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Report Summary

Safety and Germline Transmission of rAAV2-hAAT Vector After Intramuscular Injection in the Baboon


Study Summary

Overall Study Design 
This study was performed in two parts in order to assess the potential risks of humoral immune reaction and germline 
transmission and to support the use of rAAV2-CB-hAAT gene therapy In Part A of the study four baboons that were dosed 
with the rAAV2-CB-hAAT vector expressing human AAT showed no detectable serum levels of hAAT but rather had levels 
of anti-hAAT antibodies that rose gradually over several weeks after the initial injection 
Based on this information an alternative strategy was applied in Part B of the study to determine if the humoral immune 
response seen in Part A was due to the presence of the human AAT gene In Part B the baboon AAT cDNA which was 
tagged with a 10-amino acid c-myc epitope that is shared between human and baboon was used The baboon AAT cDNA 
bAAT had been cloned from baboon liver mRNA by reverse transciptase-PCR The bAAT fragment between designed 
5-EcoRI and 3-Xbal sites with a second fragment with designed 5-XbaI and 3 - Not I sites encoding 10 amino acids of 
the c- myc tag was used to replace the hAAT cDNA in rAAV2-CB-hAAT to generate vectors expressing a c-myc-bAAT 
fusion This epitope should not be immunogenic in baboons and yet it should still be identifiable with anti-c-myc antibodies 
reacting on Western blot with a serum protein of about 54 kD The rAAV construct to express this new fusion protein 
includes the rAAV-CB-AT backbone ITRs promoter pA signal and the coding sequence of the baboon AAT cDNA with the 
c-myc tag at the C-terminal end The C-terminus was used to avoid interference with the amino-terminal signal peptide 
In order to test the concept that this fusion protein could be secreted properly and then be detected with anti-c-myc 
antibodies cultures containing 5 x 105 293 cells per well were transfected with the rAAV-CB-bAAT-c-myc construct using 
calcium phosphate transfection When various dilutions of the supernatant were then analyzed by immunoblotting with an 
anti-c-myc antibody in the presence of baboon serum the fusion protein could be readily detected in the background of the 
baboon serum 
Four baboons were injected with the new vector construct rAAV2-CB-bAAT-c-myc 2 at the high dose of approximately 1 x 
1013 vg and 2 at a ten-fold lower dose No anti-AAT antibodies were observed in these animals There were no obvious 
adverse effects in these animals and only mild local injection site inflammation noted on histopathological analysis 
Real-time PCR of organs harvested at 4 months after injection indicated no significant signals for vector DNA within the 
gonads of injected animals Vector expressed hAAT could be readily detected by immunostaining of the muscle sample 
harvested at the time of sacrifice These studies indicated that it is likely that efficient transgene expression was achieved in 
these animals without signficant toxicity or significant risk of gonadal transfer 
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