Gene Delivery Approaches Viral Methods

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Viruses are a natural delivery system for delivering their deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) to the host cell. For their own survival they must deliver their DNA or RNA materials, which must be copied successfully within the host cell. Using this delivery system, vector virology has been used to construct several efficient vectors for gene therapy trials. Detailed studies of the viral genome have helped scientists to eliminate disease-causing viral genes before using their natural delivery power for transferring a therapeutic gene of interest. However, the risk factors associated with the integration of the viral genome into patient cells, which can cause unwanted side effects, need always to be critically evaluated before using natural viral particles.

Therapeutic gene transfer vectors have been developed from different groups of viruses, each one with its own set of advantages and disadvantages (Table 18.1). Depending upon the targeted disease and type of treatment needed, one has to decide which vector is suitable for a given application. It is important to keep in mind that development of a viral vector in the laboratory is often done in order to produce a replication-incompetent virus, so that the virus can deliver its genetic contents but cannot replicate. This requires dedicated approaches in which large amounts of virus can be grown through the use of cell lines that carry missing genes or helper viruses that provide the necessary genes needed for viral replication. Without these approaches it would not be possible to replicate the replication-incompetent viruses. Overall gene therapy approaches using

Table 18.1. Comparison chart of major gene delivery vectors.

Vector

Insert size

Transduction efficiency

Major advantages

Major disadvantages

Retrovirus (MMLV)

< 8 Kb

High

Stable expression of dividing cells; low immunogenicity

Poor in vivo delivery; unable to transfect nondividing cell; safety concern of insertional mutagenesis

Lentivirus

< 8-9 Kb

High

High and stable expression of both dividing and non-dividing cells

Potential safety concern for generating human disease

Adenovirus

< 7.5 Kb

High

Transfects nearly all cell types, both dividing and nondividing; substantial clinical experience

Strong immune reaction; transient expression

Adeno associated virus

< 4.5 Kb

High

Stable expression; infect wide range of cells; no significant immune response

Capability of carrying small DNA size; Safety concern of insertional mutagenesis; little clinical experience

Herpes simplex virus

< 20 Kb

Low

Capacity to carry large DNA fragment

Neuronal cell specificity; transient expression; Cytotoxic; Safety concern to generate human infections

Liposome

>20 Kb

Low

Larger DNA carrying capacity; low or no immunogenicity; good safety profile; cell specific targeting

Inefficient in vivodelivery; transient expression

Naked DNA

< 20 Kb

Low

Simple and cheap; low or no immune reaction; very safe

Very short duration of expression; poor ex vivoand in vivodelivery

Ballistic DNA

< 20 Kb

Low

Simple; low or no immune reaction; safe

Transient expression; local cellular damage

viral vectors have focused on four major groups of viruses: Retrovirus, Adenovirus, Adeno Associated Virus (AAV) and Herpes Simplex Virus (HSV). Each of these is discussed in the following sections.

Retrovirus

Retroviruses are a class of enveloped viruses, which contain a single-stranded RNA molecule as their genome. After infecting a host cell, the viral genome is reverse transcribed (RNA to DNA) by reverse transcriptase into double-stranded DNA, which integrates into the host genome and is expressed (Fig. 18.2). The retroviral genome consists of little more than the genes essential for viral replication. The prototype and simplest genome is that of the Moloney Murine Leukemia Virus (MoMLV), in contrast to the highly complex genomes of the HTLV (human T-cell leukemia virus) and HIV (human immunodeficiency virus) retro-

Gene Delivery Viral Method

Figure 18.2. Schematic diagrams showing basic differences of three major gene delivery approaches: retrovirus (left), adenovirus (center) and liposome (right). Retrovirus, containing a RNA genome, attaches to a host cell by binding to a specific cell surface receptor and a double-stranded DNA is synthesized from the RNA genome by a process called reverse transcription. This DNA integrates into the host genome ensuring sustained expression of the transgene. Adenovirus is comprised of a double-stranded DNA genome. After entering the cell by a receptor mediated endocytosis process, the viral DNA is released within the cell, which then leads to a synthesis of proteins using host cell machinery. The DNA does not integrate into the host genome. In liposomal delivery the plasmid DNA containing the therapeutic gene is encased in a lipid bilayer, which upon reaching a host cell, gets fused with the cellular membrane and releases the DNA within the cell. This DNA then synthesizes its protein products along with cellular DNA.

Figure 18.2. Schematic diagrams showing basic differences of three major gene delivery approaches: retrovirus (left), adenovirus (center) and liposome (right). Retrovirus, containing a RNA genome, attaches to a host cell by binding to a specific cell surface receptor and a double-stranded DNA is synthesized from the RNA genome by a process called reverse transcription. This DNA integrates into the host genome ensuring sustained expression of the transgene. Adenovirus is comprised of a double-stranded DNA genome. After entering the cell by a receptor mediated endocytosis process, the viral DNA is released within the cell, which then leads to a synthesis of proteins using host cell machinery. The DNA does not integrate into the host genome. In liposomal delivery the plasmid DNA containing the therapeutic gene is encased in a lipid bilayer, which upon reaching a host cell, gets fused with the cellular membrane and releases the DNA within the cell. This DNA then synthesizes its protein products along with cellular DNA.

viruses. The basal retroviral genome can be divided into three transcriptional units: gag, pol and env. The gag region encodes genes that comprise the capsid (the virus capsule) proteins; the pol region encodes the reverse transcriptase and integrase proteins; and the env region encodes the proteins needed for receptor recognition and envelope anchoring. An important feature of the retroviral genome is the long terminal repeat (LTR) regions, which play an important role in initiating viral DNA synthesis, as well as regulating transcription of the viral genes. The recombinant viral vector that was used for the first human clinical gene therapy trial (for severe combined immunodeficiency disease) in 1990 was based on the retrovirus MoMLV [10].

Subsequently, the well-characterized genome and lifecycle of retroviruses has made it possible to utilize them in designing gene delivery vectors for many different purposes [11]. First, retroviruses satisfy one of the major prerequisites for gene therapy applications, which is long-term and relatively stable expression of the transferred gene, due to the fact that their genome is integrated into the host genome. Also, their integration into the host cell does not alter normal cell function. They satisfy the prerequisite of safety for gene delivery since their cis-acting elements (i.e., viral genes and the genes responsible for reverse transcriptase, integrase, and packaging signal) can be separated from the coding sequences. Removing these genes from the vector and causing them to be expressed in the packaging cell line allows production of replication-deficient virus particles ex vivo and helps to ensure safety. A major disadvantage of using retroviruses as a gene transfer tool is that they only transduce cells that divide shortly after viral infection [12, 13]. Thus, this vector has limited applications to important target organs like the liver, skeletal muscle, hematopoietic stem cells, and neuronal cells. Another potential drawback of retroviruses is the chance of insertional muta-genesis as the virus genome randomly integrates inside the host genome.

Lentivirus

Lentiviruses are a subclass of retroviruses of which HIV are the most recently discovered members. HIV based vectors show promise for future use in clinical studies, but their complex biology and mechanisms of pathogenesis complicate their choice for clinical applications [14]. Like other members of the retroviral family, the HIV genome also contains the gag, pol and env genes. In addition, six other genes are contained within their genome: the tat, rev, vpr, vpu, vif, and nef genes, which code for nonstructural regulatory proteins. Lentiviruses have the capability to infect both proliferating and non-proliferating cells, which is considered advantageous since a much larger range of cell types can be targeted for therapeutic applications. Early results using reporter genes have shown promising in vivo expression in muscle, liver, and neuronal cells [15-17]. However, exceptions to this ability to transduce non-proliferative tissues have been reported, suggesting that lentivirus vectors cannot infect all non-dividing cells [18]. A major concern in using HIV vectors is the strong possibility of genetic recombination between infectious HIV and the lentiviral vector itself, which would result in the lentiviral vector acting as an infectious HIV particle. One possible method to overcome this risk would be to introduce a suicide gene (see below) into the HIV vector genome. This risk could also be avoided by using equine infectious anemia virus (EIAV) and feline immune deficiency virus (FIV) as vectors, since neither of these is associated with human disease. These lentiviruses do not infect human cells, but the gene determining the host range can be substituted by another gene, allowing the viruses to infect and deliver genes to human tissue.

Adenovirus

Adenoviruses are non-enveloped viruses containing a linear double-stranded DNA genome. There are currently 47 distinct serotypes and as many as 93 different varieties of adenovirus, all of which generally infect the human eye, upper respiratory tract, and gastrointestinal epithelium. The adenoviral genome is about 35 kilobases (kb), and is comprised of four regulatory transcription units, E1, E2, E3, and E4 [19]. For developing an inactivated adenovirus vector, the E1 and E3 genes are deleted from the genome and then supplied in trans (gene elements provided in another plasmid/ complimentary strand), either by a helper virus or plasmid, when packaging the virus. Graham et al. [20] first developed a cell line, which enabled the production of recombinant adenovirus in a helper-free environment by integrating E1 into the cell genome. Since then, adenoviral vectors have received much attention as gene transfer agents and currently offer a wide variety of gene therapy applications.

The exact mechanism by which an adenovirus targets a host cell is poorly understood. However, in-ternalization occurs via receptor-mediated endocyto-sis followed by release from an endosome. A primary receptor of the immunoglobulin gene family, CAR (coxsackie adenoviral receptor) has been identified as specific for adenovirus. After endosomal release, the viral capsid undergoes disassembly outside the nucleus (Fig. 18.2). Nuclear entry of the viral DNA is completed upon capsid dissociation, but the viral DNA does not integrate into the host genome; instead it replicates as episomal elements in the nucleus of the host cell. Also, since the viral DNA lacks the ability to integrate, it cannot bring about mutagenic effects caused by random integration into the host genome. As a consequence, their use as a vector is devoid of the risk of generating insertional mutagenesis. Adenoviral vectors have high transduction efficiency, are capable of containing DNA inserts of up to 8 kb, have extremely high viral titers, and infect both replicating and differentiated cells. Disadvantages of adenoviral vectors include: (i) gene expression is transient since the viral DNA does not integrate into the host, (ii) expression of viral proteins that are present in the adenoviral vector leads to an immune response, and (iii) adenoviral vectors are extremely common human pathogens and in vivo delivery may be hampered by prior host-immune response to one viral type.

Adeno-Associated Virus

Adeno-associated viruses (AAV) are non-pathogenic satellite viruses of other human viruses and require co-infection with either adenovirus or herpes simplex virus (HSV) for their replication [21, 22]. A unique feature of the DNA molecule contained within the virion is the presence of inverted terminal repeat (ITR) at both ends. These ITRs are important in the site-specific integration of AAV DNA into a specific site in human chromosome 19. The ability of wild-type AAV to selectively integrate into chromosome 19 makes them attractive candidates for the production of a gene therapy vector. For production of virus vectors, cells are first infected with the wild-type adenovirus or HSV and then both the recombinant AAV vector plasmid DNA and the non-rescuable AAV helper plasmids are co-transfected into the cells. The cells produce mature recombinant AAV vectors as well as wild-type aden-ovirus or HSV. The wild-type adenovirus or HSV is then removed by either density gradient centrifugation or heat inactivation.

These vectors have the potential to produce a therapeutic gene transfer tool with site-specific integration and the ability to infect multiple cell types. Unfortunately, this has not been the case to date for these vectors. Current research focuses on how to regain the missing site-specific integration sequences that are lost when the nonessential genes are removed from the viral genome. These vectors do offer some advantages over other vector systems, including minimization of immune response, stability, and ability to infect a variety of dividing and non-dividing cells. Unfortunately, a major drawback is their inability to incorporate genes larger than 5 kb. Also, these vectors and must be closely screened for adenoviral or HSV contamination [23].

Herpes Simplex Virus

The Herpes simplex virus (HSV) genome consists of one double-stranded DNA molecule, which is 120-200 kb in length. The virus itself is transmitted by direct contact and replicates in the skin or mucosal membranes before infecting cells of the nervous system. It exhibits both lytic and latent function. It is thought that it will be possible to modify this virus for gene therapy, to produce a vector that exhibits only the latent function for long-term gene maintenance and has expression with specific tendency towards cells of the central nervous system.

Key regions of the HSV genome, which must be removed to produce a replication-deficient HSV vector, include the ICP4, ICP0, and ICP27 [24]. The following must be taken into consideration before developing an engineered HSV vector suitable for gene therapy: (i) the vector must be non-cytotoxic to nerve cells, as well to other cell lines; (ii) the vector must be unable to carry out the lytic cycle; (iii) the vector should be able to establish latency; and (iv) there must be persistent and sufficient levels of the desired gene expression once latency has been obtained.

The HSV vectors currently under study have a number of advantages as gene transfer reagents. Because of the large size of the virus, recombinant HSV vectors are capable of containing inserted genes of around 20 kb. They give an extremely high titer, similar to the adenoviruses, and they have been shown to express the transgene for a long period of time in the central nervous system, as long as the lytic cycle does not occur. Major disadvantages for the HSV systems include: (i) expression does appear to be transient with the current vector systems, suggesting either lytic infection or viral protein expression, (ii) transduction efficiency is relatively low such that not many target cells show expression of the desired gene, and (iii) they are far from complete and require much additional engineering to be become efficient vectors for gene therapy.

Amplicon-Based HSV Vectors

Amplicon-based vectors rely upon the natural occurrence of defective interfering (DI) particles. DI parti cles arise during HSV propagation and are infectious agents, which lack portions of the HSV genome for replication. It was observed that plasmid DNA could be packaged into DI particles if the plasmid DNA contained HSV packaging signals. Such DI particles were referred to as DI vectors. Therefore, amplicon-based HSV or DI vectors rely on two basic components: (i) the amplicon, which contains HSV packaging signals and regions where desired genes can be cloned, and (ii) DI particle production. Following insertion of the desired genes into the amplicon, the amplicon is trans-fected into any cell line, which allows for efficient HSV propagation. Following amplicon transfection, the cells are infected with helper HSV virus. The DI vector is then generated along with helper virus during the viral propagation process. The system can be manipulated so that the ratio of DI particles to helper virus is high.

Sindbis Virus

The use of virus based expression vectors for gene therapy and vaccine applications is increasing, with a number of diverse virus types and approaches. Alphaviruses, especially Sindbis virus and Semiliki Forest Virus (SFV) are also potential candidates as a gene transfer vector. The viral genome consists of positive single-stranded RNA about 12 Kb that codes for several structural and non-structural proteins. After the virus enters a cell, the nonstructural proteins (NSP1-4) get translated. Then the NS proteins function to produce the sense strand, from which large amounts (~105) of new genomic and subgenomic RNAs coding for the virus structural proteins get transcribed. Assembly of the structural proteins resulted in the production of progeny viruses. The viruses pass their entire life cycle in the cytoplasm.

Sindbis viral vectors offer obvious advantages as a gene delivery vector in having extremely high transgene expression in a variety of mammalian and insect cell lines. Though this virus has a RNA genome, the viral genome does not integrate, which minimizes the chance of chromosomal recombination to occur in the host genome. Further, relatively small sized genome and rapid production of high-titer virus are advantageous in using this virus as a delivery tool. Typical disadvantages for Sindbis virus is that they have a relatively short-term expression and have a strong cytotoxic effect on host cell. However, both of these characteristics can be utilized in applications such as cancer therapy [25, 26].

Hybrid Vectors

Gene researchers are now studying a class of recombinant virus vectors called "conditional viral vectors" in the hope of designing vectors that selectively deliver transgenes to the site of the disease. This is one of the greatest challenges of gene therapy. The purpose of these vectors is specifically to track tumor cells and kill them by delivering a payload of genes. Using a combination of tissue-specific and tumor-specific elements, researchers have demonstrated that viruses can achieve preferential killing of target tumor cells. Several studies showing the use of adenovirus [27], adeno-associated virus [28, 29] and HSV-1 virus [30, 31] have been reported. In all of these studies, it has been shown that by combining different viral and regulatory elements from different viruses and other genomes, it is possible to obtain regulated transgene expression in a target-specific manner. Greater details of gene therapy applications that involve combining two virus systems (hybrid vector) or combining various specific elements like promoters, enhancers, and insulators within a viral system, can be found elsewhere [32].

Nonviral Methods

The gene therapy field remains divided about optimal delivery methods due to the inherent strengths and weaknesses of different systems. The majority of cancer gene therapy approaches still employs viralbased vectors to express suitable target genes inside cancer cells [33]. However, virus-based approaches cannot overcome certain limitations of expression, such as target specificity, immune response, and safety concerns regarding secondary malignancies and recombination to form replication competent virus. These limitations in fact reinforced efforts to develop other non-infectious in vivo gene delivery procedures. As an outcome, several methods have been developed in the past few years, including the use of different plasmid-expression vectors that consist of tissue-specific transcriptional control elements and various other regulatory elements for time-limited vector replication inside tumor cells. Such nonviral vectors, while not as efficient as their viral counterparts for in vivo delivery, can avoid triggering an immune response, which is perhaps the biggest obstacle to successful gene therapy [34].

So far, the usefulness of nonviral vectors for gene therapy has been limited by their poor transfection efficiencies. However, their relatively easy production, non-pathogenicity and lack of adventitious contami nants are important advantages (Table 18.1). To develop successful nonviral vectors, the following three cellular barriers need to be overcome: (1) DNA needs to be transported to the cytoplasm, either directly through the plasma membrane or by escape from en-dosomes; (2) DNA needs to be translocated to the nucleus, and (3) for many applications the DNA has to be integrated into the genome or must be extra-chromosomally replicated. In this review, we will discuss only those major nonviral methods that hold promise for application in in vivo gene delivery. Other details are reviewed elsewhere [23].

Liposomes

To be useful for clinical application, the methods of gene introduction must be compatible with therapy. This need has led to the development of nonviral lipo-some-mediated gene transfer techniques (Fig. 18.2). Liposomes were first described in 1965 as a model of cellular membranes, and were quickly applied to the delivery of substances into cells [35]. Methods are reviewed in detail elsewhere [36]. Liposomes entrap DNA by one of two mechanisms, which has resulted in their classification as either cationic liposomes or pH-sensitive liposomes. Cationic liposomes are positively charged and interact with the negatively charged DNA molecules to form a stable complex. Cationic liposomes consist of a positively charged lipid and a co-lipid. Commonly used colipids include dioleoyl phosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPC). Colipids, also called helper lipids, are in most cases required for stabilization of liposome complex. A variety of positively charged lipid formulations are commercially available, and many other are under active development. One of the most frequently used cationic liposomes for delivery of genes to cells in culture is Lipofectin, which is a mixture of N-[1-(2, 3-dioleyloyx) propyl]-N-N-N-trimethyl ammonia chloride (DOTMA) and DOPE. DNA and Lipofectin interact spontaneously to form complexes that have a 100% loading efficiency, i.e., if enough Lipofectin is available, all DNA molecules form complex with the Lipofectin. It is assumed that the negatively charged DNA molecule interacts with the positively charged groups of DOTMA. The lipid:DNA ratio and overall lipid concentration used in forming these complexes are extremely important for efficient gene transfer and vary with each application. Lipofectin has been used to deliver linear DNA, plasmid DNA, and RNA to a variety of cells in culture [37]. Following intravenous administration of

Lipofectin-DNA complexes, both the lung and liver showed marked affinity for uptake of these complexes and transgene expression.

Negatively charged or pH-sensitive liposomes entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. In some cases, these liposomes are destabilized by low pH and hence are called pH-sensitive. To date, cationic liposomes have proved to be much more efficient for gene delivery in vitro and in vivo than pH-sensitive liposomes. However, pH-sensitive liposomes have the potential to be much more efficient for in vivo DNA delivery than their cationic counterparts, and should be able to do so with reduced toxicity and interference from serum proteins.

Liposomes offer several advantages in delivering genes to cells: (1) liposomes can complex with negatively and positively charged molecules; (2) they hold a greater degree of protection of the DNA from degrading processes; (3) liposomes can carry large pieces of DNA, potentially as large as a chromosome; and (4) liposomes potentially can be specifically targeted to any cells or tissues. In addition, liposomes avoid the issues of immunogenicity and replication competent virus contamination. The ability to synthesize chemically a wide variety of liposomes has provided a highly adaptable and flexible system capable of gene delivery, both in vitro and in vivo. Current limitations regarding in vivo application of liposomes revolve around low transfection efficiency and transient gene expression. Also, liposomes display a low degree of cellular toxicity and appear to be inhibited by serum components. As liposome technology is better understood, it should be possible to produce reagents with improved in vivo gene delivery into specific tissues.

Naked DNA

A DNA molecule that is not attached or encapsulated by a liposome or virus capsid is referred to as naked DNA. The simplest way of delivering DNA to cells is by direct injection utilizing a syringe. Surprisingly, some tissues exhibit transgene expression following naked plasmid DNA injection, including thymus, skin, cardiac muscle, and skeletal muscle. Of these tissues, long-term transgene expression was observed only in striated muscle [38]. Using this approach, intramascular DNA vaccines are currently being researched as a preventive tool for cancer and infectious diseases. The other way to transfer a segment of DNA inside the cell is by elec-troporation, a process where the application of an elec trical impulse leads to cellular membrane breakdown, giving the negatively charged DNA molecule space to enter the cell. Electroporation has been used for many years in transfecting cells, but has not been used extensively in gene therapy. With the recent development of new devices and procedures, it is now possible to minimize cell damage in vitro. For efficient gene transfer, electroporation depends upon the nature of the electrical pulse, distance between the electrodes, ionic strength of the suspension buffer, and nature of the cells. Best results have often been obtained with rapidly proliferating cells. Successful application of this procedure is very difficult, particularly in terms of in vivo applications, but it has proved useful to some ex vivo applications like local gene therapy applications on the skin.

The greatest advantages of intramuscular plasmid DNA injection are simplicity, low cost, and incorporation of relatively large genes of 2 kb to 19 kb. The application of this technique is still restricted to the skin, thymus, and striated muscle.

Ballistic DNA Injection

Ballistic DNA injection, which is also known as particle bombardment or micro-projectile gene transfer, or the gene-gun, was first developed for gene transfer into plant cells. Since its initial introduction, it has been modified to transfer genes into mammalian cells both in vitro and in vivo [39]. Plasmid DNA that encodes the gene of interest is used to coat micro-beads, which are then accelerated by motive force to penetrate cell membranes. Specifically, the plasmid DNA is precipitated onto 1-3 micron-sized gold or tungsten particles. These particles are then placed onto a carrier sheet, which is inserted above a discharge chamber. At discharge, the carrier sheet accelerates toward a retaining screen, allowing the particles to continue toward the target surface. The force can be generated by a variety of means; the most common include high-voltage electronic discharge, spark discharge, or helium pressure discharge.

Ballistic DNA injection has been successfully used to transfer genes to a wide variety of cell lines and also to the epidermis, muscle, and liver. In vivo applications have predominantly focused on the liver, skin, muscle or other organs, which can easily be surgically exposed. Ballistic DNA injection also offers the capacity to deliver precise DNA dosages. Unfortunately, genes delivered by this method are expressed transiently and there is considerable cell damage, which occurs at the center of the discharge site. Currently, the most excit ing application of intramuscular and ballistic plasmid DNA injection is in DNA-based immunization protocols. DNA-based vaccinations are being developed to prevent infectious diseases and cancer [40]. Cells that express a foreign protein and subsequently present that protein on their surfaces are more likely to produce a cell-mediated immune response. Such a response may be more important in fighting viral infections such as those caused by HIV, HSV, CMV, or RSV. Techniques that inject a foreign antigen usually yield an antibody-mediated response. Both the gene gun and direct intramuscular plasmid DNA injection are being used in protocols to immunize against HIV, hepatitis B and C, HSV, influenza, papilloma, tuberculosis, RSV (Rous Sarcoma virus), CMV, Lyme disease, Helicobacter pylori, malaria and Mycoplasma pulmonis.

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