Gene expression from transcriptionally disabled retroviral vectors.

Abstract

Retroviral vectors are used for the efficient transfer of foreign genes into mammalian cells. We report here the construction of murine retrovirus-based vectors carrying the full-length cDNA for human hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) and from which the enhancer sequences, the "CAAT box," and the "TATA box" in the long terminal repeats (LTRs) have been deleted.

B-cell-specific transgene expression using a self-inactivating retroviral vector with human CD19 promoter and viral post-transcriptional regulatory element

“…The highly structured WPRE may compensate the reported negative effect of LTR enhancer-promoter deletions on the architecture of the retroviral RNA. 11,17,[32][33][34] The titer-enhancing effect of WPRE observed here in the context of SINvectors is significantly greater than that previously reported for LTR-driven MLV vectors. 19 Lentiviral vector titers are less sensitive to deletions in the U3 region of the LTR;…”

“…However, when using vectors based on murine leukemia viruses (MLV), these approaches have been hampered by very low viral titers of SIN-vectors. [10][11][12][13] The life cycle of retroviruses requires export of unspliced and partially spliced RNA into the cytoplasm, but nuclear export of unspliced RNA usually fails. Mechanisms have evolved that circumvent the need for splicing, but these are associated with cis-acting elements and their corresponding viral or cellular factors mediating RNA transport.…”

Use of a herpes thymidine kinase/neomycin phosphotransferase chimeric gene for metabolic suicide gene transfer

“…24 Most of these vectors also contain a selectable marker, because production and characterization of retroviral producer cells in the absence of positive selection is cumbersome and extremely time consuming. However, the occurrence of transcriptional interference between multiple expression units within the same construct has been demonstrated in many systems [25][26][27] and justifies the current common efforts to develop "simplified," single-gene vectors. Clearly, the use of bifunctional fusion genes would represent an advantageous solution to these problems.…”

The U3 Region of Moloney Murine Leukemia Virus Contains Position-independent Cis-acting Sequences Involved in the Nuclear Export of Full-length Viral Transcripts

“…However, the employment of these vectors in the experimental works showed their low transduction efficiency (3,4). Although introducing exogenous poly(A) sites into the SIN vectors led to an increase in viral titers, this improvement was not Ͼ2-3-fold (8,13).…”

“…An improvement in retroviral vector design and safety was achieved by deleting the enhancer/promoter sequences in the U3 region of the 3Ј long terminal repeat (LTR) (which renders the vectors self-inactivating (SIN) 4 ) (3)(4)(5). Subsequent to reverse transcription in a host target cell, the SIN deletion in the U3 region of the 3Ј LTR is transposed to the U3 region of the 5Ј LTR in the proviral DNA, thereby preventing the expression of full-length vector RNA.…”