Vector-mediated delivery of a polyamide ("peptide") nucleic acid analogue through the blood-brain barrier in vivo.

Polyamide ("peptide") nucleic acids (PNAs) are molecules with antigene and antisense effects that may prove to be effective neuropharmaceuticals if these molecules are enabled to undergo transport through the brain capillary endothelial wall, which makes up the blood-brain barrier in vivo. The model PNA used in the present studies is an 18-mer that is antisense to the rev gene of human immunodeficiency virus type 1 and is biotinylated at the amino terminus and iodinated at a tyrosine residue near the carboxyl terminus. The biotinylated PNA was linked to a conjugate of streptavidin (SA) and the OX26 murine monoclonal antibody to the rat transferrin receptor. The blood-brain barrier is endowed with high transferrin receptor concentrations, enabling the OX26-SA conjugate to deliver the biotinylated PNA to the brain. Although the brain uptake of the free PNA was negligible following intravenous administration, the brain uptake of the PNA was increased at least 28-fold when the PNA was bound to the OX26-SA vector. The brain uptake of the PNA bound to the OX26-SA vector was 0.1% of the injected dose per gram of brain at 60 min after an intravenous injection, approximating the brain uptake of intravenously injected morphine. The PNA bound to the OX26-SA vector retained the ability to bind to synthetic rev mRNA as shown by RNase protection assays. In summary, the present studies show that while the transport of PNAs across the blood-brain barrier is negligible, delivery of these potential neuropharmaceutical drugs to the brain may be achieved by coupling them to vector-mediated peptide-drug delivery systems.

DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors.

Viral vectors based on adeno-associated virus (AAV) preferentially transduce cells in S phase of the cell cycle. We recently found that DNA-damaging agents increased the transduction of nondividing cells. However, the optimal concentrations were toxic to cells. Here we show that the transduction of normal human fibroblasts by AAV vectors is increased by prior exposure to DNA synthesis inhibitors, such as aphidicolin or hydroxyurea, and topoisomerase inhibitors, such as etoposide or camptothecin. Transduction efficiencies could be increased > 300-fold in stationary cultures at concentrations that did not affect cell viability or proliferative potential. Both S-phase and non-S-phase cells were affected, suggesting that cellular functions other than replicative DNA synthesis may be involved. Applying these methods to gene transfer protocols should improve prospects for gene therapy by AAV vectors.

Delivery of antisense oligodeoxyribonucleotides against the human epidermal growth factor receptor into cultured KB cells with liposomes conjugated to folate via polyethylene glycol.

Antisense oligodeoxyribonucleotides targeted to the epidermal growth factor (EGF) receptor were encapsulated into liposomes linked to folate via a polyethylene glycol spacer (folate-PEG-liposomes) and efficiently delivered into cultured KB cells via folate receptor-mediated endocytosis. The oligonucleotides were a phosphodiester 15-mer antisense to the EGF receptor (EGFR) gene stop codon (AEGFR2), the same sequence with three phosphorothioate linkages at each terminus (AEGFR2S), a randomized 15-mer control of similar base composition to AEGFR2 (RC15), a 14-mer control derived from a symmetrized Escherichia coli lac operator (LACM), and the 5'-fluorescein-labeled homologs of several of the above. Cellular uptake of AEGFR2 encapsulated in folate-PEG-liposomes was nine times higher than AEGFR2 encapsulated in nontargeted liposomes and 16 times higher than unencapsulated AEGFR2. Treatment of KB cells with AEGFR2 in folate-PEG-liposomes resulted in growth inhibition and significant morphological changes. Curiously, AEGFR2 and AEGFR2S encapsulated in folate-PEG-liposomes exhibited virtually identical growth inhibitory effects, reducing KB cell proliferation by > 90% 48 hr after the cells were treated for 4 hr with 3 microM oligonucleotide. Free AEGFR2 caused almost no growth inhibition, whereas free AEGFR2S was only one-fifth as potent as the folate-PEG-liposome-encapsulated oligonucleotide. Growth inhibition of the oligonucleotide-treated cells was probably due to reduced EGFR expression because indirect immunofluorescence staining of the cells with a monoclonal antibody against the EGFR showed an almost quantitative reduction of the EGFR in cells treated with folate-PEG-liposome-entrapped AEGFR2. These results suggest that antisense oligonucleotide encapsulation in folate-PEG-liposomes promise efficient and tumor-specific delivery and that phosphorothioate oligonucleotides appear to offer no major advantage over native phosphodiester DNA when delivered by this route.

Combination gene therapy for liver metastasis of colon carcinoma in vivo.

The efficacy of combination therapy with a "suicide gene" and a cytokine gene to treat metastatic colon carcinoma in the liver was investigated. Tumor in the liver was generated by intrahepatic injection of a colon carcinoma cell line (MCA-26) in syngeneic BALB/c mice. Recombinant adenoviral vectors containing various control and therapeutic genes were injected directly into the solid tumors, followed by treatment with ganciclovir. While the tumors continued to grow in all animals treated with a control vector or a mouse interleukin 2 vector, those treated with a herpes simplex virus thymidine kinase vector, with or without the coadministration of the mouse interleukin 2 vector, exhibited dramatic necrosis and regression. However, only animals treated with both vectors developed an effective systemic antitumoral immunity against challenges of tumorigenic doses of parental tumor cells inoculated at distant sites. The antitumoral immunity was associated with the presence of MCA-26 tumor-specific cytolytic CD8+ T lymphocytes. The results suggest that combination suicide and cytokine gene therapy in vivo can be a powerful approach for treatment of metastatic colon carcinoma in the liver.

Chronic myeloid leukemia gene therapy delivered by cell penetrating peptides : from bench to clinic

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

In vivo gene expression: DNA electrotransfer

The use of electric pulses to deliver therapeutic molecules to tissues and organs in vivo is a rapidly growing field of research. Electrotransfer can be used to deliver a wide range of potentially therapeutic agents, including drugs, proteins, oligonucleotides, RNA and DNA. Optimization of this approach depends upon a number of parameters such as target organ accessibility, cell turnover, microelectrode design, electric pulsing protocols and the physiological response to the therapeutic agent. Many organs have been successfully transfected by electroporation, including skin, liver, skeletal and cardiac muscle, male and female germ cells, artery, gut, kidney, retinal ganglion cells, cornea, spinal cord, joint synovium and brain. Electrotransfer technology is relevant in a variety of research and clinical settings including cancer therapy, modulation of pathogenic immune reactions, delivery of therapeutic proteins and drugs, and the identification of drug targets by the modulation of normal gene expression. This, together with the capacity to deliver very large DNA constructs, greatly expands the research and clinical applications of in vivo DNA electrotransfer.

Etr1-1 gene expression alters regeneration patterns in transgenic lettuce stimulating root formation

We have evaluated the transformation efficiency of two lettuce ( Lactuca sativa L.) cultivars, LE126 and Seagreen, using Agrobacterium tumefaciens- mediated gene transfer. Six- day- old cotyledons were co- cultivated with Agrobacterium cultures carrying binary vectors with two different genetic constructs. The first construct contained the beta- glucuronidase gene ( GUS) under the control of the cauliflower mosaic virus 35S promoter ( CaMV 35S), while the second construct contained the ethylene mutant receptor etr1- 1, which confers ethylene insensitivity, under the control of a leaf senescence- specific promoter ( sag12). Tissues co- cultivated with the GUS construct showed strong regeneration potential with over 90% of explants developing callus masses and 85% of the calli developing shoots. Histochemical GUS assays showed that 85.7% of the plants recovered were transgenic. Very different results were observed when cotyledon explants were co- cultivated with Agrobacteria carrying the etr1- 1 gene. There was a dramatic effect on the regeneration properties of the cultured explants with root formation taking place directly from the cotyledon tissue in 34% of the explants and no callus or shoots observed initially. Eventually callus formed in 10% of cotyledons and some organogenic shoots were obtained ( 2.86%). These results indicate that the ethylene insensitivity conferred by the etr1- 1 gene alters the normal pattern of regeneration in lettuce cotyledons, inhibiting the formation of shoots and stimulating root formation during regeneration.

Assessment of epidermal cell viability by near infrared multi-photon microscopy following ballistic delivery of gold micro-particles

The use of gene guns in ballistically delivering DNA vaccine coated gold micro-particles to skin can potentially damage targeted cells, therefore influencing transfection efficiencies. In this paper, we assess cell death in the viable epidermis by non-invasive near infrared two-photon microscopy following micro-particle bombardment of murine skin. We show that the ballistic delivery of micro-particles to the viable epidermis can result in localised cell death. Furthermore, experimental results show the degree of cell death is dependant on the number of micro-particles delivered per unit of tissue surface area. Micro-particles densities of 0.16 +/- 0.27 (mean +/- S.D.), 1.35 +/- 0.285 and 2.72 +/- 0.47 per 1000 mu m(2) resulted in percent deaths of 3.96 +/- 5.22, 45.91 +/- 10.89, 90.52 +/- 12.28, respectively. These results suggest that optimization of transfection by genes administered with gene guns is - among other effects - a compromise of micro-particle payload and cell death. (c) 2005 Elsevier Ltd. All rights reserved.

Drug delivery strategies for the treatment of Helicobacter pylori infections

Helicobacter pylori is one of the most common pathogenic bacterial infections, colonising an estimated half of all humans. It is associated with the development of serious gastroduodenal disease - including peptic ulcers, gastric lymphoma and acute chronic gastritis. Current recommended regimes are not wholly effective and patient compliance, side-effects and bacterial resistance can be problematic. Drug delivery to the site of residence in the gastric mucosa may improve efficacy of the current and emerging treatments. Gastric retentive delivery systems potentially allow increased penetration of the mucus layer and therefore increased drug concentration at the site of action. Proposed gastric retentive systems for the enhancement of local drug delivery include floating systems, expandable or swellable systems and bioadhesive systems. Generally, problems with these formulations are lack of specificity, limited to mucus turnover or failure to persist in the stomach. Gastric mucoadhesive systems are hailed as a promising technology to address this issue, penetrating the mucus layer and prolonging activity at the mucus-epithelial interface. This review appraises gastroretentive delivery strategies specifically with regard to their application as a delivery system to target Helicobacter. As drug-resistant strains emerge, the development of a vaccine to eradicate and prevent reinfection is an attractive proposition. Proposed prophylactic and therapeutic vaccines have been delivered using a number of mucosal routes using viral and non-viral vectors. The delivery form, inclusion of adjuvants, and delivery regime will influence the immune response generated. © 2005 Bentham Science Publishers Ltd.

REST mediates resolution of HIF-dependent gene expression in prolonged hypoxia

The hypoxia-inducible factor (HIF) is a key regulator of the cellular response to hypoxia which promotes oxygen delivery and metabolic adaptation to oxygen deprivation. However, the degree and duration of HIF-1α expression in hypoxia must be carefully balanced within cells in order to avoid unwanted side effects associated with excessive activity. The expression of HIF-1α mRNA is suppressed in prolonged hypoxia, suggesting that the control of HIF1A gene transcription is tightly regulated by negative feedback mechanisms. Little is known about the resolution of the HIF-1α protein response and the suppression of HIF-1α mRNA in prolonged hypoxia. Here, we demonstrate that the Repressor Element 1-Silencing Transcription factor (REST) binds to the HIF-1α promoter in a hypoxia-dependent manner. Knockdown of REST using RNAi increases the expression of HIF-1α mRNA, protein and transcriptional activity. Furthermore REST knockdown increases glucose consumption and lactate production in a HIF-1α- (but not HIF-2α-) dependent manner. Finally, REST promotes the resolution of HIF-1α protein expression in prolonged hypoxia. In conclusion, we hypothesize that REST represses transcription of HIF-1α in prolonged hypoxia, thus contributing to the resolution of the HIF-1α response.

Somatic cell gene therapy for diabetes mellitus: engineering a surrogate B-cell

Improved methods of insulin delivery are required for the treatment of insulin-dependent diabetes mellitus (IDDM) to achieve a more physiological profile of glucose homeostasis. Somatic cell gene therapy offers the prospect that insulin could be delivered by an autologous cell implant, engineered to secrete insulin in response to glucose. This study explores the feasibility of manipulating somatic cells to behave as a surrogate insulin-secreting β-cells. Initial studies were conducted using mouse pituitary AtT20 cells as a model, since these cells possess an endogenous complement of enzymes capable of processing proinsulin to mature insulin. Glucose sensitive insulin secretion was conferred to these cells by transfection with plasmids containing the human preproinsulin gene (hppI-1) and the GLUT2 gene for the glucose transporter isoform 2. Insulin secretion was responsive to changes in the glucose concentration up to about 50μM. Further studies to up-rate this glucose sensitivity into the mM range will require manipulation of the hexokinase and glucokinase enzymes. Intraperitoneal implantation of the manipulated AtT20 cells into athymic nude mice with streptozotocin-induced diabetes resulted in decreased plasma glucose concentrations. The cells formed vascularised tumours in vivo which were shown to contain insulin-secreting cells. To achieve proinsulin processing in non-endocrine cells, co-transfection with a suitable enzyme, or mutagenesis of the proinsulin itself are necessary. The mutation of the human preproinsulin gene to the consensus sequence for cleavage by the subtilisin-like serine protease, furin, was carried out. Co-transfection of human fibroblasts with wild-type proinsulin and furin resulted in 58% conversion to mature insulin by these cells. Intraperitoneal implantation of the mature-insulin secreting human fibroblasts into the diabetic nude mouse animal model gave less encouraging results than the AtT20 cells, apparently due to poor vascularisation. Cell aggregations removed from the mice at autopsy were shown to contain insulin secreting cells only at the periphery. This thesis provides evidence that it is possible to construct, by cellular engineering, a glucose-sensitive insulin-secreting surrogate β-cell. Therefore, somatic cell gene therapy offers a feasible alternative for insulin delivery in IDDM patients.

Formulation and characterisation of cationic microparticles for the delivery of DNA vaccines

The aim of this research was to formulate a novel biodegradable, biocompatible cationic microparticle vector for the delivery of DNA vaccines. The work builds upon previous research by Singh et al which described the adsorption of DNA to the surface of poly (D,L-lactide-co-glycolide) (PLG) microparticles stabilised with the surfactant cetyltrimethyl ammonium bromide (CT AB). This work demonstrated the induction of antibody and cellular immune responses to HIV proteins encoded on plasmid DNA adsorbed to the particle surface in mice, guinea pigs and non-human primates (Singh et aI, 2000; O'Hagan et aI, 2001). However, the use of surfactants in microparticle formulations for human vaccination is undesirable due to long term safety issues. Therefore, the present research aim was to develop an adsorbed DNA vaccine with enhanced potency and increased safety compared to CTAB stabilised PLG microparticles (PLG/CTAB) by replacement of the surfactant CTAB with an alternative cationic agent. The cationic polymers chitosan and poly (N- vinylpyrrolidone/2-dimethylaminoethyl methacrylate), dimethyl sulfate quaternary (PVP-PDAEMA) were investigated as alternative stabilisers to CTAB. From a variety of initial formulations, the most promising vector(s) for DNA vaccination were selected based on physicochemical data (chapter 3) and in vitro DNA loading and release characteristics (chapter 4). The chosen formulation(s) were analysed in greater depth (chapters 3 and 4), and gene expression was assessed by in vitro cell transfection studies using 293T kidney epithelial and C2C12 myoblast non-phagocytic cell lines (chapter 5). The cytotoxicity of the microparticles and their constituents were also evaluated in vitro (chapter 5). Stability and suitability of the formulation(s) for commercial production were assessed by cryopreparation and lyophilisation studies (chapters 3 and 4). Gene expression levels in cells of the immune response were evaluated by microparticle transfection of the dendritic cell (DC) line 2.4 and primary bone marrow derived DCs (chapter 6). In vivo, mice were injected i.m. with the formulations deemed most promising on the basis of in vitro studies and humoral and cellular immune responses were evaluated (chapter 6).

The sustained delivery of antisense oligodeoxynucleotides using biodegradable polymer microspheres

Antisense technology is a novel drug discovery method, which provides an essential tool for directly using gene sequence information to rationally design specific inhibitions of mRNA, to treat a wide range of diseases. The efficacy of naked oligodeoxynucleotides (ODNs) is relatively short lived due to rapid degradation in vivo. The entrapment of ODNs within biodegradable sustained-release delivery systems may improve ODN stability and reduce dose required for efficacy. Biodegradable polymer microspheres were evaluated as delivery devices for ODNs and ribozymes. Poly(lactide-co-glycolide) polymers were used due to their biocompatibility and non toxic degradation products. Microspheres were prepared using a double emulsion-deposition method and the formulations characterised. In vitro release profiles were characterised by an initial burst effect during the first 48 hours of release followed by a more sustained release. The release profiles were influenced by microsphere size, copolymer molecular weight, copolymer ratio, ODN loading, ODN length, and ODN chemistry. The serum stability of ODNs was significantly improved when entrapped within polymer microspheres. The cellular association of ODNs entrapped within small spheres (1-2μm) was improved by approximately 20-fold in A431 carcinoma cells compared with free ODNs. Fluorescence microscopy studies showed a more diffuse subcellular distribution when delivered as a microsphere formulation compared with free ODNs, which exhibited the characteristic punctate periplasmic distribution. For in vivo evaluation, polymer microspheres containing fluorescently-labelled ODNs were stereo-taxically administered to the neostriatum of the rat brain. Free ODN resulted in a punctate cellular distribution after 24 hours. In comparison ODN delivered using polymer microspheres were intensely visible in cells 48 hours post administration, and fluorescence appeared to be diffuse covering both cytosolic and nuclear regions. Whole-body autoradiography was also used to evaluate the biodistribution of free tritium labelled ODN and ODN entrapped microspheres, following subcutaneous administration to Balb-C mice. Polymer entrapped ODN gave a similar biodistribution to free ODN. Free ODN was distributed within 24 hours, whereas polymer released ODN was observed still presented in organs and at the site of administration seven days post administration.

CNS delivery of antisense oligodeoxynucleotides using biodegradable microspheres

Antisense oligodeoxynucleotides can selectively inhibit gene expression provided they are delivered to their target site successfully for a sufficient duration. Biodegradable microspheres have previously been developed for the potential systemic delivery of antisense oligodeoxynucleotides and offer an excellent strategy for central administration of antisense oligodeoxynucleotides, providing a sustained-release delivery system. Biodegradable microspheres were formulated to entrap antisense oligodeoxynucleotides for stereotaxic implantation into site-specific regions of the rat brain.Release profiles of antisense oligodeoxynucleotides from biodegradable microspheres over 56 days that were triphasic were observed with high molecular weight polymers. Antisense oligodeoxynucleotides loaded into microspheres (1-10μm) had a five-fold increase in cellular association with glial and neuronal cells compared to the naked molecule, which was partially due to a greater cellular accumulation as observed by a slower efflux profile. In vivo distribution studies of antisense oligodeoxynucleotides demonstrated that the use of microspheres provided a sustained-release over more than 2 days compared to 12 hours of the naked molecule. Efficacy of antisense oligodeoxynucleotides was demonstrated during locomotor activity investigations, which significantly reduced cocaine-induced locomotor activity, where no efficacy was demonstrated with microspheres, possibly attributed to antisense loading and measurements being taken during a lag phase of antisense oligodeoxynucleotide release. Biodegradable microspheres can be delivered site-specifically into the brain and provide sustained-release of antisense oligodeoxynucleotides, offering the potential of in vivo efficacy in these reagents in the brain.

Cellular delivery of hammerhead ribozymes

Hammerhead ribozymes are potent RNA molecules which have the potential to specifically inhibit gene expression by catalysing the trans-cleavage of mRNAs. However, they are unstable in biological fluids and cellular delivery poses a problem. Site-specific chemical modification of hammerhead ribozymes was evaluated as a means of enhancing biological stability. Chimeric, 2'-O-methylated ribozymes, containing only five unmodified ribonucleotides, were catalytically active in vitro (kcat = 1.46 min-1) and were significantly more stable in serum and lysosomal enzymes than unmodified (all-RNA) counterparts. Furthermore, they remained undegraded in cell-containing media for up to 8 hours. Stability enhancement allowed cellular uptake properties of radiolabelled ribozymes to be assessed following exogenous delivery. Studies in vulval and glial cell lines indicated that chimeric ribozymes became cell-associated via an inefficient process, which was energy and concentration dependant. A considerable proportion of ribozymes remained bound to cell-surface components, however, a small proportion (<1%) were internalised via mechanisms of adsorptive and / or receptor mediated endocytosis. Fluorescent microscopy indicated that ribozymes were localised within endosomal / lysosomal vesicles following cell entry. This was confirmed by immuno-electron microscopy, which allowed the detection of biotin-labelled ribozymes within the cell ultrastructure. Despite the predominant localisation within endocytic vesicles, a small proportion of internalised ribozymes appeared able to exit these compartments and penetrate target sites within the nucleus and cytoplasm. The ribozymes designed in this report were directed against the epidermal growth factor receptor mRNA, which is over-expressed in a malignant brain disease called glioblastoma multiforme. In order to examine the fate of ribozymes in the brain, the distribution of FITC-labelled ribozymes was examined following intra-cerebro ventricular injection to mice. FITC-ribozymes demonstrated high punctate pattern of distribution within the striatum and cortex, which appeared to represent localisation within cell bodies and dendritic processes. This suggested that delivery to glial cells in vivo may be possible. Finally, strategies were investigated to enhance the cellular delivery of ribozymes. Conjugation of ribozymes to anti~transferrin receptor antibodies improved cellular uptake 3-fold as a result of a specific interaction with transferrin receptors. Complexation with cationic liposomes also significantly improved cell association, however, some toxiclty was observed and this could be a limitation to their use. Overall, it would appear that hammerhead ribozymes can be chemically stabilised to allow direct exogenous administration in vivo. However, additional delivery strategies are probably required to improve cellular uptake, and thus, allow ribozymes to achieve their full potential as pharmaceutical agents. KEYWORDS: Catalytic