Abstract
We have developed retroviral replicating vectors (RRV) derived from Moloney murine gammaretrovirus with an amphotropic envelope and an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-transgene cassette downstream of the env gene. During long-term (180 days) replication of the vector in animals, a bulge of 7 adenosine residues (A's) in the J-K bifurcation domain sometimes serially added A's. Therefore, vectors with 4-12 A's in the A bulge in the J-K bifurcation domain were generated, and the impact of the variants on transgene protein expression, vector stability, and IRES sequence upon multiple infection cycles was assessed in RRV encoding yeast-derived cytosine deaminase and green fluorescent protein in vitro. For transgene protein expression, after multiple infection cycles, RRV-IRES with 5-7 A's gave roughly comparable levels, 4 and 8 A's were within about 4-5-fold of the 6 A's, whereas 10 and 12 A's were marked lower. In terms of stability, after 10 infection cycles, expansion of A's appeared to be a more frequent event affecting transgene protein expression than viral genome deletions or rearrangement: 4 and 5 A's appeared completely stable; 6, 7, and particularly 8 A's showed some level of expansion in the A bulge; 10 and 12 A's underwent both expansion and transgene deletion. The strong relative translational activity of the 5 A's in the EMCV IRES has not been reported previously. The 5A RRV-IRES may have utility for preclinical and clinical applications where extended replication is required.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 59-70 |
| Number of pages | 12 |
| Journal | Human gene therapy methods |
| Volume | 27 |
| Issue number | 2 |
| DOIs | |
| State | Published - Apr 1 2016 |
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ASJC Scopus subject areas
- Molecular Medicine
- Applied Microbiology and Biotechnology
- Genetics(clinical)
- Genetics
- Pharmacology
Cite this
Extensive Replication of a Retroviral Replicating Vector Can Expand the A Bulge in the Encephalomyocarditis Virus Internal Ribosome Entry Site and Change Translation Efficiency of the Downstream Transgene. / Lin, Amy H.; Liu, Yanzheng; Burrascano, Cynthia; Cunanan, Kathrina; Logg, Christopher R.; Robbins, Joan M.; Kasahara, Noriyuki; Gruber, Harry; Ibañez, Carlos; Jolly, Douglas J.
In: Human gene therapy methods, Vol. 27, No. 2, 01.04.2016, p. 59-70.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Extensive Replication of a Retroviral Replicating Vector Can Expand the A Bulge in the Encephalomyocarditis Virus Internal Ribosome Entry Site and Change Translation Efficiency of the Downstream Transgene
AU - Lin, Amy H.
AU - Liu, Yanzheng
AU - Burrascano, Cynthia
AU - Cunanan, Kathrina
AU - Logg, Christopher R.
AU - Robbins, Joan M.
AU - Kasahara, Noriyuki
AU - Gruber, Harry
AU - Ibañez, Carlos
AU - Jolly, Douglas J.
PY - 2016/4/1
Y1 - 2016/4/1
N2 - We have developed retroviral replicating vectors (RRV) derived from Moloney murine gammaretrovirus with an amphotropic envelope and an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-transgene cassette downstream of the env gene. During long-term (180 days) replication of the vector in animals, a bulge of 7 adenosine residues (A's) in the J-K bifurcation domain sometimes serially added A's. Therefore, vectors with 4-12 A's in the A bulge in the J-K bifurcation domain were generated, and the impact of the variants on transgene protein expression, vector stability, and IRES sequence upon multiple infection cycles was assessed in RRV encoding yeast-derived cytosine deaminase and green fluorescent protein in vitro. For transgene protein expression, after multiple infection cycles, RRV-IRES with 5-7 A's gave roughly comparable levels, 4 and 8 A's were within about 4-5-fold of the 6 A's, whereas 10 and 12 A's were marked lower. In terms of stability, after 10 infection cycles, expansion of A's appeared to be a more frequent event affecting transgene protein expression than viral genome deletions or rearrangement: 4 and 5 A's appeared completely stable; 6, 7, and particularly 8 A's showed some level of expansion in the A bulge; 10 and 12 A's underwent both expansion and transgene deletion. The strong relative translational activity of the 5 A's in the EMCV IRES has not been reported previously. The 5A RRV-IRES may have utility for preclinical and clinical applications where extended replication is required.
AB - We have developed retroviral replicating vectors (RRV) derived from Moloney murine gammaretrovirus with an amphotropic envelope and an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-transgene cassette downstream of the env gene. During long-term (180 days) replication of the vector in animals, a bulge of 7 adenosine residues (A's) in the J-K bifurcation domain sometimes serially added A's. Therefore, vectors with 4-12 A's in the A bulge in the J-K bifurcation domain were generated, and the impact of the variants on transgene protein expression, vector stability, and IRES sequence upon multiple infection cycles was assessed in RRV encoding yeast-derived cytosine deaminase and green fluorescent protein in vitro. For transgene protein expression, after multiple infection cycles, RRV-IRES with 5-7 A's gave roughly comparable levels, 4 and 8 A's were within about 4-5-fold of the 6 A's, whereas 10 and 12 A's were marked lower. In terms of stability, after 10 infection cycles, expansion of A's appeared to be a more frequent event affecting transgene protein expression than viral genome deletions or rearrangement: 4 and 5 A's appeared completely stable; 6, 7, and particularly 8 A's showed some level of expansion in the A bulge; 10 and 12 A's underwent both expansion and transgene deletion. The strong relative translational activity of the 5 A's in the EMCV IRES has not been reported previously. The 5A RRV-IRES may have utility for preclinical and clinical applications where extended replication is required.
UR - http://www.scopus.com/inward/record.url?scp=84964861725&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84964861725&partnerID=8YFLogxK
U2 - 10.1089/hgtb.2015.131
DO - 10.1089/hgtb.2015.131
M3 - Article
C2 - 26918465
AN - SCOPUS:84964861725
VL - 27
SP - 59
EP - 70
JO - Human gene therapy methods
JF - Human gene therapy methods
SN - 1946-6536
IS - 2
ER -