载体的现状及其对基因表达的影响。     

Retroviral vectors for human gene delivery

The potential for gene therapy to cure a wide range of diseases has lead to high expectations and a great increase in research efforts in this area. At present, viral vectors are the most efficient means of delivering a corrective gene into human cells. While a number of different viral vectors are under development, retroviral vectors are currently the most common type used in clinical trials today. However, the production of retroviral vectors for gene therapy applications faces a number of challenges. Of primary concern is the low titre of vector stocks produced by packaging cells in culture and the inherent instability of retroviral vector activity. The problems facing large-scale retroviral vector production are outlined in this review and the research efforts by a number of groups who have attempted to optimise production methods are presented.     

Designing gene delivery vectors for cardiovascular gene therapy

Genetic therapy in the cardiovascular system has been proposed for a variety of diseases ranging from prevention of vein graft failure to hypertension. Such diversity in pathogenesis requires the delivery of therapeutic genes to diverse cell types in vivo for varying lengths of time if efficient clinical therapies are to be developed. Data from extensive preclinical studies have been compiled and a certain areas have seen translation into large-scale clinical trials, with some encouraging reports. It is clear that progress within a number of disease areas is limited by a lack of suitable gene delivery vector systems through which to deliver therapeutic genes to the target site in an efficient, non-toxic manner. In general, currently available systems, including non-viral systems and viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV), have a propensity to transduce non-vascular tissue with greater ease than vascular cells thereby limiting their application in cardiovascular disease. This problem has led to the development and testing of improved vector systems for cardiovascular gene delivery. Traditional viral and non-viral systems are being engineered to increase their efficiency of vascular cell transduction and diminish their affinity for other cell types through manipulation of vector:cell binding and the use of cell-selective promoters. It is envisaged that future use of such technology will substantially increase the efficacy of cardiovascular gene therapy.     

Pulmonary gene delivery using polymeric nonviral vectors

Pulmonary delivery provides an easy and well tolerated means of access for the administration of biomacromolecules to the pulmonary epithelium and could therefore be an attractive approach for local and systemic therapies. A growing number of reports, which are summarized in this review, mirror the viability of pulmonary gene delivery. Special attention has been paid to the biological barriers in the lung that must be overcome for successful delivery, and which can be divided into anatomic, physical, immunologic, and metabolic barriers. In light of these barriers, successful nonviral polymer-based formulations of therapeutic genes are presented depending on the chemical nature of the polymer. In addition to polyethyleneimine-based nonviral vectors, which have been most intensively studied for pulmonary gene delivery in the past, other polymeric, dendritic, and targeted materials are also described here, including novel and biodegradable polymers. As new materials need in vitro or ex vivo testing before in vivo application, sophisticated models for all three approaches have been illustrated. Although pulmonary siRNA delivery enjoys popularity in clinical trials, pulmonary gene delivery has so far not been translated into clinical applications. With this review, potential hurdles are demonstrated, but novel approaches that may lead to optimized systems are described as well.     

Lentiviral vectors for gene delivery into cells

Human immunodeficiency virus type I (HIV) is the etiologic agent of acquired immunodeficiency syndrome or AIDS. Vectors based upon HIV have been in use for over a decade. Beginning in 1996, with the demonstration of improved pseudotyping using vesicular stomatitis virus (VSV) G protein along with transduction of resting mammalian cells, a series of improvements have been made in these vectors, making them both safer and more efficacious. Taking a cue from vector development of murine leukemia virus (MLV), split coding and self-inactivating HIV vectors now appear quite suitable for phase I clinical trials. In parallel, a number of pre-clinical efficacy studies in animals have demonstrated the utility of these vectors for various diseases processes, especially neurodegenerative and hematopoietic illnesses. These vectors are also appropriate for the study of other viruses (specifically of viral entry) and investigation of the HIV replicative cycle, along with straightforward transgene delivery to target cells of interest. Vectors based upon other lentiviruses have shown similar abilities and promise. Although concerns remain, particularly with regards to detection and propagation of replication-competent lentivirus, it is almost certain that these vectors will be introduced into the clinic within the next 3-5 years.     

Subretinal gene delivery using helper-dependent adenoviral vectors

This study describes the successful delivery of helper-dependent adenoviral vectors to the mouse retina with long term and robust levels of reporter expression in the retina without apparent adverse effects. Since these vectors have a large cloning capacity, they have great potential to extend the success of gene therapy achieved using the adeno-associated viral vector.     

Poly-l-glutamic acid derivatives as multifunctional vectors for gene delivery. Part A. Synthesis and physicochemical evaluation

This paper describes the synthesis and evaluation of a series of multifunctional poly-l-glutamic acid derivatives that can be used as vectors for gene delivery. They readily form polyelectrolyte complexes with DNA, resulting in a reduced surface charge and size of the DNA. The formation of a polymer-DNA complex and the stability toward serum albumin was analyzed by ethidium bromide fluorescence measurements and agarose gel retardation studies. Most polymers, except those with more than 80% imidazoles, are able to condense calf thymus DNA, thus forming complexes with sizes varying between 105 and 172 nm. The surface charge of the complexes was determined at different charge ratios by zeta potential measurements. The buffering properties of the polymers were determined via titration studies. The results show that the polymers are able to buffer the endosomal environment, although to a smaller extent than polyethyleneimine. The first part of this study is devoted to the synthesis and the physicochemical evaluation of the multifunctional polymers and their use as carriers for genetic information. The second part, to be published subsequently, discusses the biological evaluation of the polymers and their complexes with DNA.     

Use of blood outgrowth endothelial cells as virus-producing vectors for gene delivery to tumors

Cell-based delivery of therapeutic viruses has potential advantages over systemic viral administration, including attenuated neutralization and improved viral targeting. One of the exciting new areas of investigation is the potential ability of endothelial-lineage cells to deliver genes to the areas of neovascularization. In the present study, we compared two types of endothelial-lineage cells [outgrowth endothelial cells (OECs) and culture-modified mononuclear cells (CMMCs), also known as "endothelial progenitor cells"] for their ability to be infected with adenovirus and to home to the areas of neovascularization. Both cell types were isolated from peripheral blood of healthy human donors and expanded in culture. We demonstrate that OECs are more infectable and home better to tumors expressing VEGF on systemic administration. Furthermore, we used an adenoviral/retroviral chimeric system to convert OECs to retrovirus-producing cells. When injected systemically into tumor-bearing mice, OECs retain their ability to produce retrovirus and infect surrounding tumor cells. Our data demonstrate that OECs could be efficient carriers for viral delivery to areas of tumor neovascularization.     

Novel self-assembling conjugates as vectors for agrochemical delivery

Background

Modern agricultural practises rely on surfactant-based spray applications to eliminate weeds in crops. The wide spread and indiscriminate use of surfactants may result in a number of deleterious effects that are not limited to impacts on the crop and surrounding farm eco-system but include effects on human health. To provide a safer alternative to the use of surfactant-based formulations, we have synthesised a novel, self-assembling herbicide conjugate for the delivery of a broad leaf herbicide, picloram.

Results

The synthesized self-assembling amphiphile–picloram (SAP) conjugate has three extending arms: a lipophilic lauryl chain, a hydrophilic polyethylene glycol chain and the amphiphobic agrochemical active picloram. We propose that the SAP conjugate maintains its colloidal stability by quickly transitioning between micellar and inverse micellar phases in hydrophilic and lipophilic environments respectively. The SAP conjugate provides the advantage of a phase structure that enables enhanced interaction with the hydrophobic epicuticular wax surface of the leaf. We have investigated the herbicidal efficiency of the SAP conjugate compared against that of commercial picloram formulations using the model plant Arabidopsis thaliana and found that when tested at agriculturally relevant doses between 0.58 and 11.70 mM a dose-dependent herbicidal effect with comparable kill rates was evident.

Conclusion

Though self-assembling drug carriers are not new to the pharmaceutical industry their use for the delivery of agrochemicals shows great promise but is largely unexplored. We have shown that SAP may be used as an alternative to current surfactant-based agrochemical formulations and has the potential to shift present practises towards a more sustainable approach.

     

Combination dual responsive polypeptide vectors for enhanced gene delivery

Variation in amino acid sequences on a disulfide-linked polypeptide backbone generates differing pK(a) vectors for DNA delivery, which release nucleic acids under reducing conditions and transfect cells with greater efficacy than non-reducible or non-variable pK(a) analogues.     

Polymer gene delivery vectors encapsulated in thermally sensitive bioreducible shell

Stable, nanosized polyelectrolyte complexes between rationally designed thermally sensitive block copolymers and plasmid DNA (polyplexes) were formed and their in vitro transfection efficiency was tested. The polyplexes were further stabilized through encapsulation into a biodegradable polymer shell. Although reduced as compared to that of the corresponding polyplexes, the encapsulated systems still show acceptable transfection efficiency. That opens the possibility to tune the balance between the safe transport and efficient delivery of DNA into the cells.     

Efficient gene delivery into dendritic cells by fiber-mutant adenovirus vectors

Recent studies have demonstrated the usefulness of dendritic cells (DCs) genetically modified by adenovirus vectors (Ad) to immunotherapy, while sufficient gene transduction into DCs is required for high doses of Ad. The RT-PCR analysis revealed that the relative resistance of DCs to Ad-mediated gene transfer is due to the absence of Coxsackie-adenovirus receptor expression, and that DCs expressed adequate alpha(v)-integrins. Therefore, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob can efficiently transduce and express high levels of the LacZ gene into DCs. The gene delivery by fiber-mutant Ad was more efficient than that by conventional Ad in both murine DC lines and normal human DCs (NHDC). Furthermore, NHDC transduced with fiber-mutant Ad and conventional Ad at 8000-vector particles/cell resulted in a 70-fold difference in beta-galactosidase activity. We propose that alpha(v)-integrin-targeted Ad is a very powerful tool with which to implement DC-based vaccination strategies.     

Biodegradable polymeric vectors for gene delivery to human endothelial cells

Endothelial cells are an important cell type to both cardiovascular disease and cancer, as they play critical roles in vascular function and angiogenesis. However, effective and safe gene delivery to primary endothelial cells in the presence of serum proteins is known to be particularly challenging. A library of biodegradable poly(beta-amino esters) was synthesized for use as potential vectors. Promising vectors were optimized for high efficacy and low cytotoxicity to human umbilical vein endothelial cells (HUVECs) in serum. Vector parameters including polymer type, polymer weight, and DNA loading were varied, and biophysical properties including particle size, zeta potential, and particle stability over time were studied. While many of the poly(beta-amino ester) vectors have similar biophysical properties in the presence of buffer, their biophysical properties changed differentially in the presence of serum proteins, and the properties of these serum-interacting particles correlated to transfection efficacy. Leading poly(beta-amino ester) vectors were found to transfect HUVECs in the presence of serum significantly higher (47 +/- 9% positive, n = 10) than the best commercially available transfection reagents including jetPEI (p < 0.001) and Lipofectamine 2000 (p < 0.01). These results demonstrate the potential of a new class of biomaterials, poly(beta-amino esters), for effective human endothelial cell gene therapy.     

Directed evolution of adeno-associated virus yields enhanced gene delivery vectors

Adeno-associated viral vectors are highly safe and efficient gene delivery vehicles. However, numerous challenges in vector design remain, including neutralizing antibody responses, tissue transport and infection of resistant cell types. Changes must be made to the viral capsid to overcome these problems; however, very often insufficient information is available for rational design of improvements. We therefore applied a directed evolution approach involving the generation of large mutant capsid libraries and selection of adeno-associated virus (AAV) 2 variants with enhanced properties. High-throughput selection processes were designed to isolate mutants within the library with altered affinities for heparin or the ability to evade antibody neutralization and deliver genes more efficiently than wild-type capsid in the presence of anti-AAV serum. This approach, which can be extended to additional gene delivery challenges and serotypes, directs viral evolution to generate 'designer' gene delivery vectors with specified, enhanced properties.     

Novel nonviral vectors for gene delivery: Synthesis and applications

Summary We have synthesized novel cationic lipids for gene delivery bearing an ester bond between the lipid moiety and the polyamine head. We have found that an intramolecular rearrangement occurs during purification of one of the products. The rearrangement led to a cyclic lipopolyamine which was active for DNA gene transfer. The formation of cyclization products depends on the spacer found between the lipid and the polyamine. The introduction of arginine in the linker position prevents the formation of cyclic products. Linear as well as cyclic analogues showed high-efficiency gene transfer when tested in vitro for luciferase gene expression as compared to dioctadecylamidoglycyl spermine or lipofectamine and also in vivo in the Lewis lung carcinoma model. The introduction of arginine in the linker position promoted increased transfection activity, demonstrating that a diversity of linkers, such as short peptides or glycosides, can be introduced into cationic lipids for targeted gene transfer.