Volume 12, Issue 1, Pages 77-86 (July 2005) Selective survival of peripheral blood lymphocytes in children with HIV-1 following delivery of an anti-HIV gene to bone marrow CD34+ cells  Greg M. Podsakoff, Barbara C. Engel, Denise A. Carbonaro, Chris Choi, Elzbieta M. Smogorzewska, Gerhard Bauer, David Selander, Susan Csik, Kathy Wilson, Michael R. Betts, Richard A. Koup, Gary J. Nabel, Keith Bishop, Steven King, Manfred Schmidt, Christof von Kalle, Joseph A. Church, Donald B. Kohn  Molecular Therapy  Volume 12, Issue 1, Pages 77-86 (July 2005) DOI: 10.1016/j.ymthe.2005.02.024 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 1 (A) Maps of the retroviral vectors and positions of Q-PCR primers and probes. Diagrams of the proviral genomes of the RVNL3-huM10 and RVNL3-FX retroviral vectors are shown. The Moloney murine leukemia virus 5′ and 3′ long terminal repeats (5′-LTR, 3′-LTR) and packaging sequence (Ψ) are indicated, as are the humanized huM10 gene (HuM10) and FX gene (FX) and the positions of the Q-PCR primers (A, B) and probe (P). (B) Clinical protocol design. Molecular Therapy 2005 12, 77-86DOI: (10.1016/j.ymthe.2005.02.024) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Gene marking analysis by Q-PCR in peripheral blood leukocytes at early time points after re-infusion of transduced CD34+ cells. Peripheral blood samples were obtained 5, 30, and 120 min after infusion of the CD34+ cells in subject Rev 102 and after 1 and 7 days in subjects Rev 101 and Rev 102. PBMC and granulocytes were separated and evaluated by Q-PCR for the level of the huM10 and FX genes. The values for huM10 in PBMC, huM10 in granulocytes, FX in PBMC, and FX in granulocytes are shown, as is the average for all of these values at each time point. The dashed horizontal line indicates the lower limit for detection by Q-PCR and samples without detectable vector gene sequences are plotted as not detectable (n.d.). Molecular Therapy 2005 12, 77-86DOI: (10.1016/j.ymthe.2005.02.024) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Gene marking in peripheral blood leukocytes over 2 years. Peripheral blood samples were obtained every 1–3 months after re-infusion of transduced CD34+ cells, separated into PBMC and granulocyte fractions, and assayed to measure the level of the huM10 or FX genes by Q-PCR. The values for huM10 in PBMC, huM10 in granulocytes, FX in PBMC, and FX in granulocytes are shown. The dashed horizontal line indicates the lower limit for detection by Q-PCR and samples without detectable vector gene sequences are plotted as not detectable (n.d.). Molecular Therapy 2005 12, 77-86DOI: (10.1016/j.ymthe.2005.02.024) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 4 HIV-1 viral load and the frequency of PBMC with the huM10 gene. The results of Q-PCR for the frequency of the huM10 gene in PBMC are shown over the 2 years after re-infusion of transduced CD34+ cells. HIV-1 viral loads are represented by the line graph (black boxes connected by black line) representing HIV-1 RNA genome copies/ml plasma. (A) Key clinical events indicate the period when the HAART therapy was held (HAART Held) and when a new therapeutic regimen was started (New HAART). For subject 101, marking values for samples of FACS-sorted CD4+ (4), CD8+ (8), CD4+/CD45RA+ (RA), and CD4+/CD45RO (RO) cells are indicated by boxes. (B) Gene marking and HIV-1 viral load for Rev 102. Molecular Therapy 2005 12, 77-86DOI: (10.1016/j.ymthe.2005.02.024) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 5 LAM-PCR analysis of the clonality of gene-marked cells. LAM-PCR was performed on DNA from PBMC samples collected at the indicated time points from (A) subject Rev 101 and (B) subject Rev 102. Samples were obtained at the indicated months after transduction (Months Post-TDXN). Negative controls included normal human genomic DNA (Human Genomic) and a sample of water processed through the procedure from the first linear amplification reaction (1st Linear H2O). A positive control in B consisted of a DNA sample from a clonal cell line with known retroviral insertion sequences. Molecular Therapy 2005 12, 77-86DOI: (10.1016/j.ymthe.2005.02.024) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions