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

A Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout Clones

Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines.

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.

Layered double hydroxide nanomaterials as potential cellular drug delivery agents

This paper briefly reviews the recent progress in using layered double hydroxide (LDH) nanomaterials as cellular delivery agents. The advantages of LDHs as cellular delivery agents are summarized, and the processes of interaction/de-intercalation of anionic drugs (genes) into/from LDH nanoparticles are discussed. Then the cellular delivery of LDH-drug (gene) nanohybrids and subsequent intracellular processes are presumably proposed. At the end, some challenges and remarks for efficient delivery of drugs (genes) via LDH nanoparticles are provided to the best of our knowledge.

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.

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

Comparison and characterisation of ribozyme delivery in cultured glial and neuronal cells in vivo and in vitro

Ribozymes are short strands of RNA that possess a huge potential as biological tools for studying gene expression and as therapeutic agents to down-regulate undesirable gene expression. Successful application of ribozymes requires delivery to the target site in sufficient amounts for an adequate duration. However, due to their large size and polyanionic character ribozymes are not amenable to transport across biological membranes. In this study a chemically modified ribozyme with enhanced biological stability, targeted against the EGFR mRNA has been evaluated for cellular delivery to cultured glial and neuronal cells with a view to developing treatments for brain tumours. Cellular delivery of free ribozyme was characterised in cultured glial and neuronal cells from the human and rat. Delivery was very limited and time dependent with no consistent difference observed between glial and neuronal cells in both species. Cellular association was largely temperature and energy-dependent with a small component of non-energy dependent association. Further studies showed that ribozyme cellular association was inhibited with self and cross competition with nucleic and non-nucleic acid polyanions indicating the presence of cell surface ribozyme-binding molecules. Trypsin washing experiments further implied that the ribozyme binding surface molecules were protein by nature. Dependence of cellular association on pH indicated that interaction of ribozyme with cell surface molecules was based on ionic interactions. Fluoresence studies indicated that, post cell association, ribozymes were sequestered in sub-cellular vesicles. South-Western blots identified several cell surface proteins which bind to ribozymes and could facilitate cellular association. The limited cellular association observed with free ribozyme required the development and evaluation of polylactide-co-glycolide microspheres incorporating ribozyme for enhanced cellular delivery. Characterisation of microsphere mediated delivery of ribozyme in cultured glial and neuronal cells showed that association increased by 18 to 27-fold in all cell types with no differences observed between cell lines and species. Microsphere mediated delivery was temperature and energy dependent and independent of pH. In order to assess the potential of PLGA micro spheres for the CNS delivery of ribozyme the distribution of ribozyme entrapping microspheres was investigated in rat CNS after intracerebroventricular injection. Distribution studies demonstrated that after 24 hours there was no free ribozyme present in the brain parenchyma, however microsphere entrapped ribozyme was found in the CNS. Microspheres remained in the ventricular system after deposition and passed from the lateral ventricles to the third and fourth ventricle and in the subarachnoid space. Investigation of the influence of microsphere size on the distribution in CNS demonstrated that particles up to 2.5 and O.5f.lm remained in the ventricles around the choroid plexus and ependymal lining.

Influence of microbial antigen formulation and delivery route on the immune response

Recent technological advances have resulted in the production of safe subunit and synthetic small peptide vaccines. Unfortunately, these vaccines are weakly or non-immunogenic in the absence of an immunological adjuvant (agents that can induce strong immunity to antigens). In addition, in order to prevent and/or control infection at the mucosal surface, stimulation of the mucosal immune system is essential. This may be achieved via the common mucosal immune system by exposure to antigen at a mucosal surface remote from the area of infection. Initial studies investigated the potential of multiple emulsions in effecting oral absorption and the subsequent immune responses to a lipopolysaccharide vaccine (LPS) after immunisation. Nasal delivery of LPS was carried out in parallel work using either aqueous solution or gel formulations. Tetanus toxoid vaccine in simple solution was delivered to guinea pigs as free antigen or entrapped in DSPC liposomes. In addition, adsorbed tetanus toxoid vaccine was delivered nasally free or in an aerosil gel formulation. This work was extended to investigate guinea pigs immunised by various mucosal routes with a herpes simplex virus subunit vaccine prepared from virus infected cells and delivered in gels, multiple emulsions and liposomes. Comparable serum antibody responses resulted but failed to produce enhanced protection against vaginal challenge when compared to subcutaneous immunisation with alhydrogel adjuvanted vaccine. Thus, immunisation of the mucosal surface by these methods may have been inadequate. These studies were extended in an attempt to protect against HSV genital challenge by construction of an attenuated Salmonella typhimurium HWSH aroA mutant expressing a cloned glycoprotein D-l gene fused to the Es-cherichia coli lac z promoter. Preliminary work on the colonisation of guinea pigs with S. typhimurium HWSH aroA mutants were carried out, with the aim of using the guinea pig HSV vaginal model to investigate protection.

Insulin gene therapy for the treatment of diabetes mellitus

Currently available treatments for insulin-dependent diabetes mellitus are often inadequate in terms of both efficacy and patient compliance. Gene therapy offers the possibility of a novel and improved method by which exogenous insulin can be delivered to a patient. This was approached in the present study by constructing a novel insulin-secreting cell line. For the purposes of this work immortalized cell lines were used. Fibroblasts and pituitary cells were transfected with the human preproisinulin gene to create stable lines of proinsulin- and insulin-secreting cells. The effect of known β-cell secretagogues on these cells were investigated, and found mostly to have no stimulatory effect, although IBMX, arginine and ZnSO4 each increased the rate of secretion. Cyclosporin (CyA) is currently the immunosuppresant of choice for transplant recipients; the effect of this treatment on endogenous β-cell function was assessed both in vivo and in vitro. Therapeutic doses of CyA were found to reduce plasma insulin concentrations and to impair glucose tolerance. The effect of immunoisolation on insulin release by HIT T15 cells was also investigated. The presence of an alginate membrane was found to severely impair insulin release. For the first implantation of the insulin-secreting cells, the animal model selected was the athymic nude mouse. This animal is immunoincompetent, and hence the use of an immunosuppressive regimen is circumvented. Graft function was assessed by measurement of plasma human C peptide concentrations, using a highly specific assay. Intraperitoneal implantation of genetically manipulated insulin-secreting pituitary cells into nude mice subsequently treated with a large dose of streptozotocin (STZ) resulted in a significantly delayed onset of hyperglycaemia when compared to control animals. Consumption of a ZnSO4 solution was shown to increase human C peptide release by the implant. Ensuing studies in nude mice examined the efficacy of different implantation sites, and included histochemical examination of the tumours. Aldehyde fuchsin staining and immunocytochemical processing demonstrated the presence of insulin containing cells within the excised tissue. Following initial investigations in nude mice, implantation studies were performed in CyA-immunosuppressed normal and STZ-diabetic mice. Graft function was found to be less efficacious, possibly due to the subcutaneous implantation site, or to the immunosuppresive regimen. Histochemical and transmission electron microscopic analysis of the tumour-like cell clusters found at autopsy revealed necrosis of cells at the core, but essentially normal cell morphology, with dense secretory granules in peripheral cells. The thesis provides evidence that gene therapy offers a feasibly new approach to insulin delivery.

Evaluation of biodegradable polymer microspheres and an anionic dendrimer for the potential delivery of antisense oligonucleotides

Antisense oligonucleotides (AODNs) can selectively inhibit individual gene expression by binding specifically to rnRNA. The over-expression of the epidermal growth factor receptor (EGFR) has been observed in human breast and glioblastoma tumours and therefore AODNs designed to target the EGFR would be a logical approach to treat such tumours. However, poor pharmacokinetic/pharmacodynamic and cellular uptake properties of AODNs have limited their potential to become successful therapeutic agents. Biodegradable polymeric poly (lactide-co-glycolide) (P(LA-GA)) and dendrimer delivery systems may allow us to overcome these problems. The use of combination therapy of AODNs and cytotoxic agents such as 5-fluorouracil (5-FU) in biodegradable polymeric formulations may further improve therapeutic efficacy. AODN and 5-FU were either co-entrapped in a single microsphere formulation or individually entrapped in two separate microsphere formulations (double emulsion method) and release profiles determined in vitro. The release rates (biphasic) of the two agents were significantly slower when co-entrapped as a single microsphere formulation compared to those obtained with the separate formulations. Sustained release over 35 days was observed in both types of formulation. Naked and microsphere-loaded AODN and 5-FU (in separate formulations) were tested on an A431 vulval carcinoma cell line. Combining naked or encapsulated drugs produced a greater reduction in viable cell number as compared with either agent alone. However, controls and Western blotting indicated that non-sequence specific cytotoxic effects were responsible for the differences in viable cell number. The uptake properties of an anionic dendrimer based on a pentaerythritol structure covalently linked to AODNs (targeting the EGFR) have been characterised. The cellular uptake of AODN linked to the dendrimer was up to 3.5-fold higher in A431 cells as compared to naked AODN. Mechanistic studies suggested that receptor-mediated and adsorptive (binding protein-mediated) endocytosis were the predominant uptake mechanisms for the dendrimer-AODN. RNase H cleavage assay suggested that the dendrimer-AODN was able to bind and cleave the target site. A reduction of 20%, 28% and 45% in EGFR expression was observed with 0.05μM, 0.1μM and 0.5μM dendrimer-AODN treatments respectively with a reduction in viable cell number. These results indicated that the dendrimer delivery system may reduce viable cell number by an antisense specific mechanism.