Outside of these regions there was >98.2% sequence homology between our determined 
sequence and Genentech's predicted sequence. (The number would probably be above 99%, but one of 
the sequencing primers provided poorer quality sequence information that the other 23 primers.) 
Sequence of the VEGF coding region was determined on both' strands and it was in 100% 
agreement with the predicted sequence. 
An annotated sequence showing the primer map and the approximate extents of sequence 
information derived from each is included in the Appendix. There is extensive overlap in our sequence 
information from this set of primers which thereby increased the accuracy of our determination. Also 
shown are the key features of the phVEGFi 65 vector using the numbering system from the predicted 
Genentech sequence. 
III.B. Vectors 
No vectors will be used to deliver the above-described plasmid. Instead, the plasmid will be 
applied to the hydrogel polymer coating of a standard angioplasty balloon, as was the case in our pre- 
clinical animal testing (please see accompanying manuscripts "Arterial Gene Transfer Using Pure DNA 
Applied Directly to a Hydrogel-Coated Aiigioplasty Balloon," and "Therapeutic Angiogenesis Following 
Arterial Gene Transfer of Vascular Endothelial Growth Factor in a Rabbit Model of Hindlimb Ischemia," 
both of which are included in the Appendix to this proposal). The catheter and method utilized for this 
delivery mode are also described in detail below. 
III.C. Plasmid Preparation 
The material to be administered to the patient will be limited to the plasmid DNA described 
above. As stated, no adjunctive delivery vectors will be employed. The plasmid DNA will be prepared 
shortly prior to gene transfer in the Medical Center's Human Gene Therapy (HGT) Laboratory; this 
laboratory, to be located in the Medical Center, will be used exclusively for growing and preparing the 
plasmid to be used in this protocol. It will be fully equipped with a 37° C incubator, Eppendorf 
microcentrifuge, hydraulic thermostat-controlled orbit environment shaker, Sorvall Super T-21 
microcomputer-controlled tabletop Superspeed refrigerated centrifuge -20° safety refrigerator/freezer, 
lyophilizer, and vacuum pump. These instruments - including the rotor to be used in the Sorvall centrifuge 
- will thus be exposed exclusively to the plasmid DNA intended for human administration in this protocol. 
Certain pecautions to ensure the purity of the final product will be adopted 59 These include, but are not 
limited to, use of newly acquired glassware; use of plasticware which is purchased sterile and used once; 
use of aseptic conditions; and maintenance of records throughout the production procedure detailing 
completion of each step and results of all analyses. Risks to laboratory workers will be minimized by use 
of double gloving, aerosol-free conditions, and possible use of a fume hood. 
DNA will be prepared from cultures of phVEGFi 65 -transformed E.coli by the Qiagen method 
according to the directions of the manufacturer (Qiagen, Inc., Chatsworth, CA). Briefly, cultures will be 
grown in 500 ml of LB medium with 100 fig/ml of ampicillin. Cells will be harvested at a density of 1.0 
to 1.5 (A600 units/ml) and prepared with a Qiagen-tip 2500. Following elution from the Qiagen column, 
the plasmid will be extracted with Triton X-114 to remove endotoxin. Following this procedure, the 
plasmid DNA will be ethanol precipitated, dried on a Speed Vac, and stored in vials, reconstituted in sterile 
saline. We anticipate yields of 1.5 to 2.5 mg of plasmid DNA. We have selected this method of plasmid 
preparation in lieu of a CsCl gradient for several reasons. First, previous deliberations of the RAC have 
suggested that ethidium bromide not be used for human DNA preparation "...because of safety 
concerns..."; approval was made by the RAC, instead, for a column method of separation 60 Second, in 
preliminary phone conversations with the FDA, it was explicitly indicated to us that "...investigators are 
being advised against the use of CsCl..." for the same reason. Third, C-aplen et al have previously 
reported that plasmid DNA prepared for human administration using Qiagen preps resulted in "...DNA 
qualities comparable to separation twice through caesium chloride gradients without exposure of the DNA 
to toxic agents such as ethidium bromide or phenol/chloroform." 59 in this regard, we have performed 
preliminary tests on phVEGFi 65 prepared by CsCl, Qiagen, and Promega ("Wizard") protocols. The 
260/280 ratios were 1.6, 1.8, and 1.8 (CsCl, Qiagen, and Promega, respectively); protein content was 
negligible or non-detectable using either of these purification protocols as determined by the Bradford dye- 
protein assay from Bio-Rad. Sequence determinations of each plamid disclosed no differences in the 
VEGF sequence among any of the three preps. Fourth, there is in fact published data demonstrating 
improved efficiency with Qiagen plasmids versus those prepared from cesium chloride 61, a finding that 
was attributed to a larger portion of fragmented DNA visualized by electron microscopy in the cesium 
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