Gene Therapy for CF using Canonic Liposome Mediated Gene Transfer: Phase I Trial 
plastic embedded lung tissue was prepared and studied 72 hours following DOTMA/DOPE 
administration of plasmids containing either CFTR or E. coli Lac Z. Sections from animals were 
stained with either hemotoxylin and eosin or methylene blue; these sections revealed essentially 
normal airway and airspace histology with negligible inflammation. An occasional, mild 
mononuclear cell infiltrate such as that shown in Figure 6 was observed in <5% of airways, 
and appeared to be the result of lipid/DNA administration. In addition, although the in vivo rat 
lung experiments w-ere designed to deliver lipid/DNA conjugates in a highly effective manner, 
because of the small size of the animals used, and the requirement for obtaining large cross- 
sectional tissue samples exhibiting high level expression of reporter genes or CFTR (e.g., large 
segments of efficiently transfected trachea for mounting in Ussing Chambers, see below) the in 
vivo approach used in our preclinical studies was necessarily traumatic. Two post-operative 
deaths were observed in 42 animals which underwent anesthesia and surgery and were directly 
attributable to technical aspects of the procedure (i.e., anesthesia related deaths due to difficulty 
titering appropriate levels of inhaled general anesthesia in the setting of respiratory depression 
during instillation of a very large volume [approximately l/10th total lung capacity] of 
lipid/DNA complex solution). The human protocol eliminates all such risks associated with 
anesthesia and surgical administration of lipid/DNA conjugates (i.e., no inhaled anesthesia, no 
surgical dissection, no cannulation of the trachea, and very small relative volumes of 
administered lipid DNA/conjugates, as described in Section III, below). If nasal administration 
as described in this protocol leads to appropriate correction of the CF defect, after the 
development of additional preliminary data, a modified version of our animal protocol for CF 
lung administration would be proposed. Such an approach, for example, could involve non- 
surgical, non-anesthesia-dependent administration (i.e., with very small volumes of lipid/DNA 
conjugates delivered to specific lung segments via a bronchoscope) or aerosolized administration 
of lipid/CFTR cDNA. Prior to administration of lipid/CFTR conjugates (by aerosolization, 
instillation, or any other mechanism) to the lower airways of CF lungs, therefore, a conservative 
first step in the interest of maximizing patient safety is topical administration to nasal airway 
epithelium; this approach is the one proposed in the current protocol (see study methodology, 
Section HI). As with the present protocol, all human experiments will be performed after full 
approval of the FDA regarding both safety and efficacy. 
n.A.5. Expression of Functional CFTR in Rat Trachea 
The tracheas of 19 animals treated with DOTMA/DOPE and plasmids containing human 
CFTR or nuclear targeted Lac Z were studied in Ussing Chambers for evidence of human CFTR 
chloride channel activity. Of the seven Lac Z transfected tracheas, none had a measurable 
response to forskolin, indicating very low levels of endogenous rat CFTR activity (Fig. 7 and 
8). However, in ten of twelve CFTR treated tracheas, forskolin produced a small, rapid increase 
in the 1^ (approximately 7.0+2.0^A per cm 2 )^ < .03). Bumetanide (which blocks the basolateral 
Na + -K + -2C1' cotransporter) reversed the forskolin effect (a result compatible with overexpression 
of endogenous CFTR), and produced a further inhibition of the I K in the CFTR transfected 
tracheas. In a slight modification of the above protocol, designed to enhance detection of 
endogenous CFTR, indocin was used to block prostaglandin induced basal Cl' secretory activity 
to augment the response to forskolin. In three tracheas treated with the Lac Z gene (complexed 
with either DOTMA/DOPE or DMRIE/DOPE) the mean I K increase following forskolin 
treatment was 2.11^A/cm 2 +0.71, while in 12 tracheas treated with CFTR cDNA, the mean 
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Recombinant DNA Research, Volume 18 
