4.2.3 Recent history of substance abuse unless evidence is provided of an 
ongoing therapeutic intervention (i.e. medical therapy or counseling) to 
control such abuse. 
4.2.4 Pregnancy 
4 . 3 Exclusion Criteria - Donor 
4.3.1 Untreated or inadequately treated medical condition (e.g., cardiopulmonary 
disease, acute infection) which, in the judgment of the Principal 
Investigator, precludes apheresis. 
4.3.2 Serologic positivity for either Epstein Barr virus or Cytomegalovirus if 
and only if the recipient twin tests seronegative for the corresponding virus. 
LYMPHOCYTE PROCESSING 
The anti-HIV targeting gene introduced into CD8-I- lymphocytes was generated by recombinant 
DNA technology and will produce a chimeric protein consisting of the extracellular portion of 
CD4 fused to the zeta subunit of the T cell receptor. The gene is packaged into a retroviral 
particle as a means for efficient delivery into lymphocytes. 
The retroviral particles are produced by Cell Genesys, Inc. Cells generating the recombinant 
retroviral particle are grown in culture under defined and controlled conditions. The 
supernatants are filtered and frozen. The CD8-1- lymphocytes derived from the donor twin will 
be transduced and expanded by Biotechnology Research Institute (BRI) as outlined below. 
5 . 1 Isolation and Enrichment of Peripheral Blood Lymphocytes From Donor 
Lymphapheresis (performed by the NIH Blood Bank Apheresis Unit according to 
standard procedures) will be used to obtain lymphocytes from the donor using an 
automated cell separator. Apheresis will commence at 8:00 A.M. so that processed 
cells may reach BRI before 1:00 P.M. that same day. Lymphocytes will be harvested 
and the remaining red cells and plasma returned to the donor as follows: Whole blood 
is withdrawn from one venipuncture site at a rate of 40-60 ml/min and channeled into 
the cell separator, where cellular and plasma fractions are separated by centrifugation. 
The lymphocytes are harvested into a component bag, and the red cells and plasma are 
re-infused into the donor via a second venipuncture site. Anticoagulation is achieved 
using ACD-A at a whole blood to anticoagulant ratio of 10: 1. Maximum extracorporeal 
blood volume is 300 ml (Fenwal, COBE models). Two to four hours are required to 
process 10 liters of whole blood. 
A minimum of 5 x 10^ total peripheral blood mononuclear cells (PBMC) is required. 
The pheresis procedure should be continued until the requisite yield is obtained. 
5.2 Shipping Procedure to Biotechnology Research Institute 
The lymphopacks from each apheresis will be transported to Biotechnology Research 
Institute as outlined in Appendix B. 
5.3 Cell Separation and Enrichment of CD8 T Cell Sub-populations 
Peripheral blood mononuclear cells from the lymphopacks will be fractionated into 
populations enriched for CD8-expressing cells. CD8 enrichment will be achieved by 
positive selection using an immunoaffinity-based system with a CD8-specific 
monoclonal antibody. CD4 depletion will be performed and the resulting 
Recombinant DNA Research, Volume 19 
