Sequences in the coding region of clotting factor VIII act as dominant inhibitors of RNA accumulation and protein production.
A variety of retroviral vectors for transduction and expression of clotting factor VIII (FVIII) were constructed by using truncated forms of a FVIII cDNA lacking part or all of the nonessential B-domain sequences. Both the titer of virus and FVIII protein production from the vectors was about 2 orders of magnitude lower than the virus titer and protein production from identical retroviral vectors containing other cDNAs, including clotting factor IX. These decreases could be entirely explained by an observed 100-fold lower accumulation of vector RNAs containing the FVIII sequences in comparison to vectors containing other cDNA sequences. Deletion analysis of one of the FVIII vectors demonstrated that diffuse sequences within the FVIII coding region had a deleterious effect upon vector titer and RNA accumulation. One inhibitory signal could be localized to a 1.2-kb stretch of DNA, but further localization was not possible as additional size reduction abolished the activity. These results indicate that expression of FVIII is regulated by signals within FVIII coding sequence that result in decreased RNA accumulation and FVIII protein production. Alteration of these inhibitory signals to permit high-level FVIII production may be difficult due to the wide distribution of these signals within the coding region of the protein.
Lynch
C M
CM
Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
Israel
D I
DI
Kaufman
R J
RJ
Miller
A D
AD
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D004247:DNA; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007438:Introns; D051379:Mice; D012367:RNA, Viral / Q000096:biosynthesis*; D012190:Retroviridae / Q000235:genetics*; D017384:Sequence Deletion; D012867:Skin / Q000166:cytology; D014161:Transduction, Genetic
D012367:RNA, Viral; D005169:Factor VIII; D004247:DNA