Volume 5, Issue 6, Pages 731-738 (June 2002) CpG-Depleted Plasmid DNA Vectors with Enhanced Safety and Long-Term Gene Expression in Vivo  Nelson S. Yew, Hongmei Zhao, Malgorzata Przybylska, I-Huan Wu, Jennifer D. Tousignant, Ronald K. Scheule, Seng H. Cheng  Molecular Therapy  Volume 5, Issue 6, Pages 731-738 (June 2002) DOI: 10.1006/mthe.2002.0598 Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 1 Diagram of CpG-depleted plasmid DNA vectors and transgenes. CMV, cytomegalovirus immediateearly gene enhancer/promoter; HI, hybrid intron; CAT, chloramphenicol acetyltransferase cDNA; pA, bovine growth hormone polyadenylation signal; ORI, replication origin region; KAN, kanamycin resistance gene; sHFIX, synthetic human factor IX cDNA. Each symbol represents five CpG motifs. Molecular Therapy 2002 5, 731-738DOI: (10.1006/mthe.2002.0598) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 2 Comparison of the toxicity induced by unmodified and CpG-depleted pDNA vectors after systemic delivery. BALB/c mice were injected i.v. with 100 μl of cationic lipid GL-62 complexed with pGZB-sCAT, pGZA-sCAT, or pCF1-CAT (0.5:0.5 mM ratio of GL-62:pDNA; 16.5 μg of pDNA). Blood was collected and lungs were harvested 24 hours after injection. Cell differentials (leukocytes, platelets) and liver enzymes (AST, ALT) were measured from the blood of five of eight mice per group. Cytokines (IL-12 and IFN-γ) were assayed from the remaining three mice per group. The levels of CAT in the lung were measured from all mice (n = 8 mice per plasmid). Water was used as the vehicle control. Data are expressed as mean ± SEM. Molecular Therapy 2002 5, 731-738DOI: (10.1006/mthe.2002.0598) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 3 Comparison of the toxicity induced by unmodified and CpG-depleted pDNA vectors at a higher dose of complex. BALB/c mice were injected i.v. with 100 μl of cationic lipid GL-62 complexed with pGZB-sCAT, pGZA-sCAT, or pCF1-CAT (1.0:1.0 mM ratio of GL-62:pDNA; 33.0 μg of pDNA). Blood was collected and lungs were harvested 24 hours after injection. Cell differentials (leukocytes, platelets) and liver enzymes (AST, ALT) were measured from the blood of five of eight mice per group. Cytokines (IL-12 and IFN-γ) were assayed from the remaining three mice per group. The levels of CAT in the lung were measured from all mice (n = 8 mice per plasmid). Water was used as the vehicle control. Data are expressed as mean ± SEM. Molecular Therapy 2002 5, 731-738DOI: (10.1006/mthe.2002.0598) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 4 Expression kinetics and redosing of pGZB-sCAT. (A) Persistence of CAT expression from pGZB-sCAT in the lung after i.v. delivery into BALB/c mice. BALB/c mice were injected i.v. with 100 μl of cationic lipid GL-62 complexed with pGZB-sCAT, pGZA-sCAT, or pCF1-CAT (0.5:0.5 mM ratio of GL-62:pDNA; 16.5 μg of pDNA). Lungs were harvested at different days after injection, and CAT assays were done (n = 4 mice per group). (B) Increased expression upon repeat i.v. delivery of pGZB-sCAT. BALB/c mice were injected i.v. with 100 μl of cationic lipid GL-62 complexed with either pGZB-sCAT or pCF1-CAT (0.5:0.5 mM ratio of GL-62:pDNA). Some groups of mice were injected with a second dose of either GL-62:pGZB-sCAT or GL-62:pCF1-CAT at day 14 after the initial injection (arrow). Lungs were harvested at different days after injection, and CAT assays were done (n = 4 mice per group). Data are expressed as mean ± SEM. Molecular Therapy 2002 5, 731-738DOI: (10.1006/mthe.2002.0598) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 5 Persistence of transgene expression from pGZB vectors in the liver. (A) BALB/c mice were injected rapidly through the tail vein with 5 μg of pGZB-sCAT or pCF1-CAT in 2 ml of Mirus delivery solution. Livers were harvested at 1, 14, and 28 days after injection, and CAT assays were done (n = 4 mice per time point). (B) BALB/c mice were injected rapidly through the tail vein with 10 μg of pGZB-sHFIX or pCFA-HFIX in 2 ml of Mirus delivery solution. Plasma was taken at 1, 14, 28, and 42 days after injection, and the levels of HFIX were measured (n = 5 mice per time point). Data are expressed as mean ± SEM. Molecular Therapy 2002 5, 731-738DOI: (10.1006/mthe.2002.0598) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 6 Effect of CpGs in trans or in cis on the persistence of transgene expression. (A) BALB/c mice were injected i.v. with 100 μl of cationic lipid GL-62 complexed with either pGZB-sCAT alone or a 1:1 (wt/wt) mixture of pGZB-sCAT + pOri-Kan (0.5:0.5 mM ratio of GL-62:pDNA; 16.5 μg of pDNA). Lungs were harvested at 1, 7, 14, 21, and 35 days after injection, and CAT assays were done (n = 5 mice per time point). (B) BALB/c mice were injected i.v. with 100 μl of cationic lipid GL-62 complexed with pCF1-CAT, pGZA-sCAT, pGZB-sCAT, or psCFA-CAT (0.5:0.5 mM ratio of GL-62:pDNA). Lungs were harvested at 1, 7, 14, 21, and 35 days after injection, and CAT assays were done (n = 4 mice per time point). Data are expressed as mean ± SEM. Molecular Therapy 2002 5, 731-738DOI: (10.1006/mthe.2002.0598) Copyright © 2002 American Society for Gene Therapy Terms and Conditions