High-level factor VIII gene expression in vivo achieved by nonviral liver-specific gene therapy vectors.
Two liver-specific nonviral gene transfer vectors have been developed to accommodate heterologous genes. The expression cassettes contain (1) a hepatic locus control region from the apolipoprotein E (ApoE) gene (HCR), (2) a liver-specific alpha(1)-antitrypsin promoter (HP), (3) a 1.4-kb truncated factor IX first intron (I) or a synthetic minx intron (mI), (4) a multiple cloning site (MCS) for inserting cDNA sequences, and (5) a bovine growth hormone polyadenylation signal (bpA) to make pBS-HCRHPI-A or pBS-HCRHPmI-A. These vectors were first evaluated with reporter genes encoding human factor IX (hFIX) and green fluorescent protein (GFP). hFIX constructs, pBS-HCRHPI-FIXA and control pBS-HCRHP-FIXIA with the hFIX intron in its native position, produced comparable hFIX gene expression levels (0.5-5 microg/ml) 6 months after naked DNA transfer to mice, whereas the factor IX level from pBS-HCRHPmI-FIXA averaged about 50% lower. RT-PCR analysis of the mRNA indicated that introns inserted upstream from the cDNA were correctly processed and spliced. GFP expression was detected in 15-30% of the hepatocytes in pBS-HCRHPI-GFPA-treated mice. Next, a B domain-deleted human factor VIII (hFVIII) cDNA was inserted into the modified vectors. High-level hFVIII expression (up to 750 ng/ml) was achieved initially in both C57BL/6 mice and Rag2 mice. Moreover, therapeutic levels of hFVIII (20-310 ng/ml) circulated in Rag2 mice 6 months after treatment. These liver-specific gene expression cassettes can deliver a large, heterologous gene such as hFVIII cDNA to achieve high-level, persistent transgene expression after in vivo hepatic gene therapy.
D000818:Animals; D004247:DNA / Q000008:administration & dosage; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism; D005169:Factor VIII / Q000235:genetics / Q000627:therapeutic use; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007438:Introns; D008099:Liver / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D010446:Peptide Fragments / Q000235:genetics / Q000627:therapeutic use; D012333:RNA, Messenger / Q000032:analysis; D012680:Sensitivity and Specificity; D013997:Time Factors; D019076:Transgenes
C451034:B-domain-deleted factor VIII; D008164:Luminescent Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D005169:Factor VIII; D005164:Factor IX; D004247:DNA