Improvements in Vector Design

The basic arrangement described above is functional but unsatisfactory in several ways. In particular, the considerable sequence overlap that exists between the vector and packaging components means that there is a very high risk of recombination occurring that could create an infectious replication-competent retrovirus (RCR) (8). Overlap occurs because extensive sequences of the gag gene are retained in the vector to enhance the efficiency of packaging (9), although Gag protein expression itself is prevented by mutation. In addition, early vector systems retained both the 5' and 3' LTRs in the packaging construct to provide promoter and polyadenylation sequences. Finally, because most of the early MuLV-based packaging cell lines were established in murine NIH 3T3 cells, the possibility also exists for RCR generation through recombination between vector components and endogenous MuLV-like sequences present in the mouse genome.

To minimize the risk of RCR production, an early improvement in vector design was to split the packaging component, placing the Gag-Pol and Env genes on 2 separate plasmids that could be introduced separately into the packaging cell (10,11) (Fig. 4A). The risk of recombination has been further reduced by the use of heterologous Env proteins that have no homology with the parental virus but are able to be incorporated into the viral particle (a process referred to as pseudotyping), and the use of nonmurine producer cell lines. Finally, it has now been shown for MuLV vectors that the gag sequences can be removed from the vector genome without significant loss of packaging efficiency (12).

The problem of the LTR overlap that exists between the vector components has been solved through the use of heterol-ogous promoters and polyadenylation signals in the packaging constructs. This can also have the advantage of enhancing titer (13) because the MuLV LTR promoter will not always drive high-level gene expression in nonmurine producer cell lines. In the vector itself, the LTR sequences can also be significantly deleted. Heterologous promoters, frequently the cy-tomegalovirus (CMV) immediate-early promoter, have been used to replace the 5' U3 promoter (13), which is possible because the U3 sequences in the retroviral vector are derived from the 3' LTR. Even the 3' U3 sequences can be significantly deleted, as is the case with self-inactivating (SIN) vectors, as long as the sequences necessary for recognition by the integrase protein are retained (14). These features are summarized in Fig. 4B.

Improvements have also been made in the titer [number of colony-forming units (cfu) per milliliter] achieved by ret-roviral vectors. For example, in stable producer cell lines, vector production can be maximized by linking expression of the Gag-Pol and Env components to selectable markers to facilitate screening for high titer producer cells (15). Titers have also been boosted through the development of transient expression systems, which are capable of producing very high titers during a brief and very active burst of activity in a transiently transfected cell. Typically, these systems rely on the use of a highly transfectable cell line such as 293T cells, com

Figure 1 Retroviral life cycle. Retroviral infection is initiated by binding of the envelope glycoprotein embedded in the outer lipid membrane of the retrovirus to a specific cell surface receptor. This interaction triggers fusion between the viral and host cell membranes and releases the viral core into the cytoplasm of the cell. The viral RNA genome is transcribed into a DNA copy by the viral reverse transcriptase protein and is integrated into the host cell chromosome by the action of the integrase protein. The inserted provirus is flanked by complete copies of the LTR sequence, resulting from the reverse transcription process. The 5' LTR drives transcription of the retroviral genome, which gives rise to RNAs that code for the viral proteins (Gag, Pol, and Env) as well as the viral genome. Gag and Gag-Pol proteins assemble as viral core particles at the plasma membrane and package the viral RNA genome. The particles bud from the surface of the cell, taking with them a lipid envelope derived from the host plasma membrane containing the Env glycoprotein. TGN, trans-Golgi network.

Figure 1 Retroviral life cycle. Retroviral infection is initiated by binding of the envelope glycoprotein embedded in the outer lipid membrane of the retrovirus to a specific cell surface receptor. This interaction triggers fusion between the viral and host cell membranes and releases the viral core into the cytoplasm of the cell. The viral RNA genome is transcribed into a DNA copy by the viral reverse transcriptase protein and is integrated into the host cell chromosome by the action of the integrase protein. The inserted provirus is flanked by complete copies of the LTR sequence, resulting from the reverse transcription process. The 5' LTR drives transcription of the retroviral genome, which gives rise to RNAs that code for the viral proteins (Gag, Pol, and Env) as well as the viral genome. Gag and Gag-Pol proteins assemble as viral core particles at the plasma membrane and package the viral RNA genome. The particles bud from the surface of the cell, taking with them a lipid envelope derived from the host plasma membrane containing the Env glycoprotein. TGN, trans-Golgi network.

bined with strategies to maximize the production of the individual components through the use of the strong CMV promoter and treatment with the transcription enhancer sodium butyrate (13). We have also shown that titers can be further improved by the inclusion of the adenovirus VAI gene to enhance translation (Lin and Cannon, unpublished data). The combination of these various approaches enables the routine production of vector supernatants in the laboratory with titers in excess of 107 cfu/mL. Whether such transient production systems will ever be useful for large-scale production is uncertain because of potential difficulties in the scale-up and vector characterization procedures.

Although currently preferred for large-scale vector production, the use of stable producer cell lines precludes the use of cytotoxic components. These can include both the therapeutic gene product itself as well as components of the vector system. A notable example is the vesicular stomatitis virus G protein (VSV-G), which is an extremely useful fusion protein for producing pseudotyped retroviral vectors with a very broad host range (16), but is unfortunately very toxic to its host cell. One way around this problem is to use transient systems as described above (17), but an alternate strategy is to regulate its expression through use of an inducible promoter. In particular, the tetracycline-regulated Tet system (18) has been exploited to regulate VSV-G (19), where expression is suppressed in the producer cell line by the addition of tetracycline to the culture and activated by removal of the antibiotic at an appropriate time before harvesting the vectors. In this way,

Figure 2 (A) Retroviral proteins. The LTR sequences contain promoter (5') and polyadenylation (3') sequences and produce full-length and spliced transcripts. These code for 3 major polyproteins; Gag and Gag-Pol are translated from the full-length transcript and Env is translated from the spliced transcript. The full-length transcript also serves as the RNA genome. SD, splice donor; SA, splice acceptor. (B) RNA sequences. The LTRs consist of three regions, designated U3, R, and U5. The promoter and enhancer sequences are active in the 5' LTR only and are located in the U3 region, whereas the polyadenylation (poly A) site in the 3' LTR defines the R/U5 boundary. The primer binding site (PBS) and polypurine tract (PPT) are important for the process of reverse transcription, whereas the att sequences at the ends of the LTRs are necessary for integration. At the 5' region of the genome is a packaging signal that is necessary for the incorporation of the genome into viral particles.

Figure 2 (A) Retroviral proteins. The LTR sequences contain promoter (5') and polyadenylation (3') sequences and produce full-length and spliced transcripts. These code for 3 major polyproteins; Gag and Gag-Pol are translated from the full-length transcript and Env is translated from the spliced transcript. The full-length transcript also serves as the RNA genome. SD, splice donor; SA, splice acceptor. (B) RNA sequences. The LTRs consist of three regions, designated U3, R, and U5. The promoter and enhancer sequences are active in the 5' LTR only and are located in the U3 region, whereas the polyadenylation (poly A) site in the 3' LTR defines the R/U5 boundary. The primer binding site (PBS) and polypurine tract (PPT) are important for the process of reverse transcription, whereas the att sequences at the ends of the LTRs are necessary for integration. At the 5' region of the genome is a packaging signal that is necessary for the incorporation of the genome into viral particles.

the cells can be grown to an optimum density before this toxic fusion protein is expressed. However, although such cell lines are appropriate for laboratory-scale preparation, it is not clear if such a system will be sufficiently stable for industrial production.

As stated in the introduction, retroviral vectors are the most common gene delivery system to have been used in human gene therapy protocols. This is partly for historical reasons; vectors derived from MuLV were the first real vector system to be established and a relatively large amount of information about the performance of such vectors in patients is available. However, their most characteristic feature, the ability to integrate into target cells, can have a downside as well as being advantageous. In the following 2 sections, we review the key properties of retroviral vectors that have made them such attractive gene delivery vehicles and also point out their current limitations and the steps being taken to improve this vector system.

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