very small for any one patient, and is acceptable in the context of treatment for AIDS, an 
invariably lethal illness. 
C. Replication-competent helper viruses . Minimization of the risk (discussed in detail 
above in OBJECTIVES AND BACKGROUND) of pathological replication-competent 
retroviruses arising through recombination events or contamination is designed into all 
aspects of development. As discussed above, packaging cell lines express structural and 
other genes needed for production of packaged vectors from separate constructs. The 
signal for packaging is contained only in the vector, not in the packaging cell line. 
Moreover, in previous human gene therapy trials employing this vector, no difficulties 
with helper virus have emerged (2,24,25,42,55). FDA recommended procedures for 
screening all retroviral supernatant lots for adventitious virus will be followed. Tests 
that will be done on supernatants used for patient PBMC transduction include: titre, 
sterility (see cultures below), marker rescue assay, S + L* assay for ecotropic virus, 
S + L' assay for xenotropic virus, S + L' assay for amphotropic virus, 3T3 amplification 
by standard methods, titre assay for HIV. PCR and marker rescue assays will also be done 
on patient PBLs after treatment to monitor for the unlikely event of replication- 
competent virus. 
While in vivo rescue of vector by and/or recombination with HIV are perhaps remotely 
theoretically possible, there is no experimental support or precedent for such an event 
which is extraordinarily unlikely to happen or to result in a pathogenic entity. 
D. Mutation of Germ Line . Without replication competent helper virus (screened as in 
part B above), no risk of germ line transduction exists for this kind of ex vivo gene 
transfer. 
E. Aminoglycoside Antibiotic Resistance . The NeoR gene product, neomycin 
phosphotransferase, confers cellular resistance to neomycin by phosphorylating the 3' 
hydroxyl group of the aminohexose I of neomycin and its analogs. Although amikacin can 
be inactivated by this enzyme, the more commonly used gentamicin and tobramycin do 
not contain an hydroxyl at the 3' position and are not inactivated. In any case, 
transduction of the NeoR gene will occur into only a fraction of peripheral blood cells. 
Therefore, no adverse consequences are foreseeable for the use of aminoglycoside or any 
other antibiotics in patient care. 
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Recombinant DNA Research, Volume 18 
