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.
N1 - Funding Information:
We thank Nicholas A. Boyle, PhD, MBA, and Alessandro Lobbia, PhD, for critical reading of the manuscript. The original RRV system was developed with support from NIH Grants U01-NS59821 and R01-CA105171 to N.K. The work was also supported by Accelerate Brain Cancer Cure, American Brain Tumor Association, Musella Foundation, National Brain Tumor Society, Voices Against Brain Cancer, and the Department of Health and Human Services US.
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 -