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HLTR|

Reducing LTR interference g—fltri

entry site

- reverse orientation

- SIN vector

- DC SIN vector

Figure 5 Gene expression from retroviral vectors. Strategies to drive expression of 1 or 2 genes, and to minimize interference by the 5' LTR promoter. SD, splice donor; SA, splice acceptor; P, promoter; I, internal ribosome entry site; pA, polyA sequences; SIN, self-inactivating; DC, double copy.

than 1 transgene can be achieved through the use of both the LTR and an internal promoter, or by exploiting the differential splicing of the vector that occurs when the major splice acceptor site upstream of the env gene is retained (37). In addition, the expression of 2 genes can be linked by the use of an internal ribosome entry site (38).

The transcriptional activity of the LTR can in some cases be a problem, interfering with the activity of internal promoters (promoter interference) (39). These problems can be reduced by the use of SIN vectors, where extensive sequences of the LTR are deleted to effectively silence its promoter. The level of gene expression from integrated vectors can also be increased through the use of double copy vectors (40). Here, the therapeutic gene and its promoter are inserted into the 3' LTR itself, with the result that after reverse transcription, 2 copies of this expression cassette are created in the provirus.

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