chloride versus Qiagen preps 62. Finally, our experience with the Qiagen columns in the past has been 
superior to other resin systems or CsCl with regard to reproducibility. Of course, this proposed approach 
will ultimately be discussed with both the RAC and FDA and we stand ready to accept their 
recommendation on this point. 
The sealed vials will be stored in a dedicated storage freezer/refrigerator in the HGT 
Laboratory. Approximately, 1 hour prior to delivery plasmid solution will be aliquoted in individual sterile 
vials mixed under sterile conditions as follows: 0.1 ml of plasmid DNA (5mg/ml) is diluted to 250pl with 
saline at room temperature. Approximately 15 minutes before delivery, vials of plasmid will be brought to 
the Special Procedures Laboratory and, under sterile conditions, pipette-applied to the hydrogel polymer 
coating of the inflated angioplasty balloon, following which the balloon will be allowed to air dry. Based 
on the formula for the surface area of a cylinder (27trh + 27tr 2 ) we have calculated that the maximum 
amount of plasmid DNA which we can apply to the hydrogel polymer coating a balloon 5 mm. in diameter 
and 20 mm. in length is 1.07 mg. Given that this is 2.5X the dose we used in a 3.5 kg rabbit, we believe 
that 1.0 mg represents a reasonable increase in plasmid DNA for the leg of a 70 kg patient. The balloon is 
then deflated, enclosed within a sterile sheath, and directed under fluoroscopic guidance to the site of gene 
transfer. 
III.D. Plasmid Monitoring 
III.D.l. Re-sequencing. Because we recognize that sequence changes may occur at any 
time and are not ruled out by testing at a single point in time, we will routinely re-sequence the VEGF- 
coding region of aliquots from each batch of plasmid DNA generated for human application in the current 
protocol. 
III.D. 2. Single DNA Species. Plasmid batches will be routinely grown from single 
colonies of transformed E.coli. To confirm that the plasmid is a single DNA species, aliquots of plasmid 
will be removed and digested with appropriate restriction enzymes to confirm that the pattern of generated 
fragments conform to the predicted digestion pattern. 
III.D. 3. Integration. The pre-clinical in vitro and in vivo experiments (vide infra) have 
been performed using nonlinearized DNA. As indicated by previous investigators 60 ? the use of uncut 
plasmid DNA reduces the likelihood of long-term over-expression (a feature that would not appear to 
constitute a significant liability for therapeutic angiogenesis). Moreover, we would expect that the 
likelihood of stable integration of naked DNA into the host genome, including inadvertent transfection of 
germ line cells, would be statistically quite low; and previous investigators 43,63 have shown that directly 
injected naked DNA exists in the unintegrated form. Nevertheless, we have monitored for this 
experimentally by probing for pUC sequences in isolated genomic DNA by Southern blot analysis and 
found no evidence of DNA integration. We therefore anticipate that DNA will not be stably integrated into 
the host cells. 
III.D. 4. Protein Contamination. To determine apparent protein contamination, aliquots 
of plasmid DNA prepared for patient administration will be evaluated by determining the ultraviolet 
absorbance at wavelengths of 260 and 280 nm on a Hewlett-Packard 8452A diode array 
Spectrophotometer. Protein content will be further evaluated using the BioRad protein assay. Prepared 
lots from which aliquots indicate significant protein contamination will be discarded, based on guidelines 
suggested to us by the FDA. 
The Limulus Amebocyte Lysate (LAL) gel-clot method will be used to quantitatively assess bacterial 
endotoxin levels. This testing will be performed by Microbiologic Associates, Inc., an independent 
laboratory that has provided contact research for a number of approved gene therapy protocols. Again, 
pepared lots of plasmid will be employed only if they meet FDA guidelines regarding endotoxin levels. 
To ensure that ethanol precipitation has been sufficient to eliminate contamination with adventitious 
agents such as bacteria, fungi, or mycoplasma, representative aliquots of each batch of plasmid DNA, 
including sterile vials selected at random from among those to be used for catheter delivery, will be 
evaluated by our Clinical Microbiology Laboratory under the direction of Dr. Jeff Griffiths, Medical 
Director of the Microbiology Laboratory, using standard microbiological techniques to exclude 
contamination by adventitious agents. Each batch of plasmid DNA solution will be tested for contaminants 
and toxicity and used according to previously established FDA guidelines. 
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Recombinant DNA Research, Volume 20 
