Gene therapy consists of methods which attempt to repair or replace defective genes responsible for disease, or to add genes to a therapeutic effect. To achieve this, two episomally maintained recombinant viral vectors have shown promising results: integration-deficient lentiviral vectors (IDLVs), and adeno-associated virus (AA V) vectors. The non-integrating nature of these vectors improves their safety profile but also limits transgene retention as nuclear episomes generally get lost during cell division. In the present study, the establishment of stable replicating episomes via transduction with AA V and IDL V gene therapy vectors was examined in CHO cells. Different DNA elements and cell culture conditions were evaluated, and in particular the effects of (i) DNA elements called S/MARs (scaffold/matrix attachment regions) which are involved in chromatin organisation, transcription and replication, and (ii) induction of transient cell cycle arrest in transfected and transduced cell populations. In the case of both AA V and IDL V vectors, the incorporation of S/MAR elements into vector transcription units had only marginal effects on the establishment of stable transgene- positive cell populations, either with or without induction oftransient cell cycle arrest. However, a striking general result was observed in cell populations transduced with IDL Vs and subjected to a transient cell cycle arrest soon after transduction. Under these conditions, following release from cell cycle arrest and in the absence of any selection pressure, substantial populations (10-25%) of proliferating and stably transduced cells emerged and were maintained over at least 100 population doublings. This establishment of stable transduction was seen only with IDLVs, was crucially . dependent on the induction of a period of transient cell cycle arrest, occurred independently of the presence of S/MAR elements, and resulted in transgene-positive cell populations which could be isolated and propagated as stable clonal cell lines. In these polyclonal and clonal IDL V -transduced cell lines, the existence of non-integrated vector genomes in the form of multi-copy nuclear episomes was confirmed by evidence from linear amplification -mediated PCR, deep sequencing, Southern blotting and FISH (fluorescent in situ hybridisation). 2 The cumulative evidence suggests that transduction of eHO cells with IDL Vs followed by a short period of induced cell cycle arrest leads to the establishment of stable IDL V- based nuclear episomes which are transcriptionally active and undergo replication and segregation during cell division without the need for antibiotic-based or other positive selection pressure. Preliminary investigations were also done to test the capacity of combined IDL V transduction and transient cell cycle arrest to establish stable episome Hel.a cells and murine haematopoietic stem cells. However, further experiments are required either to optimise the protocol in these cells or to find other clinically relevant cell types in which the protocol can be implemented. The transfer of this technology to a variety of clinically relevant human stem or progenitor cell populations could improve the safety profile of a range of gene therapy strategies currently under investigation. 3

Globoid cell leukodystrophy (GLD) is a rare lysosomal storage disorder (LSD) due to the deficiency of the lysosomal enzyme Galactocerebrosidase (GALC). The enzymatic deficiency results in intracellular storage of undegraded metabolites in the nervous system, leading to progressive dysmyelination. We are testing the feasibility and efficacy of a gene therapy strategy based on hematopoietic stem/progenitor cells (HSPC) and lentiviral vectors (LV) in the murine model of GLD. Differently from what observed with other lysosomal enzymes, GALC gene transfer and expression in HSPC causes apoptosis and functional impairment of the transduced cells due to an inbalance of the intracellular content in bioactive sphingolipids consequent to de novo enzyme expression. Differentiated cells of the myeloid and lymphoid lineages are not affected by GALC expression, suggesting a unique sensitivity of HSPC to enzyme toxicity.

A key factor in the success of gene therapy is the development of delivery systems that arc capable of efficient gene transfer in a variety of tissues without causing any pathogenic effects. This thesis is aimed at developing gene therapy strategies for the treatment of neurodegenerativc diseases, mainly Spinal Muscular Atrophy (SMA). Integration-deficient lentiviral vectors (lDLVs),. based on HIV -1 through the use of class I integrase mutation, fail to integrate as proviruses and are instead converted into episomal circles, which are metabolically stable. These vectors are of particular use as they support expression of transgenes in quiescent cells and exhibit a decreased risk of insertions I mutagenesis. I have embarked on an extensive study to compare the transduction efficiency of IDLVs and matched integrating lentivectors in spinal cord tissues in vitro and in vivo. My results demonstrate similar efficiency of eGFP expression from both vector forms in most cell types analysed, including motor neurons, inter-neurons and astroglia. After intraspinal injection of IDLY expressing eGFP into adult rat and mouse lumbar cord in vivo, transduction was mainly neuronal, with both motor and inter-neurons being efficiently targeted. Intraspinal injection of mouse embryos revealed that IDLY can completely transduce motor neurons. Additionally, IDLYs produced strong expression of mouse survival motor neuron gene (mSmn; SMA determining gene) in the injected cords. My data also revealed that IDLV-mediated express ion of a growth factor or tetanus toxin fragment C (TTC) rescued motor neuron cultures from death caused by removal of exogenous trophic support. Moreover, integration proficient lentiviral vectors encoding for mSmn restored the number of gems in type I SMA fibroblasts. Taken together, the results of this study strongly implicate that TDLVs could be efficient and safe tools for cord transduction in therapeutic strategies such as for treatment of SMA.

Gene therapy with human adenovirus type S (AdS) has been extensively explored for the treatment of diseases resistant to traditional therapies. Following intravenous administration, Ad is rapidly cleared from systemic blood circulation with a half life of 2 minutes, and more than 99 % of the injected dose is sequestered in the liver. The resulting innate and adaptive immune responses dramatically affect the kinetics and toxicity profile of the vector. These issues currently restrict the use of Ad-based vectors, particularly for clinical gene therapy protocols that involve systemic administration. We propose that such limitations can be improved by engineering artificial lipid envelopes around Ad. We previously designed a variety of artificial lipid bilayer envelopes around the viral capsid. Zwitterionic and cationic lipid formulations can efficiently envelop Ad, however this resulted in a significant reduction of gene expression in vitro due to the inadequate escape of the enveloped virus from the endosomal compartment. In this thesis, pH-sensitive lipid-envelopes to enhance the virus release from the endosome following endocytosis were explored. Also, different viral envelopment methodologies (sonication and extrusion) were compared in terms of percentage of virion envelopment and efficiency of gene expression. The artificially enveloped Ad were characterised physicochemically by dot blots, dynamic light scattering and atomic force microscopy. Biologically, the gene expression of enveloped Ad in different pH-sensitive enveloped Ad was studied in vitro and in vivo. The critical role of blood components during systemic administration of Ad was investigated by looking at the interaction of enveloped Ad in cationic, non-pH-sensitive (DOTAP:Chol) or anionic, pH-sensitive (DOPE:CHEMS) lipid bilayers with several different blood components. When Ad was enveloped by cationic bilayers, significantly high levels of viral uptake in HepG2 cells were achieved, independent of any blood coagulation factor, whereas, the levels of cellular uptake and gene expression were similar to naked Ad vectors when an anionic lipid envelope was used. In vitro experiments also showed that artificial envelopment of Ad completely altered the affinity towards both human and murine red blood cells. After intravenous administration into mice, real-time PCR and transgene expression studies indicated that cationic lipid envelopes significantly reduced hepatocyte transduction compared to anionic envelopes. AL T / AST serum levels and liver histology showed that envelopment also improved hepatotoxicity profiles compared to naked Ad. Furthermore, envelopment in DOTAP:Chollipid bilayers significantly increased lung accumulation compared to DOPE:CHEMS enveloped or naked Ad. These results suggest that artificial envelopes for Ad significantly alter the interactions with blood components and divert viral particles from their natural liver tropism resulting in reduced hepatotoxicity. Finally, we sought to explore further opportunities that the artificially enveloped virus constructs could offer, by designing a previously unreported gene therapy vector by simultaneous envelopment of Ad and siRNA within lipid bilayers. Such a dual-activity vector can possibly offer efficacious therapy for different genetic disorders where both turning on and switching off genes would be needed. Dynamic light scattering, transmission electron microscopy and atomic force microscopy were used to characterize these vectors. Agarose gel electrophoresis, ribo-green assays and dot blots showed that siRNA and Ad can be enveloped together within lipid bilayers at high envelopment efficiency. Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors. In summary, the studies in this thesis contribute to greater understanding of the mechanisms, obstacles and opportunities offered by artificial lipid envelopment of Ad and how these affect the biological activity of these promissing gene therapy vectors in vitro and in vivo.

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Gene therapy is a promising tool in the treatment of genetic disorders. Viral vectors have a natural ability to genetically alter cells, however, concern over their immunogenic and mutagenic effects has led to the development of safer non-viral alternatives, such as ultrasound and microbubble mediated gene transfection (UMGT). UMGT causes cellular bioeffects leading to uptake of genetic material; however transfection efficiencies in vivo are poor. It was the aim of this project to investigate key factors that affect UMGT in vivo. A Model Protocol was created with techniques capable of characterising the acoustic response and stability of microbubbles, as well as their population statistics and ability to transfect the heart of six to eight week old, CD1, female mice with a luciferase plasmid. This was used to investigate the effect of needle gauge and microbubble formulation on transfection. It was found that the needle gauge used to systemically deliver SonoVue microbubbles affected microbubble number and transfection. 29G needles caused a decrease in microbubble concentration and transfection, whilst 25G needles caused no microbubble destruction and resulted in significantly greater transfection. Albumin microbubbles are more effective than lipid microbubbles at transfection, but are not optimised. It was found that altering the dextrose and albumin content of an albumin microbubble affected their acoustic and transfection properties. A link between the attenuation behaviour of a microbubble and its transfection efficiency was found, suggesting that optimising acoustic behaviour could improve transfection. DNA-loaded, cationic lipid microbubbles have shown greater transfection ability than neutral microbubbles due to co-localisation of DNA at the site of microbubble activation. Albumin microbubbles have not been fully investigated for this effect. A novel, potentially safe, microbubble formulation was created and fluorescence techniques used to demonstrate its loading capability. This project has identified needle gauge and microbubble acoustic behaviour as key factors affecting the effectiveness of UMGT.