Recombinant DNA Advisory Committee- 9/12-13/94 
that no patients participating in any of the gene therapy studies would be excluded from 
future studies. (2) General design of the vector. Dr. Samulski explained that AAV is a 
defective, nonpathogenic human virus. This virus has a strict requirement for a helper 
virus in order to go through a lytic infection. Without a helper, the virus persists in host 
cells that makes it an attractive vehicle for gene transfer. Most of the viral coding 
sequences (96%) have been deleted with only the 145 base pair inverted terminal repeats 
(1TK) remaining at both ends of the vector. The ITR serves as the promoter to initiate 
gene transcription. Dr. Samulski asked if the ITR at the opposite end of the CFTR gene 
initiates transcription of an antisense RNA The investigators wrote in a response that 
several lines of evidence indicate that this phenomenon, if it occurs, does not block CFTR 
expression. (3) Overall production process. Why is the wild-type adenovirus used as the 
helper to produce this vector? Since the investigator is using 293 cells for production of 
AAV, it is not necessary to use the wild-type adenovirus helper. An Ela-deleted 
adenovirus will be sufficient since the 293 cells already have Ela sequences. The 
investigators noted that in the present procedure, the adenovirus will be inactivated by 
heat treatment. Dr. Samulski commented that the Ela- deleted adenovirus helper offers 
another level of biosafety since the vector will not be mobilized in the presence of a wild- 
type AAV and a defective adenovirus. (4) Primate studies. The investigator detected 
positive vector sequences in the liver of a monkey after a dose of 10 11 AAV particles. 
How widespread is the presence of vector sequences in the liver and what types of liver 
cells express the AAV sequences? How does the vector spread to fiver since it is 
administered to the lung? (5) In vivo rescue of AAV-CFTR recombinant. The 
investigator presented data in monkeys showing that after administration of the vector to 
the lung, the monkey was challenged with adenovirus and wild-type AAV in the nose. 
Mobilization of the vector was localized to the nose only. Since the protocol proposes 
vector delivery to the nasal passages, does the investigator have any results pertaining to 
the spread of vector after delivery to the nose and challenge with adenovirus and AAV? 
Dr. Samulski said that the probability for all three viruses to infect the same host cells in 
order to effect a productive infection is very small. (6) Efficiency and site-preference of 
AAV-CFTR vector integration in a CF bronchial epithelial cell fine. What is the nature 
of vector integration? Does the integrated vector express its gene? What is the nature of 
the integration site? The investigator addressed these questions in writing. Overall, Dr. 
Samulski was pleased to see that the new vector system had progressed to the stage of a 
clinical trial in human subjects. It is a biologically safer type of vector since it is derived 
from a defective nonpathogenic virus. 
Review-Dr. Straus 
Dr. Straus commented on the outstanding review by Dr. Samulski. AAV is a potentially 
useful vector, and the investigator has performed excellent preclinical studies and 
presented a well written proposal. Dr. Straus pointed out his major concern regarding the 
persistence of the AAV vector. AAV is a very hardy virus that persists in the 
environment. This property does offer an easy means to inactivate other contaminating 
viruses by a heating procedure in the production process. Studies performed in the late 
1960’s indicated that children with adenovirus respiratory infections shed AAV for a long 
period of time in their stool. Dr. Straus stressed that persistent shedding of virus is the 
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Recombinant DNA Research, Volume 20 
