Stabilization of transgene expression by incorporation of E3 region genes into an adenoviral factor IX vector and by transient anti-CD4 treatment of the host.
Complex interactions between replication deficient adenoviral vectors (Ad5) and the immune system of the host influence the stability of transgenes in vivo. Vector-infected cells are attacked by diverse cellular immune mechanisms which limit transgene persistence. On the other hand, the products of several E3 region genes of wild-type adenovirus can suppress host immune reactions by interference with the expression of MHC class I molecules and by other mechanisms. We have developed an adenoviral vector for human factor IX (Ad5E3+FIX) which carries the E3 region of wild-type adenovirus, and an E3-deleted vector of otherwise similar structure (ad5 delta E3FIX). Intravenous injection of Ad5E3+FIX in C57BI/6 mice resulted in expression levels up to 6000 ng/ml of recombinant human factor IX in the mouse plasma and in enhanced transgene stability as compared with the vector Ad5 delta E3FIX. Whereas expression from E3-deleted vectors was essentially turned off 8 weeks after the gene transfer, the vector Ad5E3+FIX3+FIX supported transgene expression with therapeutic levels of human factor IX in the mouse plasma for > 4 months. The enhanced stability of the vector Ad5E3+FIX appears to be a consequence of efficient E3 region-mediated suppression of the host's antivector immune response. As an additional approach to improving transgene stability the influence of transient CD4+ T cell depletion of the host was investigated. CD4+ cytotoxic T lymphocytes contribute to the clearance of adenovirus-infected cells and play a pivotal role in the activation of CD8+ cytotoxic T cells and as helper T cells in the formation of human adenovirus neutralizing antibodies (HANA). Transient anti-CD4 treatment of the host limited to the time of vector injection resulted in a significant prolongation of transgene expression from the factor IX vector Ad5E3+FIX and a luciferase vector Ad5Luc. The combination of transient anti-CD4 treatment of the host and integration of a complete E3 region in an adenoviral vector resulted in markedly improved transgene stability after gene transfer to the liver (therapeutic factor IX levels for > 6 months).
Poller
W
W
Medical University Clinic, University of Würtzburg, Germany.
Schneider-Rasp
S
S
Liebert
U
U
Merklein
F
F
Thalheimer
P
P
Haack
A
A
Schwaab
R
R
Schmitt
C
C
Brackmann
H H
HH
D000256:Adenoviridae; D017503:Adenovirus E3 Proteins / Q000235:genetics*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000917:Antibody Formation; D015704:CD4 Antigens / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002478:Cells, Cultured; D017931:DNA Primers; D004730:Endothelium, Vascular; D005164:Factor IX / Q000096:biosynthesis* / Q000235:genetics*; D015870:Gene Expression; D005805:Genes, MHC Class I; D005822:Genetic Vectors; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis; D006801:Humans; D008212:Lymphocyte Depletion; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D016176:T-Lymphocyte Subsets / Q000276:immunology; D014162:Transfection; D014471:Umbilical Veins
D017503:Adenovirus E3 Proteins; D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D017931:DNA Primers; D015395:Histocompatibility Antigens Class I; D011994:Recombinant Proteins; D005164:Factor IX