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CHAPTER 2 MOLECULAR BIOLOGY: PRESENT AND FUTURE(cont’4)

Retroviral Vectors. The Moloney murine leukemia virus (MoMuLV) was the first virus used in developing a vector system for gene transfer. Parts of the retroviral genome that are involved in the replication of the MoMuLV—gag, pol, and env—are removed and replaced by a gene of interest. What remains of the retrovirus are the long terminal repeats harboring regulatory elements, integration signals, and transcription promoters, and a packaging signal. To produce retroviral vectors carrying the gene of interest, one must use a packaging cell line harboring the gag, pol, and env genes, which have previously been incorporated into the genome of such packaging cells. The vectors are produced in high titers in the packaging cells, purified, and injected into the organism (in vivo gene therapy) or put in contact with cells collected from the patient and maintained in cell culture (ex vivo gene therapy). Retroviral vectors have the ability to integrate their genomic material into the host genome as a result of the presence of the remaining retroviral regulatory sequences in the form of the long terminal repeats. Once integrated, the inserted gene can be expressed to produce the desired protein.
Advantages of retroviral vectors are that they are well characterized, they can be produced in high titers, and they have a high efficiency of infection. Disadvantages are the limited size of DNA (7–8 kb) that retrovirus vectors can accommodate, the requirement that the target cells be in cell division to allow for the integration of the vectors, and the potential for insertional mutagenesis due to retroviral vector integration at random in the genome. The latter feature has the potential to interrupt important genes in the cell, with serious consequences that include oncogenesis through the activation of protooncogenes or inactivation of tumorsuppressor genes.
Lentiviruses are a group of retroviruses that have the ability to infect and integrate their genome even in cells that are not dividing. Thus, the use of lentiviruses as gene therapy vectors is broader than that of other retroviruses and is under investigation.
Adenoviral Vectors. The human adenoviruses are nonenveloped DNA viruses with a linear double-stranded DNA of ~36 kb, encapsulated in an icosahedral capsoid measuring 70 to 100 nm in diameter. Adenoviruses are known pathogens in humans, and most, if not all, adult humans have been exposed to adenoviruses and have antibodies against adenovirus antigens.
An adenovirus does not depend on host-cell division for its replication, and the chromosome rarely integrates into the genome, remaining episomal in most cases. Integration seems to occur mainly in the presence of high levels of infection in dividing cells, but this event does not contribute significantly to the utility of these viruses as vectors. Adenoviral vectors have a broad spectrum of cell infectivity that includes virtually all postmitotic and mitotic cells; they also can be produced in high titers.
The genome of the adenovirus encodes ~15 proteins. Viral gene expression occurs in a coordinated fashion and is mainly directed by the E1A and E1B genes, localized within the 5′ region of the adenoviral genome. These genes have transactivation functions for the transcription of various viral and host-cell genes. Because E1 genes are involved in adenoviral replication, their removal renders the virus replication incompetent. The removal also creates room for insertion of a foreign gene of therapeutic interest. An exogenous DNA can also replace the E3 region, which produces a product enabling the virus to evade the immune system. Packaging cells carrying adenoviral genes that provide transcomplementation functions are required to produce defective adenoviral vectors. Packaging cells of the NIH-293 cell lineage are human embryo kidney (HEK) cells that have been previously transformed with type 5 adenovirus. These cells retain the E1A and E1B regions of the viral genome covalently linked to their genomic DNA.
Disadvantages of the adenoviral vectors include the short duration of transgene expression, because the vector usually does not integrate stably into the host-cell genome. Further, the size of the inserted foreign DNA sequence is limited, and cellular and humoral immune responses are typically triggered against the adenoviral particles or against the host cell that eventually expresses adenoviral proteins, thus limiting the longevity of the adenovirus vector.
Adenoassociated Viral Vectors. Adenoassociated virus (AAV) is a small nonenveloped, nonpathogenic DNA virus belonging to the Parvoviridae family. The AAV genome is a single-strand DNA molecule of 4681 bases, including two inverted long terminal repeats (ITRs). ITRs are 145-base-long palindromic sequences involved in the regulation of the AAV cell cycle. They are located in the 5′ and 3′ terminal portions of the viral genome and serve as origins and initiators for DNA replication. Flanked by the ITRs, two large open-reading frames code for a regulatory protein and a structural protein, called rep and cap, respectively. The protein coding sequence located in the 5′ region (rep gene) encodes four nonstructural proteins involved in the genomic replication. The 3′ region contains the cap gene, which encodes three structural proteins required for the formation of the viral capsid. AAV is capable of replication in a cell only in the presence of a helper virus (adenovirus or herpes virus) that provides by transcomplementation the helper factors that are essential for its replication. In the absence of a helper virus, the AAV genome preferentially integrates into a specific site on the short arm of chromosome 19, between q13.3 and qter, called AAVS1. The ITRs as well as a rep transcript play an important role in this process, which results in a latent infection in mitotic as well as in postmitotic cells. Episomal virus and insertion in nonspecific sites has been documented.
Among the advantages of AAV as a potential gene vector in human gene therapy are the lack of relation to human diseases, broad infectivity spectrum, and ability to stably integrate into the host genome. This integration can occur in cells that are not dividing, although at a lower frequency than in dividing cells. The site-directed integration is also a most favorable property of AAV.
DNA-BASED VACCINATION
Immunization with transfer of genetic material represents a novel approach to vaccination.57,58 The technology involves transferal of a gene (encoding an antigenic protein cloned in an expression vector) to a host, leading to the induction of an immune response. Direct gene transfer may be undertaken using either viral vectors or recombinant plasmid DNA. Viral vectors have the disadvantages of being derived from pathogens (like traditional vaccines based on attenuated virus), and, therefore, are of limited interest for the purpose of immunization. In contrast, DNA plasmids encoding antigens are more frequently used because they do not have the inconvenience of classic vaccines: they are safe, inexpensive, easy to produce, heat stable, and amenable to genetic manipulation.
Currently, two main delivery systems are available for gene vaccination. Plasmid DNA is injected intramuscularly, or DNA is coated onto gold beads and transferred into the epidermis or dermis by a bioballistic process (gene gun). The intramuscular injection is the most widely used method for immunization, and it consists of direct injection of naked DNA into skeletal muscle. Plasmid DNA in some instances is injected into muscle directly in saline solution or after injection of toxins or a local anesthetic to cause necrosis and regeneration of the injected muscle, thereby increasing the expression of the encoded antigen and amplifying the immunologic response. It is unclear whether the elevated antigen in regenerating muscle cells is due to an increased expression of the antigen gene or to the antigen contained within antigen-presenting cells that are recruited to the site of tissue damage. Humoral and cell-mediated immune responses have been induced by the direct intramuscular injection of plasmid DNA endocrine immunogens. An antibody response was first reported against an influenza virus protein in mice, and specific cytotoxic T-cell responses were also detected in different systems (i.e., human immunodeficiency virus infection and hepatitis B) after genetic immunization. Protective immunity was first demonstrated in mice injected intramuscularly with nucleoprotein DNA of influenza virus. In this model, researchers have indicated that both CD4+ and CD8+ T cells contributed to the protection. Protective immune responses have also been demonstrated in mice against Leishmania major, Plasmodium yoelii, Mycobacterium tuberculosis, dengue virus, and herpes simplex virus.
The bioballistic (gene gun) method uses a helium gas pressuredriven device to deliver gold particles coated with plasmid directly into the skin. When gene vaccines are administered by gene gun technology, most of the plasmid DNA is taken up by keratinocytes and some dermal fibroblasts; they become transfected and produce the encoded antigen. Humoral responses using bioballistic approaches were demonstrated using plasmids encoding human growth hormone and human a-antitrypsin.
The nature of the immune response elicited by these DNA vaccination approaches is not clearly understood. In an initial study, the suggestion was made that DNA vaccination elicits a cell-mediated immune response, because passive transfer of serum from immune mice did not engender protective immunity. Depletion experiments demonstrated that both CD4+ and CD8+ cell populations were involved in host protection against infection. However, some investigators have also reported an induction of immunoglobulins after gene gun–mediated immunization.
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