elimination of viral virulence, the capacity of the virus to cause disease. Expression of foreign genes from viral vectors may be controlled by homologous or heterologous promoters and enhancers chosen to support efficient or cell-type-specific transcription, depending on the goals of the experiment. Such genes can be built directly into the viral genome or introduced by recombination in cells, as described above (see “Engineering Mutations into Viral Genomes”). The viral vector genome generally carries deletions and sometimes additional mutations. Deletion of some viral sequences is often required to overcome the limitations on the size of viral genomes that can be packaged in virus particles.
When viral vectors are designed for therapeutic purposes, it is essential to prevent their reproduction as well as destruction of target host cells. The deletions necessary to accommodate a foreign gene may contribute to such disabling of the vector. For example, the E1A protein-coding sequences that are always deleted from adenovirus vectors are necessary for efficient transcription of viral early genes; in their absence, viral yields from cells in culture are reduced by about 3 to 6 orders of magnitude (depending on the cell type). Removal of E1A-coding sequences from adenovirus vectors is therefore doubly beneficial, although it is not sufficient to ensure that the vector cannot reproduce or induce damage in a host animal. Adenovirus-associated virus vectors are not lytic, obviating the need for such manipulations. As discussed in detail in Volume II, Chapter 9, production of virus vectors that do not cause disease can be more difficult to achieve.
A summary of viral vectors is presented in Table 3.1, and examples are discussed below.
DNA Virus Vectors
One goal of gene therapy is to introduce genes into terminally differentiated cells. Such cells normally do not divide, and they cannot be propagated in culture. Moreover, the organs they comprise cannot be populated with cells infected by viruses ex vivo. DNA virus vectors have been developed to overcome some of these problems.
Table 3.1 Some viral vectors
| Virus | Insert size | Integration | Duration of expression | Advantages | Potential disadvantages |
|---|---|---|---|---|---|
| Adeno-associatedvirus | ~5 kb | No | Long | Nonpathogenic, episomal, infects nondividing and dividing cells, broad tropism, low immunogenicity | Small transgene capacity, helper virus needed for vector production |
| Adenovirus | ~8–38 kb | No | Short | Broad tropism, efficient gene delivery, infects nondividing and dividing cells, large cargo capacity | Transient, immunogenic, high levels of preexisting immunity |
| Baculovirus | No known upper limit | No | Short | High levels of protein synthesis, recombinant viruses easily made, more than one protein can be made in same cells | Insect cells typically used, no replication in mammalian cells, human type protein glycosylation not 100% efficient, paucimannose structures present |
| Gammaretroviru s (murine leukemia virus) | 8 kb | Yes | Short | Stable integration, broad tropism possible via pseudotyping, low immunogenicity, low preexisting immunity | Risk of insertional mutagenesis, poor infection of nondividing cells, faulty reverse transcription |
| Herpes simplex virus | ~50 kb | No | Long in central nervous system, short elsewhere | Infects nondividing cells, large capacity, broad tropism, latency | Virulence, persistence in neurons, high levels of preexisting immunity, may recombine with genomes in latently infected cells |
| Lentivirus | 9 kb | Yes | Long | Stable integration, transduces nondividing and dividing cells | Potential insertional mutagenesis; none detected in clinical trials |
| Rhabdovirus | ~4.5 kb | No | Short | High-level expression, rapid cell killing, broad tropism, lack of preexisting immunity | Virulence, highly cytopathic, neurotropism, immunogenic |
| Vaccinia virus | ~30 kb | No | Short | Wide host range, ease of isolation, large capacity, high-level expression, low preexisting immunity | Transient, immunogenic |
Figure 3.14 Adenovirus vectors. High-capacity adenovirus “gutless” vectors contain only the origin-of-replication-containing inverted terminal repeats (ITR), the packaging signal (blue arrows), the viral E4 transcription unit (red arrow), and the transgene with its promoter. Additional DNA flanking the foreign gene must be inserted to allow packaging of the viral genome (not shown). A helper virus (bottom) is required to package the recombinant vector genome. Two loxP sites for cleavage by the Cre recombinase have been introduced into the adenoviral helper genome (red arrowheads). Infection of cells that produce Cre leads to excision of sequences flanked by the loxP sites so that the helper genome is not packaged.
Adenovirus vectors were originally developed for the treatment of cystic fibrosis because of the tropism of the virus for the respiratory epithelium. Adenovirus can infect terminally differentiated cells, but only transient gene expression is achieved, as infected cells are lysed. Yields of particles are high and these viruses can infect many replicating and non-replicating cell types. In the earliest vectors that were designed, foreign genes were inserted into the E1 and/or E3 regions. As these vectors had limited capacity, genomes with minimal adenovirus sequences have been designed (Fig. 3.14). This strategy allows up to 38 kb of foreign sequence to be introduced into the vector. In addition, elimination of most viral genes reduces cytotoxicity and the host immune response to viral proteins, simplifying multiple immunizations. Considerable efforts have been made to modify the adenovirus capsid to target the vectors to different cell types. For example, the fiber protein, which mediates adenovirus binding to cells, has been altered by insertion of ligands that bind particular cell surface receptors. Such alterations could increase the cell specificity of adenovirus attachment and the efficiency of gene transfer, thereby decreasing the dose of virus that need be administered.
Adenovirus-associated virus has attracted much attention as a vector for gene therapy. This virus requires a helper virus for replication; in its absence the genome remains episomal and persists, in some cases with high levels of expression, in many different tissues.
