Modularity of Components

The 3 basic components of a retroviral vector—the structural core particle and its enzymes, the envelope-embedded fusion protein, and the vector genome itself—can be viewed as discrete components that in many cases can be ''mixed and matched'' for custom applications. This flexibility enables the exploitation of the natural variations in host range of different viruses and, in some cases, may allow transduction of a cell that is resistant to standard vector combinations. The clearest example is in the use of different envelope proteins to pseudotype the vector particles. For example, when compared with MuLV vectors containing the amphotropic MuLV Env protein, pseudotyping with VSV-G, RD114, 10A1, or gibbon ape leukemia virus (GALV) Env proteins enhances the transduction of primary human hematopoietic cells (26-31).

Figure 4 Improvements in vector design. (A) Packaging construct. The Gag-Pol and Env proteins are separated onto 2 different plasmids, and safety is further increased by the use of heterologous envelope proteins. Expression is maximized from the packaging construct through the use of a non-LTR promoter (CMV) and linkage to a selectable marker (sm). (B) Vector construct. The U3 sequences are replaced at the 5' LTR and minimized in the 3' LTR. Following reverse transcription, the deleted U3 sequences (AU3) are copied into both the 5' and 3' LTRs of the provirus. The 5' LTR therefore has greatly reduced promoter activity. This is the basis of self-inactivating (SIN) vectors.

Figure 4 Improvements in vector design. (A) Packaging construct. The Gag-Pol and Env proteins are separated onto 2 different plasmids, and safety is further increased by the use of heterologous envelope proteins. Expression is maximized from the packaging construct through the use of a non-LTR promoter (CMV) and linkage to a selectable marker (sm). (B) Vector construct. The U3 sequences are replaced at the 5' LTR and minimized in the 3' LTR. Following reverse transcription, the deleted U3 sequences (AU3) are copied into both the 5' and 3' LTRs of the provirus. The 5' LTR therefore has greatly reduced promoter activity. This is the basis of self-inactivating (SIN) vectors.

In addition, different core and vector components can also be used. The combination of GALV cores with MuLV or GALV vector genomes has been reported to enhance the trans-duction of certain human cell lines (32), and both hybrid retroviral/lentiviral genomes (33) and cores (34) have been constructed that may offer new properties. Finally, the vector LTRs can also be manipulated to improve gene expression in certain cell types. The related myeloproliferative sarcoma virus (MPSV) LTR has been used in place of the MuLV LTR to enhance gene expression in embryonic stem cells (35), and the enhancer elements of the native LTR can be replaced with more specific sequences in order to optimize gene expression in a given host cell (see Section IV.B).

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