Sustained human factor VIII expression in hemophilia A mice following systemic delivery of a gutless adenoviral vector.
Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicity, diminished immunogenicity, and an increased coding capacity compared with early generation vectors. Using hemophilia A, a deficiency in clotting factor VIII (FVIII), as a model disease, we generated and evaluated a gutless vector encoding human FVIII. The FVIII gutless vector grew to high titer and was reproducibly scaled-up from vector seed lots. Extensive viral DNA analyses revealed no rearrangements of the vector genome. A quantitative PCR assay demonstrated helper virus contamination levels of <2%, with the best preparation containing 0.3% helper virus. We compared the gutless vector with an E1/E2a/E3-deficient (Av3) early generation vector encoding an identical FVIII expression cassette following intravenous administration to hemophilia A mice. Gutless vector-treated mice displayed 10-fold higher FVIII expression levels that were sustained for at least 9 months. In contrast, mice treated with the Av3 vector displayed FVIII levels below the limit of sensitivity of the assay at 3 months. Assessment of hepatotoxicity by measuring the serum levels of liver enzymes demonstrated that the gutless vector was significantly less toxic than the Av3 vector at time points later than 7 days. At the highest dose used, both vectors caused a transient 10-fold increase in liver enzymes 1 day after vector administration, suggesting that this increase was caused by direct toxicity of the input capsid proteins. These data demonstrate that the gutless vector displayed increased duration and levels of FVIII expression, and was significantly less toxic than an analogous early generation vector.
Reddy
P Seshidhar
PS
Genetic Therapy, Inc. (A Novartis Company), 9 West Watkins Mill Road, Gaithersburg, MD 20878, USA.
Sakhuja
Kiran
K
Ganesh
Shanthi
S
Yang
Lijuan
L
Kayda
Dawn
D
Brann
Terrence
T
Pattison
Scott
S
Golightly
Doug
D
Idamakanti
Neeraja
N
Pinkstaff
Anne
A
Kaloss
Michele
M
Barjot
Catherine
C
Chamberlain
Jeffrey S
JS
Kaleko
Michael
M
Connelly
Sheila
S
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005169:Factor VIII / Q000008:administration & dosage / Q000235:genetics*; D005260:Female; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006467:Hemophilia A / Q000235:genetics* / Q000628:therapy; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction; D014161:Transduction, Genetic
D017931:DNA Primers; D005169:Factor VIII