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University of Groningen

Reconstituted viral envelopes as delivery vehicles for nucleic acidsde Jonge, Jørgen Martin

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Publication date:2007

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C H A P T E R 1

INTRODUCTION

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SCOPE OF THIS STUDY

The success of gene therapy and the therapeutic use of RNA interference (RNAi) relies on the efficient, safe and specific cellular delivery of nucleic acids. Despite extensive efforts, several obstacles impede successful application of viral and non-viral delivery strategies. The aim of the study described in this thesis was to develop and characterize a delivery device for nucleic acids, based on reconstituted influenza virus envelopes (virosomes) that combines the delivery efficiency of the viral vectors and the safety of the non-viral cationic liposomal systems. Next to efficient and safe delivery properties of these vectors, the pharmaceutical quality is of crucial importance for successful and widespread application. Therefore a procedure was developed for formulation of virosomes as a dry powder in order to enhance their shelf-life.

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THERAPY AT THE GENETIC LEVEL

Gene therapy

Over the past decades, the discovery and development of recombinant DNA technology and the sequencing of the human genome have opened the possibility to battle numerous diseases at the genetic level. These achievements have advanced the development of the scientific field of gene therapy, which has been acknowledged for its high potential and has gained interest ever since. However, due to the scarcity of clinically relevant results, the enthusiasm has been tempered the last few years.

Concept. The basic principle of gene therapy is relatively simple: introduce a specific gene into a target cell to replace a defective gene or to produce a protein either to cure inherited or acquired diseases or to induce an immune response for vaccination purposes [1]. Thus, gene therapy intends to treat diseases at the root, instead of curing the symptoms as in most other therapeutic approaches and in case of genetic immunization allows for antigen synthesis by the host itself. Cancer. Currently, therapeutic strategies are mostly aimed at genetic disorders causing cancer [2]. Since this disease generally originates from multiple genetic defects, different therapeutic approaches are possible [3]. To stop the uncontrolled proliferation of cancer cells, tumor suppressor genes that induce apoptosis or cell cycle arrest, such as p53, have been targeted to tumor cells. Alternatively, in suicide therapy genes are delivered that encode enzymes which convert an injected substrate (pro-drug) into a toxic substance inducing cell-death. Another approach is to cut off the nutrient supply to tumor cells by inhibition of tumor angiogenesis (vascular growth). To this end, endothelial cells of the tumor vasculature are transduced with angiogenic inhibitors such as angiostatin, endostatin and interleukin-12. Finally, strategies have been applied that involve the recruitment of the immune system; for example, by introducing genes into the cancer cells that encode stimulatory molecules such as cytokines, a cytolitic immune response against these cells may be induced. Moreover, cancer cells express tumor-associated antigens that can be recognized by the immune system. Genetic vaccination by delivery of genes encoding the antigen to antigen-presenting cells elicits an immune response against the tumor-associated antigen. Inherited diseases. The strategy to cure inherited diseases is by corrective gene therapy. In contrast to diseases involving multiple genes, monogenic disorders, such

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as cystic fibrosis (CF, [4]), hemophilia [5] or severe combined immunodeficiency (SCID) [6,7] demand simple one-gene-correction strategies. In CF, pulmonary complications are the major cause of death. Therefore, the lung epithelial cells specifically have to be targeted for the correction of the cystic fibrosis transmembrane conductance regulator (CFTR), malfunction of which causing the sticky mucus typical for CF. In the case of the expression of clotting factors involved in hemophilia, cell-specific targeting of a gene construct is of less importance, since any cell-type that secretes these factors into the plasma is sufficient. The most successful gene therapy clinical trial thus far was based on the introduction of a gene coding for γ-c cytokine receptor in patients that suffered from inherited SCID-XI disease [6,7]. Patients could be cured from this genetic disease and developed functional immune systems. Unfortunately, some patients developed serious adverse effects in the form of T-cell leukemia [8].

Neurodegenerative disorders and other acquired diseases. More complex are the neurodegenerative disorders, such as Parkinson’s disease. This disease is characterized by the loss of dopaminergic neurons and depletion of the neurotransmitter dopamine. Experimental gene therapeutic modalities are either based on the delivery of genes encoding enzymes catalyzing the synthesis of dopamine or on decelerating the disintegration of the neurons by transduction of various growth factors [9]. Other acquired diseases that involve multiple genes for which gene therapy has been suggested are cardiovascular [10] and autoimmune and inflammatory diseases [11,12].

Infectious diseases. The introduction of genes encoding antigens into e.g. antigen presenting cells for the purpose of vaccination is a relatively new approach in the battle against infectious diseases [13]. The advantage of DNA vaccination is that activation of the humoral as well as the cellular arm of the immune system is feasible. There is increasing evidence that infectious diseases such as HIV-1, malaria and tuberculosis require activation of cellular immunity for an effective defense [13]. An additional advantage of genetic vaccination is that genetic vaccines in the form of plasmid DNA (pDNA) can be produced much faster than conventional protein-based vaccines. For example, in the light of the current pandemic threat posed by influenza virus this is considered as an advantage [14].

Obstacles. Notwithstanding the simplicity of the idea of gene therapy, practical experience has shown that there are some important hurdles to be overcome. Certain diseases require stable expression of the gene in question. In that case, episomal genes are not favourable, since they are lost upon cell-division. Integration into the host

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genome may pose as a solution; however, this technique bears the risk of insertional mutagenesis [15]. Moreover, certain genes are tightly regulated in terms of the amount of protein expressed or the time of expression. Considering this, homologous recombination would be ideal, since the diseased gene would be replaced by a functional gene, while maintaining endogenous chromosomal control elements. Yet, this kind of recombination is very inefficient [16]. Fortunately, not all gene therapy approaches require these strict demands and in quite a number of cases, low levels and/or temporary expression of a protein may well be sufficient.

RNA interference

Another approach to treat diseases at the genetic level is by exploiting the phenomenon of RNAi. The discovery of RNAi has provided a complete new understanding on how cells regulate protein synthesis and is without a doubt one of the most important recent breakthroughs in the area of cell biology. RNAi involves sequence-specific gene silencing at the RNA level either by degradation of mRNA or by translational arrest. RNAi provides a powerful tool to unravel gene function. Moreover, the potential therapeutic applications of RNAi are undoubtedly numerous [17-21]. RNA-mediated gene silencing was first observed when a gene coding for deep purple colored flowers was introduced in plants to intensify the existing purple color [22-24]. Surprisingly, this did not result in more intense purple flowers; instead, the original purple color disappeared. In another study, the nematode worm Caenorhabditis Elegans was injected with sense or anti-sense RNA’s to specifically down-regulate gene expression [25]. The results were marginal. However, when double-stranded RNA (dsRNA) was injected, efficient and specific silencing of the complementary gene was observed. This specific gene silencing was classified as RNAi and provided the missing link for the previously observed silencing in plants; namely, that dsRNA intermediates involved in expression of the purple gene induced the gene silencing.

RNAi mechanism. A central role in the RNAi pathway is attributed to a dsRNA molecule, called small interfering RNA (siRNA), that directs highly sequence-specific gene silencing at the RNA level [18,19,26]. Activation of RNAi starts with the cleavage of exogenous or endogenous dsRNA by a protein complex containing the enzyme Dicer (Fig. 1). This enzyme catalyzes the cleavage of dsRNAs into siRNAs of 21-23 nucleotides in length with 2-3 single-stranded nucleotides overhangs. Subsequently, the RNA duplex is unwound in an ATP-dependent

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manner starting at that 5’ end of the strand where base-pairing is least tight. This strand, also called the leading strand, is incorporated in a protein complex called the RNA-induced silencing complex (RISC). If the leading strand is the anti-sense strand, it guides the RISC to the complementary mRNA after which the mRNA is degraded by the cleavage activity of RISC. Subsequently, the RISC dissociates and hooks on to the next target to be silenced.

Figure 1. The RNAi pathway. synthetic siRNA or siRNA produced from dsRNA recruit the RNA induced silencing complex (RISC) to degrade mRNA complementary to the anti-sense siRNA strand (details see text). Adapted from [19].

RNAi application in mammals. Several attempts to provoke RNAi in mammalian cells by the introduction of long dsRNA were hindered by the induction of interferon responses, which are known to down-regulate total protein synthesis resulting in apoptosis [27]. This response could be avoided when instead of long

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dsRNA, siRNA molecules were used which directly bind to RISC to induce RNAi (Fig. 1 and [28]). This breakthrough also allowed the application of RNAi in mammalian cells. Later it was shown that apart from the use of synthetic siRNA, siRNA can also be encoded on a plasmid or viral vector as short hairpin RNA (shRNA). The encoded shRNA functions as a precursor for the intracellular generation of siRNAs and since it is encoded on a plasmid long-term expression can be achieved [29].

Cancer. RNAi may be exploited for therapeutic interventions in diseases which involve aberrant gene expression [30-32]. For example in cancer, proteins that regulate apoptosis, such as Bcl-2, appear to be involved in tumor growth when overexpressed. siRNAs directed against these apoptotic regulatory proteins should restore the apoptosis process in the cell [33,34]. The tumor suppressor gene p53 is mutated in many cancers. Down-regulation of the mutant form by RNAi restores the expression of the wild-type gene, which is known to be suppressed by its mutant forms [35]. These are just two examples of the numerous targets one can imagine in cancer therapy [36]. Infectious and other diseases. In infectious diseases, RNAi has been aimed at genes involved in viral replication and assembly thereby blocking the synthesis of practically all viral genes. For example, siRNAs targeted against the influenza nucleoprotein (NP) or components of the polymerase machinery reduced viral titers in the lung and provided protection against lethal influenza challenges in mice [37,38]. For HIV, targeting host-genes essential for the viral life-cycle such as the CCR5 and CXCR4 co-receptor inhibits viral infection [35,39]. In these approaches it is essential that down-regulation of the host genes does not induce any pathogenicity. In general, challenges in therapeutic modalities against viral infections include the formation of escape variants due to rapidly mutating viral genomes, which is most pronounced in RNA viruses. Moreover, RNAi suppressor molecules secreted by some viruses might impede RNAi application [40]. Other diseases that have been suggested as targets for RNAi treatment include septic shock, inflammatory diseases and neurological diseases [30-32].

Obstacles. For therapeutic purposes the knockdown of a diseased gene should be highly specific and should not cause any side effects. For this reason siRNAs should be carefully selected not to induce so called off-target effects [41,42]. A-specific down-regulation is also related to the concentration of siRNA to which cells are exposed, indicating the presence of a therapeutic window [43]. Moreover, although initially interferon responses could be avoided by the application of siRNAs,

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there are recent indications that also siRNAs induce immunomodulatrory responses [31,32,44]. These responses appear to be dependent on the sequence of the siRNA and on the route of delivery or the carrier system involved.

DELIVERY SYSTEMS

The success of gene therapy and RNAi depends on the ability to introduce genes, siRNAs or shRNA into cells, resulting in the expression of therapeutic proteins or in the activation of the RNAi pathway. Efficient delivery of nucleic acids has been a major obstacle in the development of gene therapy or RNAi protocols. During the last two decades, the development of vehicles that mediate efficient specific and safe cellular delivery of genes has been intensively researched. These carriers may either be of biological or chemical/physical origin and are generally classified as viral or non-viral systems, respectively. The knowledge obtained on these delivery devices has paved the way for the development of delivery systems for siRNA.

Viral delivery vehicles

Viruses have evolved over millions of years, optimizing their mechanisms to penetrate target cells membranes for delivery of their genetic material and exploitation of cellular mechanisms for their replication. Viral gene delivery vehicles harness these ell-entry properties, which makes them by far the most efficient delivery devices known to date [45-49]. Delivery of nucleic acids into the target cells is achieved by replacing one or more viral genes by nucleic acids of interest. Production of the recombinant viral vectors usually involves a packaging cell line which provides the genetic information for the deleted structural viral proteins. The recombinant viral genome construct containing the nucleic acid of interest and the remaining viral sequences including a packaging signal is introduced into this cell line. Upon synthesis of the structural viral proteins, recombinant virus particles assemble in which the construct containing the gene of interest is incorporated by virtue of the packaging signal. Viral vectors. The viral vectors most frequently used for gene delivery are based on enveloped viruses, such as oncogenic retroviruses, lentiviruses, herpes viruses and poxviruses or on non-enveloped viruses, such as adenoviruses or adeno-associated viruses. The different viruses have distinct characteristics that make them suitable for certain applications [45,50,51]. The oncogenic retroviruses and lentiviruses have the

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ability to integrate their genome into the host chromosome, allowing stable transcription of the transgene in contrast to other vectors that only allow transient expression. The downside of the oncogenic retroviruses is that they only transduce dividing cells. Adenoviruses and adeno-associated viruses are the most efficient transducing vectors; however, adeno-associated viruses have a very limited packaging capacity. Poxviruses and herpes viruses contain a large genome allowing for a large packaging capacity.

Drawbacks. One of the major drawbacks of retroviral vectors is related to insertional mutagenesis. This phenomenon occurred during a clinical trial with the objective to cure patients with the SCID-X1 disease. In this trial, CD34+ bone marrow cells isolated from the patients were transduced ex vivo with an oncogenic retrovirus vector encoding the γ-c cytokine receptor involved in the development of T-cells. Although after infusion of the transduced cells, the patients developed functional immune systems and recovered from their disease [6,7], some patients developed leukemia-like disease. This was due to the insertion of the oncogenic retroviral vector genome in a site activating expression of an oncogene [8]. Another study revealed that HIV also favored integration into active genes above random integration [52], which increases the risk of insertional mutagenesis.

Another important drawback is related to the immunogenicity of viral vectors, for example, in a clinical trial, a patient who had received a recombinant adenovirus for corrective gene therapy developed a systemic inflammatory response to the vector, which finally led to the death of the patient [53,54]. The immunogenicity of these vectors can be reduced by deleting all the viral genes and thus avoiding the synthesis of viral proteins in the transduced cell that induce a cellular immune response. This has been achieved in e.g. the “gutless” adeno-viral vector. However, the immune response to the vector itself is not controlled by this approach.

The two incidents described above are characteristic for the problems with viral vectors and emphasize that the safety of these vectors is not yet guaranteed and issues related to insertional mutagenesis (retroviruses, [8,52,55]), inflammation and immune response (adenoviruses, [56-59]) still pose a major challenge.

Non-viral delivery systems

Non-viral gene delivery comprises a variety of approaches that can be subdivided into chemical and physical systems [60]. Among the physical methods, injection or hydrodynamic injection (large volume and high pressure) of naked DNA are the simplest methods used. More sophisticated are the approaches involving

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electroporation, gene gun delivery, jet injection and ultrasound treatment. The chemical methods comprise cationic liposomes, cationic polymers, cationic peptides and nano-particles. Since cationic liposomes are the most extensively studied non-viral delivery devices and the most relevant for this thesis, these systems are discussed in some more detail below [61-63]. Cationic liposomes. In 1987 Felgner and co-workers discovered that liposomes containing cationic lipids could bind DNA and that the resulting complexes were taken up by cultured cells, which subsequently expressed high levels of the encoded gene [64]. These cationic liposomes were composed of the cationic lipid dioleyltrimethylammonium chloride (DOTMA) and the neutral lipid dioleoylphosphatidylethanolamine (DOPE) in a 1:1 ratio and became widely known as an efficient transfection tool for in vitro use. Ever since, numerous cationic lipids have been synthesized, varying in their polar head group, linker and hydrophobic anchor to improve transfection efficiency and reduce toxicity [65,66]. Preparation. Cationic liposomes are prepared by evaporation of an organic solvent containing cationic and neutral lipids. Hydration of the lipid film and subsequent vortexing results in the formation of multilamellar vesicles, which can be transformed into small unilamellar vesicles by sonication or extrusion. Upon addition of DNA, the cationic liposomes condense the DNA through electrostatic interactions of the positively charged lipids with the negatively charged phosphate groups of the nucleic acids forming so-called lipoplexes. Structurally, these complexes are very different from the original cationic liposomes. To date, two lipoplex structures have been observed namely, the so-called multilamellar structure Lα

C and the columnar inverted-hexagonal HII

C liquid-crystalline structure. In the first structure the DNA is sandwiched in between lipid bilayers while the second structure is characterized by tubular inverted micelles filled with DNA [67]. Which of the lipoplex structures is formed under a certain condition is primarily dictated by the nature and amount of the neutral lipid. Mechanism. The exact mechanism by which lipoplexes transfect cells is still unclear. However, structural, microscopical and transfection studies are slowly beginning to unravel this mystery. Based on such studies, Ewert et al. suggested two distinct models for transfection dependent on the structural features of the lipoplex [68]. In general lipoplexes bind to cells due to their overall positive charge and the negative charge of the plasma membrane. The Lα

C lipoplexes are taken up by endocytosis. When their average membrane charge density is above a certain threshold they destabilize the endosomal membrane, suggesting that membrane

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destabilization is driven by electrostatic forces. In contrast, the HIIC structures act

independently of their average membrane charge density and destabilize either the plasma membrane or the endosomal membrane. Their mechanism of membrane penetration likely relates to their energetically unfavourable membrane structure.

Drawbacks. In contrast to viral delivery vehicles cationic liposome systems are regarded as relatively safe, do not induce an immune response, and are well characterized and easy to produce. However, successful application of these systems has been hampered by low transfection efficiencies in vivo, mainly caused by adsorption of serum proteins to lipoplexes resulting in clearance from the blood circulation [69]. Another issue is that lipoplexes can induce a broad range of toxic effects, which are dependent on their charge ratio, nature of the cationic lipid and cell-type [62]. Other problems to be overcome are the short transgene expression [70], poor reproducibility [71] and low biological stability [72].

VIROSOMES AS DELIVERY VEHICLES

Several drawbacks of the viral and non-viral nucleic acid delivery approaches discussed in the preceding paragraphs could be overcome by the application of a hybrid form of these delivery devices. Reconstituted viral membranes or so-called virosomes are such hybrid vehicles that combine the delivery efficiency of viral vectors and the safety of cationic liposomal systems. Since they are prepared from enveloped viruses and exploit their fusogenic mechanisms, some general characteristics of these enveloped viruses are first discussed below.

Enveloped viruses as delivery experts

Enveloped viruses have developed sophisticated mechanisms to cross the membrane barrier of target cells in order to gain access to the cellular machinery, involved in viral replication. Enveloped viruses contain a protein capsid, which contains the genetic material. In turn, this capsid is enclosed in a lipid bilayer envelope. Embedded in this bilayer are so-called spike glycoproteins that protrude from the virus particle. Enveloped viruses attach to cells via spike proteins that recognize ligands on the surface of the cellular membrane and thereby distinguish between different cells. To deliver their genome to the target cell cytoplasm, the viruses fuse their membrane with the cell membrane. Enveloped viruses can be classified into those viruses that fuse at the plasma membrane, such as HIV and Sendai virus and those that fuse with

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the endosomal membrane after endosomal uptake, such as influenza virus. Fusion is induced by different triggers, dependent on the route of entry. Whereas receptor binding is the signal for fusion at the plasma membrane, a decrease in pH in the endosomal compartment is the trigger for fusion with the endosomal membrane. Fusion is mediated by the viral fusion glycoproteins. The triggers induce a conformational change in these fusion proteins that subsequently mediate fusion of the virus membrane with the target membrane [73-75]. To date, two different types of fusion proteins have been recognized and classified as either type I or type II fusion proteins. Examples of type I fusion proteins are the influenza virus hemagglutinin, the HIV gp120/41 glycoprotein and the Sendai virus F-protein. Type II fusion proteins are exemplified by dengue and tick-borne encephalitis E-protein and Semliki Forest virus E1-protein. The major difference between these two types of fusion proteins lies in the conformational changes they undergo to constitute the pre-fusion conformation [73-75]. Recently, it has been discovered that the glycoprotein B of Herpes simplex virus (HSV) and G-protein of vesicular stomatitis virus (VSV) share structural features of both the type I and II fusion proteins and may constitute a new class of fusion proteins [76].

The virosomal concept

Virosomes can be considered as enveloped viruses from which the nucleocapsids and thus the genetic material have been removed. Since the virosomal lumen is empty, there is room for the encapsulation of compounds, such as nucleic acids. When properly reconstituted, the membrane glycoproteins or spike proteins embedded in the virosomal membrane retain their original receptor-binding features and fusogenic properties. By virtue of these characteristics, virosomes are able to bind to target cell membranes and to actively transport encapsulated compounds into the target cell cytosol.

Viruses used for virosome preparation

Reconstituted viral membranes were first prepared from Sendai virus [77] and the term virosomes was later introduced by Almeida et al. in 1975 who reconstituted influenza virus membranes [78]. Thereafter, virosomes have been prepared from various enveloped viruses including, influenza virus [79,80], Sendai virus [81-84], Semliki Forest virus [85,86], VSV [87,88], Sindbis virus [89], Epstein-Barr virus [90], Human immunodeficiency virus (HIV [91]), Friend murine leukaemia virus [92], HSV [93] and New Castle disease virus [94,95]. Since the virosomes described in this

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thesis are prepared from influenza virus, this virus and in particular the mechanistic aspects of its fusion process are describes in the next section.

Influenza virus

Viral proteins. Influenza virus is a membrane enveloped virus with a diameter of approximately 100 nm (± 20) that contains 8 single strand RNA segments of negative orientation [96,97]. These segments encode 10 proteins of which the surface glycoproteins, Haemagglutinin (HA) and Neuraminidase (NA) and the ion-channel protein M2 are embedded in the virus membrane (Fig. 2). HA is the major spike protein (± 500 copies per virion) and is involved in receptor binding and membrane fusion. The protein is synthesized as a single polypeptide HA0. The mature, fusion-active, form is obtained after cleavage of HA0 by a cellular protease into the subunits HA1 and HA2, which remain linked by two disulfide bonds [97]. NA, the other surface glycoprotein, is involved in transport of the virus particles through the mucin layer lining the respiratory tract and mediates the release of newly assembled virus particles from the cellular membrane during the budding process [97]. The nucleocapsid protein (NP) and the polymerase proteins (PA, PB1, PB2) are associated with the RNA segments, which together constitute the ribonucleoprotein (RNP) complexes. The matrix protein (M1) links this complex to the virus

membrane [97]. Finally, there are two non-structural proteins (NS1 and NS2) of which NS1 has multiple functions in the viral life cycle and is a sequester of type I interferons. NS2 is involved in the export of the RNP out of the nucleus but also associates with M1 [97].

Figure 2. A schematic drawing of the influenza virus. See text for details. Adapted from: (http://www.medizin.de /gesundheit/data_images/low/174-virus.jpg).

Cell entry. Influenza virus infects a cell by a sophisticated mechanism adapted to the intrinsic pathway of a cell to take up and degrade extra-cellular

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macromolecules [98,99]. The virus enters this pathway by anchoring the HA1 subunit to sialic acid receptors, present on many different cell types. The virus is then internalized by receptor-mediated endocytosis and directed to the endosomal compartment. The endosomes are acidified by proton pumps embedded in the endosomal membrane, which is a means to release internalized compounds from their receptors [100]. The influenza virus uses this decrease in pH to trigger a conformational change in HA, which induces fusion of the viral and the endosomal membrane. After fusion, the nucleocapsid is released into the cytoplasm from where the RNP complexes subsequently migrate to the nucleus to initiate replication of the virus [73,74].

Mechanisms of HA-induced membrane fusion. The HA spike protein is a trimer constituted by HA1/HA2 heterodimers. The spike protrudes approximately 135Å from the viral membrane and is characterized by a stem region at the base of the protein and a globular head at the distal end. The stem region is primarily α-helical and the core of the stem is composed of a trimeric coiled-coil [101]. From structural data, Carr and Kim [102] proposed that at neutral pH the conformation resides in a metastable state, which can undergo structural changes without input of external energy. They referred to this structure as a spring-loaded conformation. Huang and colleagues have proposed a model for the initial steps of the conformational change in which the HA1 globular heads become protonated upon acidification of the endosome [103]. Due to the electrostatic repulsion, the globular heads would dissociate outward from the stem region allowing the penetration of water into the core region of the HA trimer. Exposure of the HA2 subunit to water (Fig 3, state A), would initiate refolding of the central coiled-coil into an extended conformation (state B). This so-called pre-fusion conformation exposes a hydrophobic fusion peptide formerly buried within the stem, which is subsequently inserted into the target membrane (state C, [73,74,104]). The fusion protein now links the viral membrane with the target membrane via a rod-like structure with the viral membrane anchor attached to the one end and the fusion peptide to the other. The conformational change further progresses to a hairpin structure, which is considered as the energetically most stable state (state D and E). In this end-stage conformation, the membrane anchor and the fusion peptide are juxtaposed pulling the two membranes in such close proximity that destabilization of the lipid bilayer is induced, which is the prelude of the formation of a fusion pore (state F, [73,74,104]).

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Figure 3. The various stages in the process of membrane fusion mediated by the conformational changes in HA. For clarity the HA1 subunit has been omitted and only the HA2 fusion protein is shown. For details see text. Adapted from www.people.virginia.edu/~jw7g/models&figures.html.

The influenza virosomal concept

Influenza virosomes exploit the unique delivery properties of influenza virus to transport, instead of the viral genetic material, a therapeutic compound to the interior of a cell. Since influenza virosomes bear the HA spike protein in their membrane, they also bind to sialic acid residues present on the cellular surface of many cells (Fig. 4). Subsequently, like native virus, the virosomes are taken up by receptor-mediated endocytosis and are transported to endosomes. Acidification of the endosomes induces the conformational change of HA discussed above and as a consequence the contents of the virosomal lumen are deposited in the cytosol. Any compound of interest that is encapsulated within the virosomal lumen is thus transported to cytoplasm of the target cell (Fig. 4).

The delivery capacity of influenza virosomes was demonstrated first by virosomes containing the polypeptide toxins gelonin or subunit A of diphtheria toxin (DTA), both known to inhibit cellular protein synthesis [105,106]. Almost complete inhibition of protein synthesis of cultured cells was observed upon exposure to virosomes with the encapsulated toxin. Control experiments in which the virosomes were fusion-inactivated by pre-exposure to low pH in the absence of target membranes [107], demonstrated that these virosomes lost their capacity to transport the encapsulated compound. Thus, virosome-mediated delivery of the toxins was

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Figure 4. Virosomes as delivery vehicles for the transport of encapsulated compounds into cells. Depicted are virosomes with encapsulated macromolecules in the virosomal lumen. See text for further explanation.

dependent on the fusogenic properties of HA. Subsequently, it was demonstrated that virosomes mediated delivery of the whole protein antigen ovalbumin (OVA) encapsulated in the virosomal lumen into the major histocompatibility complex (MHC) class I and II presentation pathway of cultured dendritic cells [108].

In vivo, virosomes are able to induce a cellular immune response. Immunization of mice with virosomes containing either a peptide derived from the influenza virus nucleoprotein, OVA or the E7 protein of the human papillomavirus induced strong specific cytotoxic T-lymphocyte (CTL) responses against the respective antigens [109-111]. This confirms that the antigens were first delivered to the cytoplasm of antigen presenting cells after which they could enter the MHC class I pathway to activate CTL’s.

Apart from the induction of CTLs, virosomes can also be used for the induction of a humoral immune response. Since influenza virosomes present the major antigen HA of the influenza virus, they can induce an immune response against the virus strain they are derived from [112,113]. Influenza virosomes produced according to the immunopotentiating reconstituted influenza virosomes (IRIV) protocol (discussed below) are currently marketed as virosomal influenza vaccines by Berna Biotech (currently Crucell) and Solvay Pharmaceuticals under the trade names

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Inflexal V® and Invivac®. These vaccines were tested in several clinical studies and were found to be safe and induce high and long lasting antibody titers [114-116]. According to the same rationale, inactivated Hepatitis A virus (HAV) coupled to IRIVs induced effective and long-lasting anti-body titers against HAV [117,117]. Since, 1994 this vaccine has been marketed by Berna Biotech under the trade name Epaxal®.

PRODUCTION OF VIROSOMES

Several approaches have been applied for the production of virosomes. One of the most frequently used methods for the preparation of virosomes is by membrane solubilization and reconstitution. Since this method is also used for the production of influenza virosomes in this thesis, it is discussed in detail below.

General concept of reconstitution

Detergents and membrane solubilization. The first step in the reconstitution procedure of viral membranes is the solubilization of the virus membrane. This is a crucial step for the functional reconstitution of membrane proteins, since it should result in solubilization of the virus membrane only and should preserve the structural integrity and thereby the biological activity of the spike proteins.

Solubilization is a process in which cone-shaped amphiphiles disrupt bilayer structures constituted of cylindrical amphiphiles [118,119]. Detergents represent a class of amphiphiles that, due to their cone-shaped structure, aggregate into micelles above a certain concentration (Fig. 5A). This concentration is defined as the critical micelle concentration (c.m.c.). Phospholipids form another class of amphiphiles. Since they are, with some exceptions, cylindrical they align to form bilayer structures that generally close into vesicles (Fig. 5B). Membrane proteins are incorporated into this bilayer by virtue of their transmembrane domain, a hydrophobic peptide sequence spanning the lipid bilayer. The insertion of a detergent into a lipid bilayer acts as a wedge and breaks up the planar structure of the membranes (Fig. 5C). By increasing the detergent concentration so-called mixed-micelles of lipids and detergent molecules with or without membrane proteins are formed, which co-exist with detergent-saturated membrane structures. However, at excess detergent concentrations only the mixed-micellar phase with or without membrane proteins is

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present. In some cases the detergent removes all the intrinsic lipids, which may result in the formation of detergent micelles containing only a membrane protein (Fig. 5D).

Figure 5. Amphiphiles and membrane solubilization. Aggregation structures of cone-shaped amphiphiles (micelle, A) and cylindrical amphiphiles (bilayer, B). Wedge-like disruption of membrane protein-containing bilayers (C) resulting in mixed micelles with or without membrane proteins at excess concentration of detergent (D).

Denaturation and aggregation are two physical processes which may cause inactivation of membrane proteins during solubilization (denaturation) and reconstitution (aggregation). The extent of these processes depends on the membrane protein in question and the applied detergent. Unfortunately, a golden rule to determine the ideal detergent for the reconstitution of membrane proteins does not exist. A particular detergent can be very successful in the solubilization of certain membrane proteins, while it fails for others. Octylglucoside (OG), for example, has been used successfully for the functional reconstitution of the G-protein of VSV [120], while it failed to preserve the fusogenic properties of HA of influenza virus [79].

The concentration of the applied detergent is another crucial factor, since practically all detergents induce some extent of denaturation at excess concentrations. For example, in two studies OG failed to preserve the fusion activity of the VSV-G protein during solubilization and reconstitution [87,121], while functional reconstitution was achieved when a strictly defined concentration of solubilizing detergent was used [120]. Another important criterion for the choice of detergent can be its c.m.c. Removal of the detergent by dialysis, for example, requires a high c.m.c. Until now, the most frequently used detergents in the reconstitution of virus membranes have been non-ionic detergents, such as octaethyleneglycol mono(n-dodecyl)ether (C12E8), Triton X-100 and OG [79,83,86,87,120,122].

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Short-chain lecithins are phospholipids with a chain length of 6-8 carbon atoms that have gained much interest over the past few years for use as solubilizing agents in the reconstitution of membrane proteins [123]. Due to their short chain length, they tend to aggregate into micelles and exhibit detergent-like properties. Several studies have shown that the short-chain lecithin diheptanoylphosphatidylcholine (DHPC) is superior to various commonly used detergents in preserving the biological activity of membrane proteins during solubilization [124,125]. Moreover, these studies revealed that protein activity was preserved over a wide range of DHPC concentrations, whereas this range is usually narrow for commonly used detergents. Based on their observations, Kessi and colleagues [124] postulated that short-chain lipids only interact with the membrane lipids and not with the membrane proteins. Accordingly, it was considered unlikely that short-chain lecithins replace the intrinsic lipids at the trans-membrane domain of the membrane proteins. Due to the conservation of the intrinsic lipids, membrane proteins would be maintained in their original surrounding, thus preventing aggregation and denaturation and preserving the biological activity.

Membrane reconstitution. Membrane reconstitution is a process in which the components of dissolved membranes are reassembled into vesicular structures. Reconstitution is achieved by removal of the detergent from the solution containing the dissolved membrane components. The most commonly used detergent removal methods are dialysis or adsorption to a hydrophobic resin. However, the application of dilution or gel exclusion chromatography has also been reported.

There is still debate about the exact mechanism involved in the reconstitution process, which appears to be rather complicated and dependent on many parameters. However, there is consensus about a model that describes the reconstitution process by the inverse model of the membrane solubilization procedure [126-129]. For reasons of clarity, this model is first discussed for vesicle formation of simple phospholipid/detergent systems (Fig. 6). Reconstitution starts with a solution containing mixed micelles (phospholipid/detergent complexes). According to the model [126-129], removal of the detergent induces fusion of the micelles resulting in the formation of planar bilayers or so-called disk-like structures. The hydrophobic rim of these disk-like structures is shielded from the water phase by the detergent. Upon further removal of the detergent, the disks are proposed to fuse to reduce the surface of the hydrophobic edges thus compensating for the decrease in detergent concentration. When the detergent becomes limiting, hydrophobic repulsion overcomes the energy necessary to induce curvature in the disks. Subsequently, the surface of the disk edges is decreased by the bending of the disk, which finally would

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Figure 6. Schematic representation of the current model on vesicle formation from mixed micelles upon detergent removal. Adapted from Leng et al., [126]

result in the closure of the vesicle. At this stage vesicles saturated with detergent are obtained, which will develop into more stable vesicles upon further detergent removal [126-129]. Within the context of the above model, the rate of detergent removal is one of the parameters that influences the reconstitution process significantly. For example, when the detergent removal is fast, the intermediate disk-like structures have less time to fuse with other disks. Consequently, the induction of curvature is the only alternative to overcome the hydrophobic repulsion at the disk rim. As a result, the disks close at an earlier stage and smaller vesicles are obtained. In contrast, slow detergent removal, would leave more time for the disks to fuse and consequently will result in the formation of larger vesicles [130-133]. Moreover, since the fusion of discs is an uncontrolled process mainly based on collisions, slow detergent removal allows more time for these uncontrolled collisions to take place, resulting in a broader size distribution of the discs and consequently a more heterogeneous vesicle population [132]. Additionally, during slow detergent removal, there is more opportunity for vesicle-vesicle fusion which may occur when vesicles are detergent saturated [133]. In the model, the charge of the detergent or incorporated lipids also plays a role in the determination of the vesicle size. Due to the electrostatic repulsion, fusion of charged disks will be less efficient and consequently smaller vesicles will be obtained [130,131,134,135]. Salt concentration and pH have also been reported to influence vesicle size [133,136]. Yet, another factor influencing the vesicle formation process is the effect of lipid composition on the rigidity of the bilayer. More rigid membranes have higher curvature energy and therefore form larger vesicles [131]. The process of reconstitution of protein-containing membranes is an even more complicated process than that of the formation lipid vesicles, since it involves an

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additional mechanism for the insertion of the membrane proteins in the lipid bilayers. The morphology of the reconstituted membranes depends a.o. on the properties of the detergent, the nature of the membrane protein and the detergent removal rate. Rigaud and coworkers determined which stage of vesicle formation is optimal for protein insertion using the detergents cholate, Triton X-100 and OG [128]. For this purpose, dissolved membrane proteins were added to a solution containing mixed micelles at various stages of the vesicle formation process. These studies revealed that cholate facilitates insertion at the micellar stage, Triton X-100 at the stage in which both vesicles and micelles are present and OG at the vesicle stage. Since the stage at which membrane proteins are inserted in the lipid bilayer determines their orientation, proteins are randomly, partially or fully orientated to the exterior, respectively [128].

Based on experiments with several membrane proteins with varying detergents and detergent removal rates, Riguad and co-workers proposed a model for reconstitution based on the classification of membrane proteins in those that easily self-aggregate and those that do not [128]. Complete solubilization of membranes results in binary complexes containing lipid and detergent and ternary complexes containing lipid, protein and detergent. When a protein has a tendency to self-aggregate, slow detergent removal would induce an early collapse of the ternary complexes, since these complexes are less stable than the binary complexes. At a later stage, liposomes are formed that coexist with the protein-rich aggregates. Depending on the stage at which the detergent facilitates protein insertion, these proteins would remain as protein-rich aggregates (e.g. using cholate), or some proteins (e.g. using Triton X-100) or all of the proteins (e.g. using OG) are inserted in the vesicle bilayer. This would result in a heterogeneous, more homogenous or completely homogenous distribution of the membrane proteins over the vesicles, respectively. However, when the detergent removal is fast both the binary and ternary complexes coalesce at the same time, resulting in a homogenous distribution of the membrane proteins. In case the membrane proteins do not self-aggregate, the binary and ternary complexes are equally stable and independent of the detergent removal rate, vesicles with a homogenous protein distribution are likely to be produced [128].

Classical procedures for influenza virosome generation

Almost 20 years ago, our group developed a method for the production of fusion-active influenza virosomes [79]. In this procedure, influenza virus is treated with the detergent C12E8, resulting in the solubilization of the virus membrane. Subsequently,

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the nucleocapsid is removed by ultracentrifugation and reconstitution of the virus membrane is achieved by detergent removal through adsorption to a hydrophobic resin (Bio-Beads SM-2). Virosomes obtained by this procedure are approximately 100 nm in diameter and their surface is decorated with spike proteins. Morphologically, the virosomes are similar to native influenza virus. Importantly, the fusogenic properties of HA are preserved during the reconstitution procedure. This is demonstrated by the fact that virosomes fuse with target membranes in a pH-dependent manner similar to that of native virus. Exposure of virosomes to low pH in the absence of target membranes abolishes fusion activity. This observation underlines that fusion is HA-dependent, since low-pH treatment in the absence of target membranes is known to induce an irreversible conformational change which renders HA fusion-inactive [107].

A modified version of the above described procedure is being used by Berna Biotech for the production of virosomes for the purpose of vaccination, which they have appropriately called immunopotentiating reconstituted influenza virosomes (IRIVs) [80,137]. In this procedure, an excess amount of exogenous lipids is added prior to the reconstitution procedure. This method results in virosomes with an HA density that is far less than that of the fusion-active virosomes. In our hands coreconstitution of excess amounts of exogenous lipids severely impaired the fusion activity of virosomes [unpublished results]. Additionally, a virosomes containing various ratio’s of HA with a pH optimum of 5.1 and HA with a pH optimum of 5.6 was prepared to investigate the influence of the HA density on the fusion activity. In this assay, the final extent of fusion decreased with decreasing densities of the HA of which the pH-optimum was used for the induction of fusion [138].

VIROSOMES AS A DELIVERY VEHICLE FOR NUCLEIC ACIDS

Encapsulation of nucleic acids during membrane reconstitution

Encapsulation of nucleic acids in the virosomal lumen during the reconstitution process can either be achieved passively or actively. With passive encapsulation the amount of material encapsulated depends on the volume entrapped in the virosomal lumen. This volume is dependent on the size and concentration of the virosomes. High concentrations result in more vesicles per volume unit and therefore in a larger entrapped volume. However, experimental conditions require detergent

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concentration and detergent to lipid ratios that limit the use of high membrane component concentrations. As a consequence, the entrapped volume is relatively small. Moreover, pDNA is a large negatively charged molecule, which is not easily packaged into the virosomal lumen due to the electrostatic repulsion within the molecule itself. Several groups have achieved passive encapsulation of nucleic acids in the lumen of Sendai virosomes. However, efficiencies were generally low [139-141]. In our hands, encapsulation of pDNA in influenza virosomes was inefficient [unpublished observations].

To overcome the charge repulsion within the DNA molecule, Ponimaskin and co-workers used positively charged poly-L-lysine, which condenses the negatively charged DNA through electrostatic interaction, thereby facilitating encapsulation [142,143] Using this method, the problem of encapsulation of the large pDNA in a small lumen is resolved. However, the process is still passive.

Cationic lipids are also known to condense nucleic acids [144] and, moreover, they can be coreconstituted in the virosomal membrane thereby facilitating active encapsulation of the condensed nucleic acid. This active encapsulation of nucleic acids was achieved for influenza virosomes as described in Chapter 3 and 4.

Other approaches for the association of nucleic acids with virosomes

Surface-attachment of nucleic acids. As an alternative for encapsulation, binding of pDNA to the surface of fusogenic influenza virosomes [145] and IRIVs [146-150] has been reported. To obtain these systems, cationic lipids are coreconstituted with the dissolved influenza membrane components. Subsequently, nucleic acids are attached to the surface of the virosomes through electrostatic binding with the incorporated cationic lipids by simple mixing of the nucleic acids with the final virosome preparation. Waelti et al. claimed that using this procedure nucleic acids are encapsulated in the virosomal lumen, however without presenting the experimental data to substantiate this statement are lacking [146].

Fusion of virus particles with liposomes containing nucleic acids. Another method of nucleic acid encapsulation involves the use of liposomes with encapsulated nucleic acids. Fusion of these liposomes with UV-treated Sendai virus or VSV results in so-called fusogenic liposomes [122,151]. However, these vehicles still contain the inactivated genetic material of the virus, which is likely to induce side effects.

Centrifugation of nucleic acids into sub-solubilized virus particles. In this procedure, Sendai virus is treated with sub-solubilizing concentrations of detergent after which nucleic acids are added to the virus suspension. Subsequently, the viral

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genome is centrifuged out of the viral envelope and during the same centrifugation step the nucleic acids are moved into the viral envelope [152]. However, results on the removal of the nucleocapsid proteins and thus the tightly associated genetic material are questionable. Nevertheless, the so-called Sendai envelope system is currently marketed by Ishihara Sangyo Kaisha ltd as GenomONE® and GenomONE-Neo® as a cellular delivery system for nucleic acids, but also for proteins and other compounds.

Post-insertion of fusion proteins into pre-formed liposomes or lipoplexes. This approach involves detergent extraction of viral fusion proteins. After extraction and purification, the G-protein of VSV has been used for either post-insertion into pre-formed liposomes containing encapsulated nucleic acids [151] or for post-insertion into pre-formed cationic lipid/pDNA complexes (lipoplexes) [153].

Virosome-related approaches. Diverging from the virosomal concept, a method has been reported, which exploits the fusogenic properties of enveloped viruses by mixing whole inactivated viruses with either liposomes containing pDNA or with lipoplexes. Using this approach, nucleic acid delivery with the help of Herpes Simplex virus, influenza virus and Sendai virus has been achieved [154,155]. Likewise, the membrane penetrating property of the non-enveloped adenovirus has also been utilized for delivery of nucleic acids to target cells. In these systems, UV-inactivated adenovirus is complexed to lipoplexes, in which the positive charge of the cationic lipids enhances cellular uptake and the adenoviral spike protein facilitates endosomal escape [156-160]. The term virosome has also appeared in studies in which complexation of cationic lipid to retroviral vectors improved their transduction efficiency [161,162]. However, this system comprises entire retrovirus particles including the viral genome and is therefore not comparable with the above described virosomes.

Applications of virosomes for delivery of nucleic acids

Virosomes or virosome-related systems produced from various viruses that have been used for delivery of different types of nucleic acids are summarized in Table 1. Most frequently, virosomes have been used for the delivery of pDNA to achieve gene expression. Many studies have been performed to demonstrate proof of principle of the virosomal concept in cultured cells using reporter genes [Table 1]. Several reports have shown in vitro delivery of pDNA that was passively encapsulated in Sendai virosomes [Table1]. In vivo these virosomes were shown to specifically deliver pDNA to mouse liver using a reporter system [143,163] and delivery to mouse brain of a

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plasmid coding for a neuropeptide was confirmed by immunostaining [164]. Delivery of passively encapsulated dsRNA in Sendai virosomes to cultured cells has also been assessed [140].

Multiple in vivo studies have been performed with the two different Sendai systems of the group of Kaneda namely, the Sendai fusogenic liposomes and Sendai envelope system. These studies show in vivo delivery of reporter plasmids to organs, such as liver, spleen, muscle and uterus in rat and mice and their efficiency has been examined in various disease models [152,165-168]. The Sendai virosome-systems have also been used for the delivery of fluorescently labeled ODNs and mRNA in cultured cells [Table 1] and for the ODN-mediated inhibition of protein expression in various disease models a.o. in cardio-vascular disease [167]. Moreover, one in vivo study assessed inhibition of protein synthesis by Sendai-mediated siRNA delivery in a tumor model [169].

VSV virosomal systems and the virosome-related systems involving whole influenza, Sendai and Herpes simplex viruses have thus far only been applied in an in vitro setting [Table 1]. Adenoviral-mediated pDNA delivery to cultured cells [Table 1] and to vascular cells in rabbit was shown using a reporter system [160].

Fusion-active virosomes prepared from influenza virus with surface-attached pDNA efficiently transfected cultured cells as was demonstrated by expression of the reporter gene β-galactosidase [145]. Despite the location of the nucleic acids on the exterior of the virosomes, they could be delivered to target cells. Most likely this is explained by the fact that fusion of virosomes with target cells is a leaky process [170]. This allows for the escape of compounds located in the endosomes and likely also for compounds attached to the surface of virosomes through the fusion pore.

A method by which pDNA and siRNA can be actively encapsulated within the lumen of influenza virosomes with the help of cationic lipids is described in Chapter 3 and 4 of this thesis. These virosomes efficiently transfect cultured cells. Delivery of either pDNA or siRNA was confirmed by EGFP expression and inhibition of EGFP synthesis, respectively [Chapter 3 and 4].

IRIVs with coreconstituted cationic lipids were used for delivery of oligodeoxynucleotides (ODNs) in vitro and pDNA to antigen-presenting cells in vivo, using the approach in which nucleic acids are attached to the IRIV surface [146,148]. Additional studies were carried out to demonstrate the induction of a humoral immune response against mumps virus antigens and a cellular immune response against cancer antigens [147,149,150].

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Virus Nucleic acid

Method of encapsulation/binding

Target Reference

cultured cells [145] in vivo, immune response against F and HN proteins of Mumps virus in mice

[147]

in vivo, induction of a CTL response against a tumor associated antigen in mice

[150]

in vivo, induction of a CTL response against an epithelial cancer marker in mice

[149]

attachment to the surface of virosomes using cationic lipids

in vitro and in vivo, delivery to antigen presenting cells

[148]

cationic lipids, encapsulation cultured cells [Chapter 3]

pDNA

virus mixed with liposomes containing pDNA

cultured cells [155]

ODN attachment to the surface of virosomes using cationic lipids


Influenza virus

siRNA cationic lipids, encapsulation cultured cells [Chapter 4] cultured cells [171]

[172] [139] [173] [174] [175] [176] [177] [178] [141] [179]

in vivo, parenchymal cells in mouse liver

[163]

passive encapsulation

in vivo, neurons in rat brain

[164]

in vitro and in vivo delivery to various cells

[166]

cultured cells [165]*

encapsulation of DNA in liposomes and subsequent fusion with Sendai virus

delivery to the cardio vascular system in vivo

[167]


[152]*

Sendai virus

pDNA

centrifugation into detergent treated virus particles

delivery to the uterus of mice in vivo

[168]

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Virus Nucleic acid

Method of encapsulation/binding

Target Reference

polylysine cultured cells [142] cationic lipids in vivo, hepatocytes in

mouse liver [143]

pDNA

virus mixed with liposomes containing pDNA


encapsulation of ODN in liposomes and subsequent fusion with Sendai virus

delivery to the cardio vascular system in vivo

[167] ODN

centrifugation into detergent treated virus particles


[152]*

dsRNA passive encapsulation cultured cells [140] mRNA encapsulation of mRNA in

liposomes and subsequent fusion with Sendai virus

cultured cells [165]*

Sendai virus

siRNA centrifugation into detergent treated virus particles

in vitro and in vivo delivery to mice tumor

[169]

encapsulation of pDNA in liposomes and post insertion of VSV-G protein

[151]

insertion of VSV-G into cationic/pDNA complexes

[153]

Vesicular stomatitus virus

pDNA

encapsulation of pDNA in liposomes and subsequent fusion with VSV

cultured cells

[180]

Herpes simplex virus

pDNA virus mixed with cationic lipids complexed with pDNA


cultured cells

[156] [157] [158] [159]

Adenovirus pDNA UV-treated virus mixed with cationic lipids complexed with pDNA

cultured cells and in vivo delivery in rabbit arteries

[160]

Table 1. Overview of virosomes and related delivery systems exploiting the membrane-penetrating properties of viruses for the introduction of nucleic acids into cells. *numerous papers have been published on the various Sendai fusogenic liposomes or Haemagglutinating virus of Japan systems (Pubmed search: Kaneda Y HVJ results in 165 hits).

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STABILIZATION OF BIOPHARMACEUTICALS

Disadvantages of biotechnological therapeutics in solution

In the preceding paragraphs various promising characteristics of virosomes have been discussed. However, the pharmaceutical properties of a drug formulation play a crucial role in its successful application. A major problem associated with virosomes and in fact with most biotechnological therapeutics, such as (recombinant) proteins, vaccines, liposomes/lipoplexes and recombinant viral vectors, is that they are produced in an aqueous solution. Dispersed in water, these therapeutics are prone to physical or chemical degradation, such as denaturation, aggregation, hydrolysis or oxidation [181]. Especially, aggregation plays an important role in lipidic formulations [182-184]. The chemical and physical degradation reactions may result in the loss of functional activity of the therapeutic in question. In other words, biotechnological therapeutics in solution have a limited shelf-life. Refrigeration or, when applicable, freezing generally prolongs the shelf-life. However, this requires the use of an undesirable cold-chain for distribution. Moreover, administration of proteineous or lipidic drugs dispersed in an aqueous solution is often restricted to parenteral injection, which restricts widespread application and is not in favour of the patient’s comfort.

Freeze-drying of biotechnological compounds

To overcome most of the above problems, theoretically, simple removal of water from the active compound could be sufficient. It is well known that various food substances can be preserved over a long period of time when they are kept in a dry state. A commonly used method to obtain biotechnological therapeutics in the dry state is freeze-drying (lyophilization). In this process, the substance is usually rapidly frozen after which water is removed by sublimation under vacuum [185]. This process, however, involves freezing and drying stresses, such as formation of ice crystals, solute concentration and removal of the hydration shell surrounding the compound. These stresses can induce denaturation, aggregation and structural damage of protein therapeutics, leakage, aggregation and fusion of lipid membranes, which may result in the loss of functional activity of the formulation [185-188]. It is known that plants use low-molecular weight solutes, such as carbohydrates to protect themselves from injury during freezing and drying stresses [189]. These

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carbohydrates have also proven to be adequate protectants during freeze-drying and storage, as has been shown for various proteins, lipoplexes and (recombinant) viruses [185,190-192]. Upon rapid freezing, the sugar molecules and part of the water molecules form an amorphous matrix, the so-called “freeze-concentrated fraction” in which the compounds are captured. The presence of a high concentration of the lyoprotective excipient isolates the drug molecules from one another during freezing, drying and storage, which avoids aggregation of the compounds [187]. The sugar molecules replace the water molecules in the hydrogen-bonding interaction with the protein during lyophilization, such that the structural integrity of the protein is preserved [193]. In addition, some sugars are also known to interact with lipids and consequently stabilize membrane structures [186,194,195]. Moreover, the process of glass formation, or in other words vitrification, slows down all chemical reactions considerably and prevents conformational changes within the therapeutic [196,197]. Thus, lyophilization of biotechnological therapeutics in the presence of an appropriate (oligo)saccharide to act as a protectant and stabilizer has the potential to increase the shelf-life, to avoid the use of a cold-chain and thereby reducing transport costs. Moreover, sugar glasses can be processed to powders or tablets, such that alternative needle-free routes of administration can be applied, such as oral, nasal, pulmonary, or dermal delivery.

Sugar glass technology

The principle of the formation of a sugar glass is illustrated by the state-diagram of a binary sugar/water system depicted in Figure 7 (reviewed in: [198,199]). The contribution of the drug in this system is neglected, since it is usually present in very low amounts. When the temperature of a solution of water and sugar of composition A, containing a low drug concentration, is lowered, water starts to crystallize at 0°C (point B). During freezing, the crystallization temperature of the remaining water decreases due to freeze-concentration of the sugar. At the eutectic temperature (Te, point C), the sugar starts to crystallize simultaneously with the water molecules. However, this only occurs when the solution is in thermodynamic equilibrium. When the solution is rapidly frozen, e.g. in liquid nitrogen, the crystallization rate of the sugar is too slow to form crystals. As a consequence, rapid cooling below the Te results in crystallization of water only and an increase in viscosity of the fluid phase. At the glass-transition temperature (Tg’, point D), the viscosity increases dramatically such that the sugar, water and drug molecules are immobilized. This temperature is thus defined as the temperature at which the maximally freeze-

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concentrated fraction turns into a glass. In this state, the sugar molecules are randomly oriented (the amorphous state) and form a vitrified matrix in which water and drug molecules are captured.

To obtain the drug in a dry amorphous sugar glass, the frozen sample is kept under vacuum and the water is removed by sublimation. In the first stage of this process, referred to as primary drying, the water crystals formed during the freeze process above Tg’ are removed (reviewed in: [198,199]). Essential during this stage is that the temperature is kept below the Tg’. Above this temperature the sugar glass turns into the rubbery state, in which the mobility of the sugar considerably increases and crystallization may occur. This is detrimental for the stabilization of the drug, since hydrogen bonds or other stabilizing interactions with the sugar are lost and the translation freedom of the drugs increases, which could cause aggregation. Moreover, the mechanical forces induced by crystallization of the sugar can damage the structure of the therapeutic, which in turn may cause loss of functional activity.

Figure 7. State diagram of a binary (water/sugar) system showing the physical changes during freeze-drying. See text for further explanation. Adapted from [198]

The remaining water molecules captured in the maximally freeze-concentrated fraction after Tg’ was passed are removed by secondary drying, which takes place when the surface of the sugar glass is freed from ice-crystals. During this stage, the temperature can be slowly increased as long as it stays below the Tg line of the sugar-water mixture. After all the water is removed, a dry sugar glass with the encapsulated drug is obtained with a glass-transition temperature of Tg characteristic for the sugar.

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To assure a long shelf-life, the lyophilized drug should be stored below the Tg of the particular sugar to avoid transition into the rubbery state. Another factor that could cause this transition is an increase in humidity, which in turn decreases the Tg. Thus, the relative humidity should be kept as low as possible (reviewed in: [198,199]).

Excipients

There are several criteria a carbohydrate should meet to fulfil the requirements for a successful stabilizer during freeze-drying and storage. One of the most important ones is that it should possess a high Tg’ which allows storage at ambient temperatures. A high Tg and thus a high Tg’ also facilitates the freeze-drying process, since the sample can be kept at a higher temperature during drying which increases the sublimation rate of water. A low hygroscopicity is also desirable, since the absorption of water lowers the Tg and increases the risk of transition to the rubbery state. Furthermore, a low crystallization rate would be favourable since, when the storage temperature is temporarily higher than the Tg’ crystallization is not directly initiated. Additionally, the absence of reducing groups within the carbohydrate is essential, since these could initiate a cascade of reactions with the therapeutic, which could severely impair its activity.

Sucrose and trehalose are two frequently used saccharides that possess most of the above properties and have been successfully used for the lyophilization of various biotechnological therapeutics [185,191,192]. In particular, trehalose is favoured for its excellent stabilizing properties at suboptimal conditions [200]. Inulins are oligosaccharides recently applied for the stabilization of biotechnological products. High-molecular weight inulins possess higher Tg’s and lower crystallization rates than trehalose [201]. In a study in which alkaline phosphatase was lyophilized using glucose, trehalose and inulin, it was shown that lyophilization with inulins allowed the most challenging temperature and relative humidity during storage [201]. Moreover, inulins have been used successfully for the lyophilization of liposomes, lipoplexes and HA of a subunit vaccine [183,202,203].

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OUTLINE OF THIS THESIS

The objective of the study described in this thesis was to develop methods for the production and stabilization of virosomes as delivery devices for pDNA and siRNA, thereby providing an alternative for the current viral and non-viral delivery systems. In Chapter 2, a novel method is described for the production of influenza virosomes based on the solubilization of the influenza virus membrane by the short-chain phospholipid dicaproylphosphatidylcholine (DCPC). In this procedure, reconstitution of the dissolved virus membrane is achieved by means of dialysis. In the study presented in Chapter 2, the optimal DCPC concentration was determined for efficient solubilization of the virus membrane. After reconstitution, the resulting virosomes were physically and morphologically characterized and fusion assays were performed to study the HA-dependent fusion activity. Moreover, the influence of the rate of detergent removal on the vesicle size was studied.

Chapter 3 deals with the encapsulation of pDNA in the lumen of influenza virosomes. To achieve this, cationic lipids and pDNA were added to the dissolved membrane components prior to the reconstitution process. The obtained DNA-virosomes were physically, morphologically and functionally (fusion-activity) characterized. Successful encapsulation of the pDNA was demonstrated by exposure of the virosomes to nucleases. The ability of virosomes to deliver encapsulated pDNA to cells was studied in transfection experiments, in which various cells lines were exposed to virosomes containing an EGFP reporter plasmid. In Chapter 4, a procedure similar to that for the production of DNA-virosomes is applied to encapsulate synthetic siRNA in the virosomal lumen. The resulting siRNA-virosomes were characterized as described above. Endosomal uptake of siRNA-virosomes by cultured cells was visualized by confocal microscopy. Cellular delivery of siRNA was investigated by measuring inhibition of synthesis of an EGFP reporter protein in cultured cells exposed to siRNA-virosomes either in a prophylactic or a therapeutic setting. Moreover, siRNA-virosomes were injected into the peritoneal cavity of mice to examine the binding/uptake by cells in vivo. In Chapter 5, a procedure is presented to stabilize influenza virosomes by freeze-drying using inulin sugar glass technology and preservation of the structural integrity and biological activity of freeze-dried virosomes is assessed. Freeze-dried virosomes were investigated for the preservation of their immunological properties after lyophilization and during storage. To investigate if the delivery properties of

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virosomes are maintained during freeze-drying and subsequent storage, the transfection efficiency of lyophilized DNA-virosomes were studied. In Chapter 6, the results of the preceding chapters are discussed in the light of pharmaceutical improvements of virosome production and stabilization and the concept of a flexible virosome platform for nucleic acid delivery is introduced. Moreover, preliminary in vivo experiments are presented. Finally, the future perspectives on the use of virosomes as nucleic acid delivery devices are discussed.

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