3023-3044 in LNL6, and to 2403-2424 in GlNa) will generate a 1401 
base pair fragment using LNL6 as a template and a 758 base pair 
fragment using G1 Na as a template. These fragments are easily 
separated on 2% agarose gels. 
The neoR gene encodes for an enzyme (NPT-II) which inactivates the 
antibiotics neomycin and Amikacin. NPT-II does not inactivate other 
aminoglycoside antibiotics (such as Gentamicin and Tobramycin) and 
many other suitable alternatives for gram negative infections are 
available for clinical use. No untoward effects have been observed of 
the expression of the neoR gene in T cells either in vitro or in mice, 
monkeys, or humans who have received T cells expressing this 
enzyme. 
5.4.3 Preparation of cells for infusion 
Cells will be prepared according to previously published methods (39- 
43). After 5-9 days in culture, the numbers and phenotypes of the 
enriched subpopulations will be determined. When the total number of 
cells reaches 3 x 10 9 - 2 x 1 0 1 1 , the CD4- and CD8-enriched fractions 
will be combined. Samples for Gram stain and microbiologic cultures 
for aerobic and anaerobic bacteria and fungus will be obtained from 
each bag prior to pooling the cells and from the pooled cells at 72 and 
48 hours and immediately prior to infusion. 
As we learn more about the separation and growth of the CD4 and 
CD8 cells, these procedures may be modified at the discretion of the 
principal investigator. Experience to date indicates that growth of 
unseparated cells from each individual patient appears to be unique in 
its kinetics and the enriched populations will probably grow differently 
from each other as well. Therefore, the ability to mix cells grown under 
different selection conditions (CD4-enriched, CD8-enriched) at the 
time of reinfusion should maximize the polyclonality in the infused cell 
population. 
5.4.4 Infusion of expanded, transduced cells into HIV-infected twin 
HIV-infected patients will be admitted to the Warren G. Magnuson 
Clinical Center inpatient wards for the first infusion. An intravenous 
catheter will be inserted into either a peripheral or, if no peripheral 
access is available, into a central vein, and approximately 3x1 0 9 - 
2x10ii expanded cells carrying the genetic marker for neomycin 
resistance will be infused over 60 minutes. Vital signs and oxygen 
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