M.J. Welsh and A.E. Smith, RAC Application 
B.3.c. What treated cells (or vector/DNA combination) will be given to patients? How will 
the treated cells be administered? What volume of cells will be used? Will there be 
single or multiple treatments? If so, over what period of time? 
The Ad2/CFTR-1 gene construct will be prepared at Genzyme, as described above. 
Appropriate aliquots of purified production lots of virus will be diluted to 50 - 200 fil in 
Tris buffered saline. There will be a single, 30 min. application of virus to each nares. The 
virus will be applied to each nares using a plexiglass applicator that will limit the area of 
application to a defined 0.5 cm 2 area and will allow the majority of virus to be removed 
and the area to be rinsed with virus-free solution after application. 
B.3.d. How will it be determined that new gene sequences have been inserted into the 
patient's cells and if these sequences are being expressed? Are these cells limited to 
the intended target cell populations? How sensitive are these analyses? 
As described above in response to point B.3., we will assess the production of mRNA by 
RT-PCR and in situ hybridization, we will assess the production of protein by 
immunocytochemistry, and we will assess the correction of the CF electrolyte transport 
defect by measurement of the transepithelial electrical potential difference across the nasal 
epithelium. We will examine the treated areas and those immediately adjacent. 
B.3.e. What studies will be done to assess the presence and effects of the contaminants? 
The Ad2/CFTR-1 will be purified and tested for the presence of biological and protein 
contaminants. The most likely contaminant in the preparation is BSA from the serum used 
to grow the virus. If detected in the vector preparation, we will examine patient serum for 
the presence of antibodies to BSA in a Patient Immune Response (PIR) assay. Genzyme 
routinely develops similar assays to measure antibody responses to other therapeutic 
proteins. However since it is likely that patients will already have antibodies to BSA this 
test is likely to be of limited value. 
B.3.f. What are the clinical endpoints of the study? Are there objective and quantitative 
measurements to assess the natural history of the disease? Will such measurements 
be used in following patients? How will patients be monitored to assess specific effects 
of the treatment on the disease? What is the sensitivity of the analyses? How 
frequently will follow-up studies be done? How long will patient follow-up continue? 
The biochemical endpoints and methods for assessment are described above in response to 
Point B.3. Because virus administration is limited to a small area of epithelium in the nose, 
we expect no effect on the clinical disease. Nevertheless, we will assess the patient's 
clinical status as described in Point B.3. 
B.3.g. What are the major beneficial and adverse effects of treatment that you anticipate? 
What measures will be taken in an attempt to control or reverse these adverse effects 
if they occur? Compare the probability and magnitude of potential adverse effects on 
patients with the probability and magnitude of deleterious consequences from the 
disease if recombinant DNA transfer is not used. 
The major beneficial effect of this study will be the acquisition of knowledge that will 
allow us and others to progress in the treatment of this disease. While it is unlikely that the 
present protocol will be of benefit to the participants, future protocols or treatments that 
may emerge as a result of this work could be of significant benefit to patients with CF. 
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Recombinant DNA Research, Volume 16 
