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High-Titer ,Retroviral Vectors for Gene Delivery

sfer Systems

Fig.1

Nonreplicating retroviral vectors contain all of the cis elements required for transcription of mRNA molecules encoding a gene of interest and packaging of these transcripts into infectious virus particles (Figure 1). The vectors typically comprise an E. coli plasmid backbone containing a pair of 600-bp viral long terminal repeats (LTRs) between which the gene of interest is inserted. The LTR is divided into three regions: The U3 region contains the retroviral promoter/enhancer, and is flanked in the 3 direction by the R region, which contains the viral polyadenylation signal (pA). The R region is followed by the U5 region, which, along with R, contains sequences that are critical for reverse transcription. Expression of the viral RNA is initiated within the U3 region of the 5 LTR and is terminated in the R region of the 3 LTR. The viral packaging signal (Y), which is required in cis for the viral RNA to be packaged into virion particles, resides between the 5 LTR and the coding sequence for the gene of interest.

To generate infectious virus particles that carry the gene of interest, specialized packaging cell lines have been constructed that contain chromosomally integrated expression cassettes for viral gag, pol, and env proteins, all of which are required in trans to make virus.2 The gag gene encodes internal structural proteins; the pol gene encodes reverse transcriptase (RT) and integrase; and the env gene encodes the viral envelope protein, which resides on the viral surface and facilitates infection of the target cell b
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