Volume 12, Issue 2, Pages 264-273 (August 2005) Transient siRNA-Mediated Attenuation of Liver Expression from an α-Galactosidase a Plasmid Reduces Subsequent Humoral Immune Responses to the Transgene Product in Mice Qiuming Chu, Macy Joseph, Malgorzata Przybylska, Nelson S. Yew, Ronald K. Scheule Molecular Therapy Volume 12, Issue 2, Pages 264-273 (August 2005) DOI: 10.1016/j.ymthe.2005.04.007 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions
Fig. 1 Coadministration of an αgal-siRNA specifically and transiently suppresses initial αgal expression from a CMV-driven vector and attenuates antibodies to αgal in BALB/c mice. (A) A CMV-driven pDNA bearing human αgal (pCMV; 10 μg) was hydrodynamically injected either alone or together with 10 μg of an siRNA against CAT or against αgal. Serum αgal expression was assayed over time by ELISA as described under Materials and Methods. Symbols indicate means ± SD; N = 14 animals/group. (B) A CMV-driven pDNA bearing pCMV-sSEAP (pSEAP; 10 μg) was hydrodynamically injected either alone or together with 10 μg of an siRNA against αgal. Serum SEAP expression was assayed over time as described under Materials and Methods. Symbols indicate means ± SD; N = 5 animals per group. Serum antibodies against αgal were determined at day 112 post-administration of a pDNA containing αgal (pCMV) either alone or with an siRNA against CAT or αgal. The number of animals with a given antibody titer are shown both (C) in tabular form and (D) as a bar graph in terms of percentages of the total number of animals in the group. Titers <200 are considered negative. The antibody distributions for the pCMV alone and pCMV + CAT-siRNA groups were not statistically different. However, the distributions of these two groups (in which 30–40% of the animals were negative) were statistically different (P < 0.0001) from that of the pCMV + αgal-siRNA group (in which 72% of the animals were negative). Molecular Therapy 2005 12, 264-273DOI: (10.1016/j.ymthe.2005.04.007) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions
Fig. 2 Coadministration of an αgal-siRNA specifically and transiently suppresses initial αgal expression from a hepatocyte-restricted promoter-driven vector in BALB/c mice. (A) A hepatocyte-restricted promoter-driven pDNA bearing αgal (pHRP; 10 μg) was hydrodynamically injected either alone or together with 10 μg of an siRNA against CAT or against αgal. Serum αgal expression was assayed over time by ELISA as described under Materials and Methods. Symbols indicate means ± SD; N = 5 animals/group. (B) Serum anti-αgal antibody titers were determined at day 112 for the three groups shown in (A); the number of animals with a particular titer are presented as a percentage of the total number of animals as in Fig. 1D. Titers <200 are considered negative. Antibody distributions for the three groups are not statistically different. Molecular Therapy 2005 12, 264-273DOI: (10.1016/j.ymthe.2005.04.007) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions
Fig. 3 Coadministration of an αgal-siRNA specifically and transiently suppresses initial αgal expression from a CMV promoter-driven vector in Fabry mice. (A) A CMV-driven pDNA bearing αgal (pCMV; 10 μg) was hydrodynamically injected either alone or together with 10 μg of an siRNA against CAT or against αgal. Serum αgal levels were assayed over time by ELISA as described under Materials and Methods. Symbols indicate means ± SD; N = 11–14 animals/group. (B) Serum anti-αgal antibody titers were determined at day 112 for the three groups shown in (A); the number of animals with a particular titer are presented as a percentage of the total number of animals as in Fig. 1D. Titers <200 are considered negative. Antibody distributions for the three groups are not statistically different. Molecular Therapy 2005 12, 264-273DOI: (10.1016/j.ymthe.2005.04.007) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions
Fig. 4 Coadministration of an αgal siRNA attenuates anti-αgal antibodies and specifically and transiently suppresses initial αgal expression from an HRP-driven vector in Fabry mice. (A) An HRP-driven pDNA bearing αgal (pHRP; 10 μg) was hydrodynamically injected either alone or together with 10 μg of an siRNA against CAT or against αgal. Serum αgal levels were assayed over time by ELISA as described under Materials and Methods. Symbols indicate means ± SD; N = 15–27 animals/group. (B) Serum anti-αgal antibody titers were determined at day 112 for the three groups shown in (A); the number of animals with a particular titer are presented as a percentage of the total number of animals as in Fig. 1D. Titers <200 are considered negative. The antibody distributions of the pHRP and pHRP + CAT-siRNA groups are not statistically different (P = 0.20), but each is statistically different from the pHRP + αgal-siRNA group (P = 0.04 and 0.004, respectively). Molecular Therapy 2005 12, 264-273DOI: (10.1016/j.ymthe.2005.04.007) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions
Fig. 5 Hydrodynamic delivery of αgal plasmids is tolerizing in mice. CMV- and HRP-driven αgal plasmids (10 μg of pCMV and pHRP, respectively) were hydrodynamically delivered either alone or together with 10 μg of an siRNA against either CAT or αgal in either (A) BALB/c or (B) Fabry mice. Serum anti-αgal antibody titers were determined at 16 weeks postadministration (W16). The animals were then challenged with human αgal protein in CFA (see Materials and Methods) and their serum anti-αgal antibody titers redetermined after an additional 3 weeks (W19). Titers of individual animals are shown before (W16) and after (W19) the αgal protein challenge. Naive groups of BALB/c and Fabry mice, i.e., not given pDNA or siRNA, were challenged with the αgal protein in CFA and their titers are shown in the leftmost graph in the upper row. The number of animals/group is shown for each individual graph. Molecular Therapy 2005 12, 264-273DOI: (10.1016/j.ymthe.2005.04.007) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions
Molecular Therapy 2005 12, 264-273DOI: (10.1016/j.ymthe.2005.04.007) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions
Molecular Therapy 2005 12, 264-273DOI: (10.1016/j.ymthe.2005.04.007) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions
Molecular Therapy 2005 12, 264-273DOI: (10.1016/j.ymthe.2005.04.007) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions
Molecular Therapy 2005 12, 264-273DOI: (10.1016/j.ymthe.2005.04.007) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions