Long-term transgene expression from plasmid DNA gene therapy vectors is negatively affected by CpG dinucleotides.
CpG-reduced, CMV-based plasmid DNA constructs encoding human alpha-galactosidase A and factor IX were injected into C57Bl/6, BALB/c, and CD1 mice using hydrodynamics-based delivery of plasmid DNA (pDNA), and gene expression was monitored for 6 months. Linearized and supercoiled pDNAs were compared for their abilities to support long-term expression and to generate immune responses to the transgene product. In all mouse strains supercoiled CpG-reduced pDNA encoding alpha-galactosidase A and factor IX generated higher and more sustained levels of circulating gene product than their supercoiled CpG-replete analogs. Linearizing supercoiled CpG-reduced pDNA did not significantly increase levels of circulating gene product beyond levels supercoiled CpG-reduced pDNA could achieve. Linearizing supercoiled CpG-replete pDNA vectors significantly increased expression compared to their supercoiled CpG-replete analogs, but the increase was short-lived or subtherapeutic. Regardless of vector, liver depot expression did not elicit significant antibody responses to human alpha-galactosidase A or factor IX. Taken together, these data suggest that a clinically acceptable hydrodynamics-based approach targeting the liver combined with CpG-reduced pDNA vectors may represent a viable option for individuals with hemophilia, a lysosomal storage disease, or other disease in which prolonged depot expression of a therapeutic protein from the liver is desirable.
Genzyme Corporation, 31 New York Avenue, Framingham, MA 01701, USA. brad.hodges@genzyme.com
Bradley L
BL
Hodges
Kristin M
KM
Taylor
Macy F
MF
Joseph
Sarah A
SA
Bourgeois
Ronald K
RK
Scheule
D000818:Animals; D018899:CpG Islands / Q000235:genetics*; D003587:Cytomegalovirus / Q000235:genetics; D019175:DNA Methylation; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism; D005164:Factor IX / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006467:Hemophilia A / Q000628:therapy; D006801:Humans; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D016464:Lysosomal Storage Diseases / Q000628:therapy; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods; D019076:Transgenes / Q000235:genetics; D000519:alpha-Galactosidase / Q000097:blood / Q000235:genetics* / Q000378:metabolism
D004278:DNA, Superhelical; D012333:RNA, Messenger; D005164:Factor IX; D000519:alpha-Galactosidase