Lipid-based systems for the intracellular delivery of genetic drugs

Currently available delivery systems for genetic drugs have limited utility for systemic applications. Cationic liposome/ plasmid DNA or oligonucleotide complexes are rapidly cleared from circulation, and the highest levels of activity are observed in `first pass' organs, such as the lungs, spleen and liver. Engineered viruses can generate an immune response, which compromises transfection resulting from subsequent injections and lack target specificity. A carrier, which can accumulate at sites of diseases such as infections, inflammations and tumours, has to be a small, neutral and highly serum-stable particle, which is not readily recognized by the fixed and free macrophages of the reticuloendothelial system (RES). This review summarizes lipid-based technologies for the delivery of nucleic acid-based drugs and introduces a new class of carrier systems, which solve, at least in part, the conflicting demands of circulation longevity and intracellular delivery. Plasmid DNA and oligonucleotides are entrapped into lipid particles that contain small amounts of a positively charged lipid and are stabilized by the presence of a polyethylene glycol (PEG) coating. These carriers protect nucleic acid-based drugs from degradation by nucleases, are on average 70 nm in diameter, achieve long circulation lifetimes and are capable of transfecting cells.     

Gene delivery systems using the Sendai virus

Fusogenic liposome (FL) is a delivery system that can transfer encapsulated materials into living cells directly through membrane fusion. FL is a promising approach for gene therapy because it can deliver various genetic materials much more efficiently than other non-viral vectors without damaging the cell. FL-mediated gene transfer consists of two independent membrane fusion phenomena; generation of a FL by fusing a Sendai virus (SV) particle with a simple liposome encapsulating DNA, and successive fusion of the FL with cell membrane. The former requires viral F protein but no other special molecule on the liposomal membrane, whereas the latter may require the receptor (sialic acid) and unidentified assistant molecule(s) on the cell membrane. Further analysis suggests that these assistant molecule(s), not the receptor, may control the fusion and govern the cell specificity of FL-mediated delivery. This review has described a detailed analysis of these fusion phenomena and discussed possible applications of FL-mediated gene delivery to human gene therapy.     

Antibodies to human immunodeficiency virus in human sera induce cell-mediated lysis of human immunodeficiency virus-infected cells

The capacity of human immunodeficiency virus (HIV) antibody-positive sera from homosexually active men without acquired immune deficiency syndrome to lyse the HIV-infected T cell lines MOLT-4f and CCRF-CEM (CEM) in cooperation with lymphocytes from normal donors was investigated. Twenty-seven HIV antibody-positive sera, most of which enhanced the killing of HIV-infected MOLT-4f and CEM target cells by normal mononuclear cells were studied in detail. HIV antibody-positive sera resulted in lysis at dilutions as high as 1/10,000. HIV antibody-negative sera did not augment lysis of infected target cells. In addition, lysis of uninfected targets was not enhanced in the presence of HIV antibody-positive sera. Because fractionation of the HIV antibody-positive sera on a protein A affinity column resulted in recovery of the activity from the IgG fraction, the extra cytotoxic activity mediated by nonimmune cells in the presence of immune sera appears to be antibody-dependent. Furthermore, the cytotoxic effector cells were in the nonrosetting fraction of lymphocytes and expressed Leu-11 (cluster designation (CD)15) antigens, which is characteristic of cells participating in antibody-dependent cellular cytotoxicity reactions. The antibody specificity of the sera, determined by radioimmunoprecipitation, provides evidence that antibody-dependent cellular cytotoxicity can occur even when there are no detectable antibodies directed against gag proteins. Sera which lacked detectable antibodies to the envelope protein gp120 by radioimmunoprecipitation did not mediate antibody-dependent cellular cytotoxicity.     

Complementary antiviral efficacy of hydroxyurea and protease inhibitors in human immunodeficiency virus-infected dendritic cells and lymphocytes

Dendritic cells are susceptible to human immunodeficiency virus (HIV) infection and may transmit the virus to T cells in vivo. Scarce information is available about drug efficacy in dendritic cells because preclinical testing of antiretroviral drugs has been limited predominantly to T cells and macrophages. We compared the antiviral activities of hydroxyurea and two protease inhibitors (indinavir and ritonavir) in monocyte-derived dendritic cells and in lymphocytes. At therapeutic concentrations (50 to 100 microM), hydroxyurea inhibited supernatant virus production from monocyte-derived dendritic cells in vitro but the drug was ineffective in activated lymphocytes. Concentrations of hydroxyurea insufficient to be effective in activated lymphocytes cultured alone strongly inhibited supernatant virus production from cocultures of uninfected, activated lymphocytes with previously infected monocyte-derived dendritic cells in vitro. In contrast, protease inhibitors were up to 30-fold less efficient in dendritic cells than in activated lymphocytes. Our data support the rationale for testing of the combination of hydroxyurea and protease inhibitors, since these drugs may have complementary antiviral efficacies in different cell compartments. A new criterion for combining drugs for the treatment of HIV infection could be to include at least one drug that selectively targets HIV in viral reservoirs.     

Characterization of the inhibitory effect of PEG-lipid conjugates on the intracellular delivery of plasmid and antisense DNA mediated by cationic lipid liposomes

Poly(ethylene glycol)-lipid (PEG-lipid) conjugates are widely used in the field of liposomal drug delivery to provide a polymer coat that can confer favorable pharmacokinetic characteristics on particles in the circulation. More recently these lipids have been employed as an essential component in the self-assembly of cationic and neutral lipids with polynucleic acids to form small, stable lipid/DNA complexes that exhibit long circulation times in vivo and accumulate at sites of disease. However, the presence of a steric barrier lipid might be expected to inhibit the transfection activity of lipid/DNA complexes by reducing particle-membrane contact. In this study we examine what effect varying the size of the hydrophobic anchor and hydrophilic head group of PEG-lipids has on both gene and antisense delivery into cells in culture. Lipid/DNA complexes were made using unilamellar vesicles composed of 5 mole% PEG-lipids in combination with equimolar dioleoylphosphatidylethanolamine and the cationic lipid dioleyldimethylammonium chloride. Using HeLa and HepG2 cells we show that under the conditions employed PEG-lipids had a minimal effect on the binding and subsequent endocytosis of lipid/DNA complexes but they severely inhibited active gene transfer and the endosomal release of antisense oligodeoxynucleotides into the cytoplasm. Decreasing the size of the hydrophobic anchor or the size of the grafted hydrophilic PEG moiety enhanced DNA transfer by the complexes.     

Liposome-mediated delivery of ribosome inactivating proteins to cells in vitro

This study describes the liposome-mediated delivery of toxins to a variety of cells in vitro. Gelonin, a potent inhibitor of protein synthesis from Gelonium multiflorum, was delivered to the cytoplasm of TLX5 lymphoma cells most effectively by phosphatidylserine vesicles. These liposomes were also capable of inhibiting protein synthesis in XC (transformed rat fibroblasts) and phytohaemagglutinin-stimulated CBA mouse lymphocytes. Phosphatidylcholine liposomes had no capacity to deliver their contents to the cytoplasm, but the addition of cholesterol to the vesicle membrane resulted in an increased capacity. Delivery events were enhanced further by the addition of mixed bovine brain gangliosides to the membrane in the ratio 5:5:1 phosphatidylcholine/cholesterol/gangliosides. The addition of cholesterol to phosphatidylserine vesicles failed to increase the inhibitory effects of the gelonin liposomes. The A chain of diphtheria toxin encapsulated in phosphatidylserine liposomes had no inhibitory effect on the level of protein synthesis in TLX5 or Daudi cells.     

Liposome-mediated delivery of DNA to carrot protoplasts

The encapsulation of DNA within liposomes and subsequent fusion of the liposomes with carrot (Daucus carota L.) protoplasts were examined to determine optimum conditions for effective liposome-mediated delivery of DNA to protoplasts. Escherichia coli [³H]DNA could be encapsulated with 50% efficiency using encapsulation volumes as low as 0.5 ml. Incorporation of liposome-encapsulated [³H]DNA by carrot protoplasts increased linearly for 2.5 h, and increasing the ratio of protoplasts to liposomes increased the total amount of radioactive label incorporated within the protoplasts. Liposome-mediated incorporation of [³H]DNA by protoplasts increased over a range of polyethylene glycol concentrations up to 20%, but Ca²⁺ did not increase liposome-mediated incorporation when present in the liposome-protoplast incubation mixture. Optimum incorporation was observed when the pH of the liposome-protoplast incubation medium was decreased to 4.8. Encapsulation experiments using DNA of the plasmid pBR322 indicated that an average of 200–1,000 intact copies of pBR322 were sequestered within each nucleus after liposome delivery.     

Liposomal delivery of CTL epitopes to dendritic cells

The induction of strong and long lasting T-cell response, CD4+ or CD8+, is a major requirement in the development of efficient vaccines. An important aspect involves delivery of antigens to dendritic cells (DCs) as antigen presenting cells (APCs) for the induction of potent antigen-specific CD8+ T lymphocyte (CTLs) responses. Protein or peptide-based vaccines become an attractive alternative to the use of live cell vaccines to stimulate CTL responses for the treatment of viral diseases or malignancies. However, vaccination with proteins or synthetic peptides representing discrete CTL epitopes have failed in most instances due to the inability for exogenous antigens to be properly presented to T cells via major histocompatibility complex (MHC) class I molecules. Modern vaccines, based on either synthetic or natural molecules, will be designed in order to target appropriately professional APCs and to co-deliver signals able to facilitate activation of DCs. In this review, we describe the recent findings in the development of lipid-based formulations containing a combination of these attributes able to deliver tumor- or viral-associated antigens to the cytosol of DCs. We present in vitro and pre-clinical studies reporting specific immunity to viral, parasitic infection and tumor growth.     

Modulation of DNA and RNA by PNA

Peptide nucleic acid (PNA), a synthetic DNA mimic that is devoid of the (deoxy)ribose-phosphate backbone yet still perfectly retains the ability to recognize natural nucleic acids in a sequence-specific fashion, can be employed as a tool to modulate gene expressions via several different mechanisms. The unique strength of PNA compared to other oligonucleotide analogs is its ability to bind to nucleic acid targets with secondary structures such as double-stranded and quadruplex DNA as well as RNA. This digest aims to introduce general readers to the advancement in the area of modulation of DNA/RNA functions by PNA, its current status and future research opportunities, with emphasis on recent progress in new targeting modes of structured DNA/RNA by PNA and PNA-mediated gene editing.     

Liposome-mediated delivery of deoxyribonucleic acid to cells: enhanced efficiency of delivery related to lipid composition and incubation conditions

Delivery of liposome-encapsulated simian virus 40 (SV40) DNA to African green monkey Related to been used as a probe to study liposome--cell interactions and to determine conditions which favor the intracellular delivery of liposome contents to cells. The efficiency of DNA delivery by various liposome preparations (monitored by infectivity assays) was found to be dependent both on the magnitude of vesicle binding to cells and on the resistance of liposomes to cell-induced leakage of contents. Acidic phospholipids were much more effective in both binding and delivery, and phosphatidylserine (PS) was the best in both aspects. The inclusion of 50 mol % cholesterol in liposomes reduces the cell-induced leakage of vesicle contents (2--5-fold) and substantially enhances the delivery of DNA to cells (2--10-fold). Following incubation of cells with negatively charged liposomes containing SV40 DNA, infectivity can be enhanced greatly by brief exposure of the cells to glycerol solutions. In contrast, only slight enhancement by glycerol was observed for SV40 DNA encapsulated in neutral or positively charged liposomes. The results of competition experiments between empty phosphatidylcholine liposomes and DNA-containing PS liposomes also suggest possible differences in the interaction of neutral and negatively charged liposome preparations with cells. Morphological studies indicate that the glycerol treatment stimulates membrane ruffling and vacuolization and suggest that the enhanced uptake of liposomes occurs by an endocytosis-like process. Results obtained with metabolic inhibitors are also consistent with the interpretation that the enhancement of liposome delivery in glycerol-treated cells occurs via an energy-dependent endocytotic pathway. Pretreatment of cells with chloroquine, a drug which alters lysosomal activity, further enhanced infectivity in glycerol-treated cells (4-fold). This observation suggests the involvement of a lysosomal processing step at some point in the expression of liposome-encapsulated DNA and, more importantly, illustrates the possibility of altering cellular mechanism to engineer more efficient delivery by liposomes. Under optimal conditions determined in this study, the efficiency of liposome-mediated SV40 DNA delivery was increased more than 1000-fold over that obtained by simply incubating cells with liposomes. It is also demonstrated that these conditions enhance delivery of other molecules, besides DNA, which are encapsulated in liposomes.     

Terminally modified,short phosphorothioate oligonucleotides as inhibitors of gene expression in cells

Phosphorothioates are excellent antisense inhibitors, which are active both in cells and in vivo. Since their affinity to complementary ribonucleic acids is rather low, long strands (⩾20-mers) are typically required to achieve the desired biological activity. However, mismatch discrimination of long inhibitors is reduced. In contrast, shorter phosphorothioates exhibit better sequence specificity, but have in most cases too low affinity for practical applications in cells. We screened a range of terminal modifiers of a 14-mer phosphorothioate sequence, which is complementary to mRNA of a representative gene, whose protein product is fluorescent (DsRed2) and easy to monitor in cells. We found that optimal combinations of 5′- and 3′-modifications include 5′-trimethoxystilbene with 3′-uracil(anthraquinone)-cap, 5′-chloic acid derivative with 3′-uracyl(anthraquinone)-cap and 5′-cholic acid derivative with three 3′-LNA moieties. In contrast to the LNA, stabilizing and activity-enhancing effects of other mentioned modifiers for PTO/RNA duplexes have not been previously reported. We observed that the 14-mer inhibitor carrying 5′-cholic acid derivative with three 3′-LNA moieties inhibits expression of DsRed2 in cells stronger than the unmodified 21-mer. Mismatch discrimination of this inhibitor was found to be comparable to that of the unmodified 14-mer.     

Gene delivery to human bone marrow mesenchymal stem cells by microporation

Electroporation has been considered one of the most efficient non-viral based methods to deliver genes regardless of frequently observed high cell mortality. In this study we used a microporation technique to optimise the delivery of plasmid DNA encoding green fluorescence protein (GFP) to human bone marrow mesenchymal stem cells (BM-MSC). Using resuspension buffer (RB) and as low as 1.5 × 10⁵ cells and 1 μg of DNA, we achieved 40% of cells expressing the transgene, with cell recovery and cell viabilities of 85% and 90%, respectively. An increase in DNA amount did not significantly increase the number of transfected cells but clearly reduced cell recovery. A face-centered composite design was used to unveil the conditions giving rise to optimal plasmid delivery efficiencies when using a sucrose based microporation buffer (SBB). The BM-MSC proliferation kinetics were mainly affected by the presence of plasmid and not due to the microporation process itself although no effect was observed on their immunophenotypic characteristics and differentiative potential. Based on the data shown herein microporation demonstrated to be a reliable and efficient method to genetically modify hard-to-transfect cells giving rise to the highest levels of cell survival reported so far along with superior gene delivery efficiencies.     

A DNA uptake-stimulating protein increases the antiproliferative effect of c-myb antisense oligonucleotide on leukemic cells

Proliferation of HL-60 and MOLT4 leukemia cells was inhibited by a c-myb antisense oligonucleotide (ASO) in the presence of a DNA uptake-stimulating protein (DNA uptake-stimulating factor, DUSF). The inhibitory effect was very mild in the absence of DUSF. Sense oligonucleotides or DUSF, alone or in combination, were found to be ineffective. Cellular expression of the c-myb protein was significantly more inhibited by the c-myb ASO in the presence than in the absence of DUSF. In the presence of DUSF, c-myb protein practically disappeared from the nuclei of HL-60 and MOLT4 cells treated with the ASO. Thus, DUSF appears to effectively stimulate the uptake of c-myb ASO into tumor cells in vitro, augmenting its antiproliferative effect by decreasing c-myb expression.     

人类免疫缺陷病毒感染者免疫功能重建不全的研究进展

约20%的人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染者在接受抗病毒治疗后,外周血CD4^＋ T细胞水平无法有效恢复,即免疫功能重建不全,但发生机制目前尚不明确。本文根据HIV感染者的临床和免疫学指标,分析发生免疫功能重建不全的风险因素;进一步从外周循环系统中T细胞损伤、细胞因子变化及肠道黏膜局部CD4^＋ T细胞删除等方面解析可能机制,其中肠道黏膜CD4^＋ T细胞删除可能与肠道微生物菌群诱导的细胞焦亡有关。虽然目前国际上有多项关于免疫功能重建不全患者治疗方案的临床试验,但由于缺乏一致的、有力的基础研究证据,导致疾病诊断指标缺乏,单独细胞因子给药治疗亦未取得突破性进展。因此,应深入探索免疫功能重建不全发生机制,进行大样本、长时间的前瞻性队列研究,制定免疫功能重建不全的临床与免疫学界定标准,从而为临床治疗提供科学依据。     

Therapeutic gene delivery and transfection in human pancreatic cancer cells using epidermal growth factor receptor-targeted gelatin nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality (1). Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released (2-5). Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect (6). Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells (7). In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.(8) Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells (9). The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes (10). In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells (11). Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.     

Liposome-mediated transfer of YAC DNA to tobacco cells

This article describes a set of protocols—for retrofitting, transformation and purification—that together enable the delivery of full-sized YAC-DNA to plant cells. To be able to equip YACs of interest with plant selectable markers, we have constructed a retrofitting vector that carriesnptII anduidA. Furthermore, we established a transformation protocol for plant protoplasts that is sufficiently efficient to support transfer of high-molecular-weight DNA. In this protocol lipofection is combined with PEG-mediated direct gene transfer. Large amounts of purified DNA are necessary for lipofection. To obtain sufficient quantities of concentrated, purified YAC-DNA, we used an optimized two-step, gel-purification method. Transient expression of a YAC-bornuidA demonstrates that both retrofitting vector and transformation protocol are effective.     

Seroprevalence of antibodies to Chlamydia spp. in human immunodeficiency virus-infected subjects in Japan

To evaluate the seropositivity of Chlamydia spp. in human immunodeficiency virus (HIV)-infected subjects in Japan, Chlamydia-specific antibodies in sera collected from 106 HIV-infected subjects were measured by the microimmunofluorescence test. The prevalence of C. pneumoniae-specific IgA, C. trachomatis-specific IgG and IgA and mean titers were significantly higher in the homosexual and heterosexual HIV-infected subjects than in the hemophilic patients and HIV-negative controls. These data indicate that the higher C. pneumoniae and C. trachomatis seroprevalence among HIV-infected subjects is probably due to an HIV risk factor, such as promiscuous sexual behavior, rather than to HIV infection itself.