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Lentiviral vector design using alternative RNA export elements.

Oh T, Bajwa A, Jia G, Park F - Retrovirology (2007)

Bottom Line: It was found that multiple copies of the constitutive transport element (CTE) originating from different simian retroviruses, including simian retrovirus type 1 (SRV-1) and type-2 (SRV-2) and Mason-Pfizer (MPV) could be used to eliminate the requirement for the rev responsive element (RRE) in the transfer and packaging plasmids with titers >106 T.U./mL (n = 4-8 preparations).The addition of multiple copies of the murine intracisternal type A particle, the woodchuck post-regulatory element (WPRE), or single and dual copies of the simian CTE had minimal effect on viral titer.Immortalized cell lines from different species were found to be readily transduced by VSV-G pseudotyped lentiviral vectors containing the multiple copies of the CTE similar to the findings in HeLa cells, although the simian-derived CTE were found to have a lower infectivity into murine cell lines compared to the other species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Kidney Disease Center, Medical College of Wisconsin, Milwaukee, WI, USA. tgohkjs@chungbuk.ac.kr

ABSTRACT

Background: Lentiviral vectors have been designed with complex RNA export sequences in both the integrating and packaging plasmids in order to co-ordinate efficient vector production. Recent studies have attempted to replace the existing complex rev/RRE system with a more simplistic RNA export system from simple retroviruses to make these vectors in a rev-independent manner.

Results: Towards this end, lentiviral transfer plasmids were modified with various cis-acting DNA elements that co-ordinate RNA export during viral production to determine their ability to affect the efficiency of vector titer and transduction in different immortalized cell lines in vitro. It was found that multiple copies of the constitutive transport element (CTE) originating from different simian retroviruses, including simian retrovirus type 1 (SRV-1) and type-2 (SRV-2) and Mason-Pfizer (MPV) could be used to eliminate the requirement for the rev responsive element (RRE) in the transfer and packaging plasmids with titers >106 T.U./mL (n = 4-8 preparations). The addition of multiple copies of the murine intracisternal type A particle, the woodchuck post-regulatory element (WPRE), or single and dual copies of the simian CTE had minimal effect on viral titer. Immortalized cell lines from different species were found to be readily transduced by VSV-G pseudotyped lentiviral vectors containing the multiple copies of the CTE similar to the findings in HeLa cells, although the simian-derived CTE were found to have a lower infectivity into murine cell lines compared to the other species.

Conclusion: These studies demonstrated that the rev-responsive element (RRE) could be replaced with other constitutive transport elements to produce equivalent titers using lentivectors containing the RRE sequence in vitro, but that concatemerization of the CTE or the close proximity of RNA export sequences was needed to enhance vector production.

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Schematic and viral titer of different lentiviral vector packaging constructs. Lentiviral vectors were produced using different transfer plasmids containing RRE (Fig. 4A) or multiple copies of SRV-1 (Fig. 4B) or MPV CTE (Fig. 4C). Viral titer was determined by end-point dilution using X-gal stained HeLa cells and p24 Gag protein ELISA. HATCHED bars = Kozak sequence (CCACCATGG); W = woodchuck post-regulatory element; C = constitutive transport element; pA = bovine growth hormone poly(A) signal; RRE = rev-responsive element; SD = splice donor; SA = splice acceptor. * p < 0.05 difference between lentiviral vectors produced with or without the rev protein.
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Figure 4: Schematic and viral titer of different lentiviral vector packaging constructs. Lentiviral vectors were produced using different transfer plasmids containing RRE (Fig. 4A) or multiple copies of SRV-1 (Fig. 4B) or MPV CTE (Fig. 4C). Viral titer was determined by end-point dilution using X-gal stained HeLa cells and p24 Gag protein ELISA. HATCHED bars = Kozak sequence (CCACCATGG); W = woodchuck post-regulatory element; C = constitutive transport element; pA = bovine growth hormone poly(A) signal; RRE = rev-responsive element; SD = splice donor; SA = splice acceptor. * p < 0.05 difference between lentiviral vectors produced with or without the rev protein.

Mentions: Minimal packaging plasmids containing the gag-pol genes from HIV-1 were cloned as described in the Materials and Methods, in which the DNA sequences attributed to the expression of the viral accessory genes were deleted. As shown in Figure 4, there did not appear to be any significant difference in viral titer using different cis-acting DNA elements in the packaging constructs. Various transfer plasmids were examined to determine their compatibility with different packaging plasmid containing the RRE or multiple sequences of the SRV-1 CTE. Our study found that there was a marked difference in p24 Gag protein levels depending on the combination of transfer and packaging plasmid used for lentivector production as the levels ranged from 47 +/- 5 ng/mL (n = 5; pCMV.gag.pol.RRE.bpA) to 858 +/- 109 ng/mL [n = 5; pCMV.gag.pol.C4(+).bpA]. p24 Gag protein measurements by ELISA has been one common method to titer lentivector preparations, and generally, the functional viral titer of lentiviral vectors is dependent upon the p24 Gag protein levels. For this reason, we cloned the consensus Kozak sequence (CCACCATGG) in front of the Gag-pol genes in the packaging construct to enhance translation of p24 Gag protein production. As shown in Figure 4A, the viral titer in HeLa cells were similar (between 2–4 × 106 T.U./mL) following the production of a lentiviral vector containing the same transfer plasmid, but using two different gag-pol constructs to express the structural and packaging genes. The interesting finding was that inclusion of the Kozak sequence (pCMV.Kozak.gag.pol.RRE.bpA) resulted in an approximate 3-fold increase of p24 Gag protein to 147 +/- 12.5 ng/mL, but the functional viral titer as determined by end-point dilution did not markedly change compared to the packaging plasmid without the Kozak sequence. Interestingly, the p24 Gag levels were 2–3-fold higher using a transfer plasmid containing multiple CTE elements with either a RRE-containing packaging plasmid or a WPRE-containing packaging plasmid. The elevated p24 Gag protein levels did not appear to affect the viral titers in a positive way. These results demonstrate that the functional titer is not necessarily a function of the p24 Gag protein levels provided that a minimal threshold of gag protein is produced, and that several different factors, such as optimizing translation start site and other cis-acting DNA elements may play a role in producing high titer vector.


Lentiviral vector design using alternative RNA export elements.

Oh T, Bajwa A, Jia G, Park F - Retrovirology (2007)

Schematic and viral titer of different lentiviral vector packaging constructs. Lentiviral vectors were produced using different transfer plasmids containing RRE (Fig. 4A) or multiple copies of SRV-1 (Fig. 4B) or MPV CTE (Fig. 4C). Viral titer was determined by end-point dilution using X-gal stained HeLa cells and p24 Gag protein ELISA. HATCHED bars = Kozak sequence (CCACCATGG); W = woodchuck post-regulatory element; C = constitutive transport element; pA = bovine growth hormone poly(A) signal; RRE = rev-responsive element; SD = splice donor; SA = splice acceptor. * p < 0.05 difference between lentiviral vectors produced with or without the rev protein.
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Related In: Results  -  Collection

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Figure 4: Schematic and viral titer of different lentiviral vector packaging constructs. Lentiviral vectors were produced using different transfer plasmids containing RRE (Fig. 4A) or multiple copies of SRV-1 (Fig. 4B) or MPV CTE (Fig. 4C). Viral titer was determined by end-point dilution using X-gal stained HeLa cells and p24 Gag protein ELISA. HATCHED bars = Kozak sequence (CCACCATGG); W = woodchuck post-regulatory element; C = constitutive transport element; pA = bovine growth hormone poly(A) signal; RRE = rev-responsive element; SD = splice donor; SA = splice acceptor. * p < 0.05 difference between lentiviral vectors produced with or without the rev protein.
Mentions: Minimal packaging plasmids containing the gag-pol genes from HIV-1 were cloned as described in the Materials and Methods, in which the DNA sequences attributed to the expression of the viral accessory genes were deleted. As shown in Figure 4, there did not appear to be any significant difference in viral titer using different cis-acting DNA elements in the packaging constructs. Various transfer plasmids were examined to determine their compatibility with different packaging plasmid containing the RRE or multiple sequences of the SRV-1 CTE. Our study found that there was a marked difference in p24 Gag protein levels depending on the combination of transfer and packaging plasmid used for lentivector production as the levels ranged from 47 +/- 5 ng/mL (n = 5; pCMV.gag.pol.RRE.bpA) to 858 +/- 109 ng/mL [n = 5; pCMV.gag.pol.C4(+).bpA]. p24 Gag protein measurements by ELISA has been one common method to titer lentivector preparations, and generally, the functional viral titer of lentiviral vectors is dependent upon the p24 Gag protein levels. For this reason, we cloned the consensus Kozak sequence (CCACCATGG) in front of the Gag-pol genes in the packaging construct to enhance translation of p24 Gag protein production. As shown in Figure 4A, the viral titer in HeLa cells were similar (between 2–4 × 106 T.U./mL) following the production of a lentiviral vector containing the same transfer plasmid, but using two different gag-pol constructs to express the structural and packaging genes. The interesting finding was that inclusion of the Kozak sequence (pCMV.Kozak.gag.pol.RRE.bpA) resulted in an approximate 3-fold increase of p24 Gag protein to 147 +/- 12.5 ng/mL, but the functional viral titer as determined by end-point dilution did not markedly change compared to the packaging plasmid without the Kozak sequence. Interestingly, the p24 Gag levels were 2–3-fold higher using a transfer plasmid containing multiple CTE elements with either a RRE-containing packaging plasmid or a WPRE-containing packaging plasmid. The elevated p24 Gag protein levels did not appear to affect the viral titers in a positive way. These results demonstrate that the functional titer is not necessarily a function of the p24 Gag protein levels provided that a minimal threshold of gag protein is produced, and that several different factors, such as optimizing translation start site and other cis-acting DNA elements may play a role in producing high titer vector.

Bottom Line: It was found that multiple copies of the constitutive transport element (CTE) originating from different simian retroviruses, including simian retrovirus type 1 (SRV-1) and type-2 (SRV-2) and Mason-Pfizer (MPV) could be used to eliminate the requirement for the rev responsive element (RRE) in the transfer and packaging plasmids with titers >106 T.U./mL (n = 4-8 preparations).The addition of multiple copies of the murine intracisternal type A particle, the woodchuck post-regulatory element (WPRE), or single and dual copies of the simian CTE had minimal effect on viral titer.Immortalized cell lines from different species were found to be readily transduced by VSV-G pseudotyped lentiviral vectors containing the multiple copies of the CTE similar to the findings in HeLa cells, although the simian-derived CTE were found to have a lower infectivity into murine cell lines compared to the other species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Kidney Disease Center, Medical College of Wisconsin, Milwaukee, WI, USA. tgohkjs@chungbuk.ac.kr

ABSTRACT

Background: Lentiviral vectors have been designed with complex RNA export sequences in both the integrating and packaging plasmids in order to co-ordinate efficient vector production. Recent studies have attempted to replace the existing complex rev/RRE system with a more simplistic RNA export system from simple retroviruses to make these vectors in a rev-independent manner.

Results: Towards this end, lentiviral transfer plasmids were modified with various cis-acting DNA elements that co-ordinate RNA export during viral production to determine their ability to affect the efficiency of vector titer and transduction in different immortalized cell lines in vitro. It was found that multiple copies of the constitutive transport element (CTE) originating from different simian retroviruses, including simian retrovirus type 1 (SRV-1) and type-2 (SRV-2) and Mason-Pfizer (MPV) could be used to eliminate the requirement for the rev responsive element (RRE) in the transfer and packaging plasmids with titers >106 T.U./mL (n = 4-8 preparations). The addition of multiple copies of the murine intracisternal type A particle, the woodchuck post-regulatory element (WPRE), or single and dual copies of the simian CTE had minimal effect on viral titer. Immortalized cell lines from different species were found to be readily transduced by VSV-G pseudotyped lentiviral vectors containing the multiple copies of the CTE similar to the findings in HeLa cells, although the simian-derived CTE were found to have a lower infectivity into murine cell lines compared to the other species.

Conclusion: These studies demonstrated that the rev-responsive element (RRE) could be replaced with other constitutive transport elements to produce equivalent titers using lentivectors containing the RRE sequence in vitro, but that concatemerization of the CTE or the close proximity of RNA export sequences was needed to enhance vector production.

Show MeSH