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Instability of retroviral vectors with HIV-1-specific RT aptamers due to cryptic splice sites in the U6 promoter.

Braun SE, Shi X, Qiu G, Wong FE, Joshi PJ, Prasad VR, Johnson RP - AIDS Res Ther (2007)

Bottom Line: We did not observe inhibition of HIV-1 replication in these transduced populations.This deletion decreased transcriptional activity of the U6 promoter.The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology, New England Primate Research Center, Harvard Medical School, One Pine Hill Drive, Southborough, MA 01772, USA. stephen_braun@hms.harvard.edu

ABSTRACT

Background: Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated.

Results: A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences.

Conclusion: The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

No MeSH data available.


Related in: MedlinePlus

Diagrams of the retroviral vectors and the shuttle-packaging scheme. A) The MMP-based vector series contains GFP transcriptionally regulated by the MPSV LTR and the control or 3 different HIV-1 specific RT aptamers transcriptionally regulated by the U6+1 promoter positioned in the antisense orientation. The forward and reverse arrows above GFP and the 3' UTR represent the PCR primers used to amplify the inhibitor cassette in transduced cells. B) Generation of high-titer producer cell lines by shuttle packaging. Vector plasmids were transiently transfected into Phoenix (GaLV) packaging cell line and supernatant was used to transduce Phoenix (amphotropic) packaging cell lines. After selecting for Phoenix (amphotropic) cells expressing GFP, supernatants were collected for titering and to transduce Phoenix (GaLV) packaging cell lines with 2 exposures. Supernatants from these Phoenix (GaLV) cells were collected for titration and to transduce the CD4+ cell line CEMx174.
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Figure 1: Diagrams of the retroviral vectors and the shuttle-packaging scheme. A) The MMP-based vector series contains GFP transcriptionally regulated by the MPSV LTR and the control or 3 different HIV-1 specific RT aptamers transcriptionally regulated by the U6+1 promoter positioned in the antisense orientation. The forward and reverse arrows above GFP and the 3' UTR represent the PCR primers used to amplify the inhibitor cassette in transduced cells. B) Generation of high-titer producer cell lines by shuttle packaging. Vector plasmids were transiently transfected into Phoenix (GaLV) packaging cell line and supernatant was used to transduce Phoenix (amphotropic) packaging cell lines. After selecting for Phoenix (amphotropic) cells expressing GFP, supernatants were collected for titering and to transduce Phoenix (GaLV) packaging cell lines with 2 exposures. Supernatants from these Phoenix (GaLV) cells were collected for titration and to transduce the CD4+ cell line CEMx174.

Mentions: Previous studies have demonstrated effective inhibition of HIV and SHIV-RT viral replication in aptamer-transduced cells [11]. Towards evaluating the in vivo efficacy of the HIV-1 RT-specific RNA aptamers, we sought to generate high-titer stable Phoenix(amphotropic) [12] and Phoenix(GaLV) [13] packaging cell lines. Therefore, we shuttled packaged the 4 MMP-GFP aptamer vectors (Figure 1a) [11] by transient transfection through Phoenix(GaLV) packaging cell lines and stable transduction of Phoenix(amphotropic) packaging cell lines (Figure 1b). Expression of GFP in the empty-, 70.15-, 70.8-, and 80.55-transduced Phoenix(amphotropic) cells averaged 14% before sorting (Table 1) and >95% after sorting. When used for packaging of other MMP-GFP vectors, the titers of the Phoenix(GaLV) cells were consistently less than 1 × 105 IU/ml (data not shown). Therefore, Phoenix(GaLV) cells were transduced with two serial exposures to supernatant from the sorted Phoenix(amphotropic) cells. Expression of GFP in the empty-, 70.15-, 70.8-, and 80.55-transduced Phoenix(GaLV) cells averaged 65% before sorting (Table 1) and >95% after sorting. Molecular analysis of the packaging cell lines after sorting (Table 1) indicated that the Phoenix(amphotropic) cells had <1 copy per cell (0.82, 0.33, 0.80, and 0.97 copies, respectively), while the Phoenix(GaLV)-empty, -70.15, -70.8, and -80.55 cells had on average 26 copies of integrated provirus per cell (29, 26, 15, and 33 copies, respectively).


Instability of retroviral vectors with HIV-1-specific RT aptamers due to cryptic splice sites in the U6 promoter.

Braun SE, Shi X, Qiu G, Wong FE, Joshi PJ, Prasad VR, Johnson RP - AIDS Res Ther (2007)

Diagrams of the retroviral vectors and the shuttle-packaging scheme. A) The MMP-based vector series contains GFP transcriptionally regulated by the MPSV LTR and the control or 3 different HIV-1 specific RT aptamers transcriptionally regulated by the U6+1 promoter positioned in the antisense orientation. The forward and reverse arrows above GFP and the 3' UTR represent the PCR primers used to amplify the inhibitor cassette in transduced cells. B) Generation of high-titer producer cell lines by shuttle packaging. Vector plasmids were transiently transfected into Phoenix (GaLV) packaging cell line and supernatant was used to transduce Phoenix (amphotropic) packaging cell lines. After selecting for Phoenix (amphotropic) cells expressing GFP, supernatants were collected for titering and to transduce Phoenix (GaLV) packaging cell lines with 2 exposures. Supernatants from these Phoenix (GaLV) cells were collected for titration and to transduce the CD4+ cell line CEMx174.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211285&req=5

Figure 1: Diagrams of the retroviral vectors and the shuttle-packaging scheme. A) The MMP-based vector series contains GFP transcriptionally regulated by the MPSV LTR and the control or 3 different HIV-1 specific RT aptamers transcriptionally regulated by the U6+1 promoter positioned in the antisense orientation. The forward and reverse arrows above GFP and the 3' UTR represent the PCR primers used to amplify the inhibitor cassette in transduced cells. B) Generation of high-titer producer cell lines by shuttle packaging. Vector plasmids were transiently transfected into Phoenix (GaLV) packaging cell line and supernatant was used to transduce Phoenix (amphotropic) packaging cell lines. After selecting for Phoenix (amphotropic) cells expressing GFP, supernatants were collected for titering and to transduce Phoenix (GaLV) packaging cell lines with 2 exposures. Supernatants from these Phoenix (GaLV) cells were collected for titration and to transduce the CD4+ cell line CEMx174.
Mentions: Previous studies have demonstrated effective inhibition of HIV and SHIV-RT viral replication in aptamer-transduced cells [11]. Towards evaluating the in vivo efficacy of the HIV-1 RT-specific RNA aptamers, we sought to generate high-titer stable Phoenix(amphotropic) [12] and Phoenix(GaLV) [13] packaging cell lines. Therefore, we shuttled packaged the 4 MMP-GFP aptamer vectors (Figure 1a) [11] by transient transfection through Phoenix(GaLV) packaging cell lines and stable transduction of Phoenix(amphotropic) packaging cell lines (Figure 1b). Expression of GFP in the empty-, 70.15-, 70.8-, and 80.55-transduced Phoenix(amphotropic) cells averaged 14% before sorting (Table 1) and >95% after sorting. When used for packaging of other MMP-GFP vectors, the titers of the Phoenix(GaLV) cells were consistently less than 1 × 105 IU/ml (data not shown). Therefore, Phoenix(GaLV) cells were transduced with two serial exposures to supernatant from the sorted Phoenix(amphotropic) cells. Expression of GFP in the empty-, 70.15-, 70.8-, and 80.55-transduced Phoenix(GaLV) cells averaged 65% before sorting (Table 1) and >95% after sorting. Molecular analysis of the packaging cell lines after sorting (Table 1) indicated that the Phoenix(amphotropic) cells had <1 copy per cell (0.82, 0.33, 0.80, and 0.97 copies, respectively), while the Phoenix(GaLV)-empty, -70.15, -70.8, and -80.55 cells had on average 26 copies of integrated provirus per cell (29, 26, 15, and 33 copies, respectively).

Bottom Line: We did not observe inhibition of HIV-1 replication in these transduced populations.This deletion decreased transcriptional activity of the U6 promoter.The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology, New England Primate Research Center, Harvard Medical School, One Pine Hill Drive, Southborough, MA 01772, USA. stephen_braun@hms.harvard.edu

ABSTRACT

Background: Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated.

Results: A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences.

Conclusion: The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

No MeSH data available.


Related in: MedlinePlus