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.     

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.     

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.     

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.     

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.     

Recombinant gene expression in human umbilical vein endothelial cells transduced by retroviral vectors

Endothelial cells are attractive targets for gene transfer because of their immediate contact with the bloodstream, and, therefore, they might serve as vehicles for therapeutic drug delivery. Recently, we and others reported that endothelial cells of animal origin efficiently express both secretory and nonsecretory recombinant proteins. We now show that human endothelial cells are also capable of expressing a recombinant gene following transduction with retroviral vectors. Human umbilical vein endothelial cells were transduced with either the N2 or the SAX vector. Following selection with G418, cells transduced by both vectors were found to express neophosphotransferase activity, the product of the neomycin resistance gene. The fact that a recombinant gene can be readily inserted and efficiently expressed into human endothelial cells suggests that these cells may be able to serve a role in human gene therapy.     

Baculovirus vectors for efficient gene delivery and expression in mammalian cells

Baculovirus expression vectors are extensively used for the delivery of foreign genes and expression of recombinant proteins in insect and mammalian cells. Modified baculoviruses containing mammalian promoter elements (BacMam viruses) for an efficient transient and stable transduction of diverse mammalian cells ensure a high level of heterologous protein expression both in vitro and in vivo. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus promoter, green or red fluorescent protein gene, SV40pA polyadenylation signal, and polylinker MCS were constructed for the delivery of genes encoding hepatitis C virus structural proteins into mammalian cells. In HEK293T and Huh7 cells, formation of glycoprotein complexes and HCV4ike particles was observed. A high efficiency of the baculovirus-medi-ated gene transfer and expression of the virus envelope proteins in mammalian cells was demonstrated using fluorescence, flow cytometry, and immunoblot techniques.     

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.     

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.     

Cell-specific delivery of polymeric nanoparticles to carbohydrate-tagging cells

Carbohydrates on cell surfaces contribute a variety of communications between the cell and its environment, and they have been assumed to act as markers for cellular recognition. In this research, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer nanoparticles, which can react with specific carbohydrates of target cells, were newly prepared to serve as novel drug carriers. A water-soluble MPC polymer bearing hydrazide groups (PMBH) was synthesized by conventional radical polymerization. The MPC polymer showed amphiphilic nature and worked as an emulsifier to form nanoparticles. The nanoparticles covered with PMBH were prepared by the solvent evaporation method and exhibited monodispersity. They were approximately 200 nm in diameter and -2.0 mV in surface potential. According to a surface analysis of the nanoparticles, phosphorylcholine and hydrazide groups were observed, and the surface was fully covered with PMBH. Unnatural carbohydrates having ketone groups on human cervical carcinoma cell (HeLa) surfaces were expressed by treatment with levulinoyl mannosamine (ManLev). When the PMBH nanoparticles were in contact with the ManLev-treated HeLa cells, they accumulated in the cells. In contrast, the nanoparticles were not observed in native HeLa cells (without unnatural carbohydrates). These results indicate that the hydrazide groups of the nanoparticles selectively reacted to the ketone groups of the carbohydrates on the cell surface. The PMBH nanoparticles immobilized with anticancer drugs such as doxorubicin or paclitaxel were in contact with either ManLev-treated or untreated HeLa cells. The viability of the ManLev-treated HeLa cells was effectively reduced, but that of the untreated cells was preserved. This indicated that the anticancer drugs were selectively delivered to the ManLev-treated cells. Nonspecific cellular uptake of the nanoparticles was effectively reduced by MPC polymer coating. Furthermore, the immobilization processes of the drugs differed because of the solubility of the drugs. In conclusion, cellular-specific drug delivery by means of the novel nanoparticles was demonstrated with the selective reaction between unnatural carbohydrates on the cell surface and the hydrazide groups bearing the phosphorylcholine polymer nanoparticles.     

Integrating retroviral cassette extends gene delivery of HSV-1 expression vectors to dividing cells

Retroviral vectors have long been used in a wide variety of gene transfer applications but have certain drawbacks, such as small cargo size, limited tropism, and low titers. HSV expression vectors overcome these disadvantages, but, because they persist in target cells as nonreplicative episomes, they are not retained in all the progeny of dividing cells. Chimeric HSV/AAV products that can mediate transgene integration in human mitotic cells have been constructed, but, to date, genetic modification of dividing cells in animal models using HSV products has not been possible. Here, we report the construction of hybrid HSV/retroviral vectors that exhibit up to 50-fold higher transgene integration efficiency compared to vectors containing only HSV-1 components. Efficient integration of a retroviral transgene cassette encoding pac in human cells required expression of the Moloney murine leukemia virus gag-pol genes, but in murine cells, could also be mediated by endogenous activities, albeit at a lower level. Gene delivery was equally efficient in BHK21, a cell line resistant to retroviral infection, and transgene retention and expression were observed to be stable for least one month in Hs683 human glioma cells. These vectors have wide applications for the genetic modification of many cell types.     

Decreased binding to proteins and cells of polymeric gene delivery vectors surface modified with a multivalent hydrophilic polymer and retargeting through attachment of transferrin

Binding of serum proteins to polyelectrolyte gene delivery complexes is thought to be an important factor limiting bloodstream circulation and restricting access to target tissues. Protein binding can also inhibit transfection activity in vitro. In this study a multivalent reactive hydrophilic polymer has been used to inhibit protein binding. This polymer is based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA) bearing pendent oligopeptide (Gly-Phe-Leu-Gly) side chains terminated in reactive 4-nitrophenoxy groups (8.6 mol%). The polymer reacts with the primary amino groups of poly(L-lysine) (pLL) and produces a hydrophilic coating on the surface of pLL.DNA complexes (as measured by fluorescamine). The resulting pHPMA-coated complexes show a decreased surface charge (from +14 mV for pLL.DNA complexes to -25 mV for pHPMA-modified complexes) as measured by zeta potential analysis. The pHPMA-coated complexes also show a slightly increased average diameter (approximately 90 nm compared with 60 nm for pLL. DNA complexes) as viewed by atomic force and transmission electron microscopy and around 100 nm as viewed by photon correlation spectroscopy. They are completely resistant to protein interaction, as determined by turbidometry and SDS-polyacrylamide gel electrophoresis analysis of complexes isolated from plasma, and show significantly decreased nonspecific uptake into cells in vitro. Spare reactive ester groups can be used to conjugate targeting ligands (e.g. transferrin) on to the surface of the complex to provide a means of tissue-specific targeting and transfection. The properties of these complexes therefore make them promising candidates for targeted gene delivery, both in vitro and potentially in vivo.     

Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors

We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105(+) endothelial cells, human CD133(+) hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133(+) cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.     

Lentiviruses as gene delivery vectors.

Lentivirus vectors are already used as effective gene delivery tools in cells from liver, retina, skeletal muscle and the central nervous system. In the past year, new and exciting data from gene transfer experiments in human hematopoietic progenitor cells have been obtained. Furthermore, new generations of HIV-1 based lentivirus vectors as well as new potentially less pathogenic HIV-2 based vectors have been described; however, old and new obstacles remain to be cleared.     

Biodegradable cationic polyester as an efficient carrier for gene delivery to neonatal cardiomyocytes

Viral-mediated gene delivery has been explored for the treatment and protection of cardiomyocytes, but so far there is only one report using cationic polymer for gene delivery to cardiomyocytes in spite of many advantages of polymer-mediated gene delivery. In this study, a cationic poly(beta-amino ester) (PDMA) with a degradable backbone and cleavable side chains was synthesized by Michael addition reaction. The toxicity of PDMA to neonatal mouse cardiomyocytes (NMCMs) was significantly lower than that of polyethyleneimine (PEI). PDMA formed stable polyplexes with pEGFP. The dissociation of the polyplexes could be triggered by PDMA degradation, and the dissociation time was tunable via the polymer/pEGFP ratio. In vitro transfection showed that PDMA was an effective and low toxic gene delivery carrier for NMCMs. The PDMA/pEGFP polyplexes transfected EGFP gene to NMCMs with about 28% efficiency and caused little death. In contrast, a significant portion of cardiomyocytes cultured with PEI/pEGFP died.     

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.     

Development of polymeric drug delivery system for recognizing vascular endothelial dysfunction

The vascular endothelium plays an important role in regulating vascular homeostasis. Damage to the endothelium can lead to cardiovascular diseases such as arteriosclerosis. Therefore, early-stage detection and evaluation of vascular endothelium dysfunction would be very important for effective diagnosis and therapy. We synthesized a polymeric drug carrier bearing an Evans blue analogue as a probing unit for endothelium injury. The polymeric carrier spontaneously formed stable nanoparticles with micelle-like structure in aqueous media and could encapsulate hydrophobic doxorubicin (DOX). The encapsulated DOX showed a sustainable release profile over a period of 10-60 h depending on the loaded DOX concentration. The polymeric carrier specifically adsorbed against the endothelium-injured site in extracted porcine aorta. These properties of the polymeric drug carrier will be suitable for specific drug delivery to endothelium dysfunctional region.