Of the remaining patients, 52X will have localized tumors. However, 38% of these patients will 
have local failures following radiation therapy (22,900). Thus, 31,900 patients per year could 
benefit from improved local -regional therapy. Patients with unresectable obstructing NSCLC that 
is resistant to radiation therapy or who have coexisting metastases have a median survival of 6 
months or less*'. The Department of Thoracic Surgery at the University of Texas M. D. Anderson 
Cancer Center has extensive experience in the treatment of lung cancer. Over 1200 patients with 
lung cancer are seen yearly and over 200 of these patients undergo resection. 
2.2.1 Measure of disease activity 
The goal of this therapy is to halt or reverse the manifestations of the disease. The 
efficacy of therapy in this group of patients will be measured by determining length of 
patient survival, length of time the affected lobe of the lung remains aerated, and 
reduction in measurable endobronchial tumor. There is no curative therapy for this stage 
of disease and thus the outcome is predictable enough to allow for an assessment of the 
results of gene therapy. The measurements that will be used are described in Section 7.0. 
2.2.2 Anticipated effect of protocol treatment 
It is anticipated that the uptake of the retroviral constructs by proliferating NSCLC 
cells will decrease the rate of proliferation of these cells. This would increase the 
length of time the affected lung would remain expanded, prevent regrowth of the 
endobronchial tumor, and prolong the patient’s survival. 
2.2.3 Alternative therapies 
Patients with unresectable endobronchial tumor recurrence that is partially or completely 
obstructing the airway and that have failed or are unable to receive external beam 
radiotherapy will be considered for this protocol. Existing therapies for this condition 
offer only short-term palliation. Most patients have recurred despite external beam 
radiotherapy. It may be possible to insert a brachytherapy catheter and administer 
additional radiotherapy. Patients receiving this treatment have a median survival of 6 
months*'. Patients failing brachytherapy would also be eligible to receive gene therapy. 
Tumor can be removed from the airway with the laser or biopsy forceps. This can be done 
in conjunction with injection of the retroviral construct thus decreasing the volume that 
must be injected. The administration of the retroviral constructs would not preclude the 
patient from receiving other palliative therapy if the tumor progresses. 
Structure and characteristics of the biological system 
2.3.1 Downregulation of activated K-ras expression with an antisense construct 
2. 3. 1.1 Preliminary studies with plasmid DNA 
The r« p21 gene product plays an important role in the intracellular 
signaling pathway of cells. Dysregulation of this pathway because of point 
mutations in regions critical to ras function confer transforming properties to 
the mutant protein. These mutations render the p21 protein gene product 
consti tuti vely active by keeping the protein in the GTP-bound state. We began 
Investigations on alterations of expression of p21**. A homozygous mutation at 
codon 61 was detected in the H460a large-cell undifferentiated NSCLC cell line 
clone; a normal glutamine residue (CAA) was substituted by histidine (CAT) 
using hybridization with specific oligonucleotide probes. Direct PCR DNA 
sequencing confirmed this. An antisense K- ras RNA construct selectively blocked 
the production of mutant p21 so the contribution of the mutated p21 protein to 
the malignant phenotype could be studied. A recombinant plasmid clone was 
constructed using a normal wildtype 2-Kb K- ras genomic DNA segment carrying 
second and third exons with flanking intron sequences subcloned into an Apr-1- 
neo expression vector in antisense orientation. The intron sequence used has a 
low degree of homology with other r« genomic sequences; it allowed specific 
inhibition of K- ras while preserving H- ras and N- ras expression. For example, 
the 241 bases flanking exon 2 and the 240 bases flanking exon 3 have no 
significant homology between K- ras and H- ras sequences. Previous studies of 
uptake of ras antisense oligonucleotides by cancer cells resulted in cell 
death instead of regulated growth, probably because functioning p21 is 
necessary for cell viability, and the oligonucleotides unselecti vely blocked 
p21 expression”. Unselect ive blockade of oncogene expression can therefore be 
harmful to both normal and cancer cells. An additional novel feature of this 
construct was the use of a f-actin promoter that can constitutively direct 
synthesis of RNA in a human tumor cell. The 2-Kb DNA insert was stably 
Integrated into H460a cells, as shown by Southern hybridization. Northern blot 
analysis detected expression of antisense RNA. Western blot analysis showed a 
Recombinant DNA Research, Volume 19 
