1. Introduction: Red blood cells can theoretically be used as a platform for therapeutic proteins because they are both long lived and naturally occur in circulation. The inherent advantages of erythrocytes lend themselves to mimicking the cell surface of the helper T-lymphocyte, which is the natural target of the HIV-1 virus. Modified blood cells could theoretically act as a decoy target for HIV. As a decoy, these cells would be capable of binding to and internalizing the retrovirus, effectively neutralizing viruses it contacts while in the blood stream. Through the addition of the CD4, CXCR4 and CCR5 proteins, erythrocytes will share many characteristic surface markers of the helper T-lymphocyte. Only the cell surface marker CD4 has been added in previous research to test this therapeutic strategy. Advances in understanding may allow the addition of the CXCR4 and CCR5 proteins, to more effectively mimic the membrane of the T-lymphocyte. A possible method for the insertion of these proteins is gene therapy using a retrovirus that targets the CD34+ stem cells that give rise to red blood cells. The proposed research develops a test of new methods of stem cell transformation can yield erythrocytes that more closely resemble helper T–lymphocytes and are subsequently more effective in neutralizing various strains of HIV-1 Will a lentiviral vector be able to insert full length copies of CD4, CXCR4 and CCR5 genes into CD34+ stem cells? Will the addition of CD4, CXCR4 or CCR5 cells surface markers interfere with differentiation into erythrocytes? Will the addition of the proteins CXCR4 and CCR5 significantly increase the red blood cells ability to absorb different varieties of HIV-1? Primary Research questions: Full length insertion of CD4 into erythrocytes was first accomplished via electroinsertion, which will fuse with HIV-1 in vitro (1) (Figure 3.) The chemokine receptors CCR5 and CXCR4 were discovered to mediate entry of HIV-1 into CD4+ cells after research into CD4- RBC’s was abandoned (2,3) It was found that CD4-negative cells (like erythrocytes) efficiently bind HIV-1 via cell surface heparins, and transfer these viruses to T-cells. (4) CXCR4 and CCR5 using strains of HIV-1 are found to exhibit differential pathogenesis in vivo (5). Efficient lentiviral vectors for gene therapy were developed and targeted to the CD34+ stem cell progenitors of erythrocytes (6,7). Review of literature: Experimental Design: (1) Purify CD34+cells from Mobilized Peripheral Blood. (2) Insert treatments via a lentiviral vector. Treatments: 1)Control, erythrocytes derived in vivo, no additions 2) Control, erythrocytes derived in vitro, no additions 3) Add CD4 Via lentivirus 4) Add CD4 and CXCR4 Via lentivirus 5) Add CD4 and CCR5 Via lentivirus 6) Add CD4, CXCR4 and CCR5 via lentivirus (3) Differentiate by placing on semisolid matrix of methylcellulose. (4) Supplement with IL-3 SCF, EPO, and GM-SCF. (5) Grow and separate mature erythrocytes. Confirm Treatments with Flow Cytometry. (6) Inject treatments with isolated X4 and R5 strains of HIV-1 that have been fluorescently labeled. (7)Place samples under a spectroflorometer to assay the amount of florescence dequenching. This will indicate how much HIV has been absorbed by each cell. Figure 1. Depiction of the HIV-1 lifecycle showing the importance of CXCR4 and CCR5 to initial binding and fusion. ( Image taken from www.thebody/nami/cycle.html) Figure 3. Results from pervious research indicating HIV-1 is absorbed into CD4 baring erythrocytes (1). Expected Results: Literature Cited: (1) Ziera, Micheal. 1991 “Full-length Cd4 electroinserted in the erythrocyte membrane: as a long lived inhibitor of infection by human immunodeficiency virus.” Proc Natl Acad Sci U. S. A. May 15:88: (see p4409-4413) (2) Deng, H. 1996 “Identification of a major co-receptor for primary isolates of HIV-1” Nature. 1996 June 20: 381 (6584): 661-6. (3) Dragic, T. “HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.” Nature 312: 667-673. (4) Gene, G. “CD4-Negative Cells Bind Human Immunodeficiency Virus Type 1 and Efficiently Transfer Virus to T Cells.” Journal of Virology, September 2000, p. 8550-8557, vol. 74, NO. 18 (5) Berkowitz, Robert D. “CCR5 and CXCR4- Utilizing Strains of Human Immunodeficiency Virus Type 1 Exhibit Differential Tropism and Pathogenesis In Vivo,” Journal of Virology, December 1998, p. 10108-10117, Vol 72, No. 12 (6) Geronimi, Fabian. “Highly Efficient Lentiviral Gene Transfer in CD34+ and Cd34+/38-/lin- Cells from Mobilized Peripheral Blood after Cytokine Prestimulation. (7) Grande, Alexis. “Transcriptional Targeting of Retroviral Vectors to the Transuded Hematopoetic Stem Cells” Blood, Vol (3 No.10 (May 15), 1999: pp3276-3285. Hematopoiesis : Figure 2. Overview of hematopoiesis: In order to Introduce specific cell markers to the human erythrocyte, lentiviruses will be employed in order to Insert the genetic information into stem cells. This must be done because mature erythrocytes lack nuclei, and are therefore incapable of producing the Protein. Figure 5. Expected results: Each treatment group is tested for florescence dequenching represented in this figure. The treated erythrocytes should show both increased uptake of the virus as well as specificity for viruses that bind the markers inserted into them. Fig 4. A. A typical wild type retrovirus: The components for the virus can be removed from the virus in a producer cell line to produce the retroviral vector shown in B. which can then act as a vector for the gene of interest without further viral replication. (Source www.gmu.edu/.../ 385-Ch10b- GeneTherapy/img019.jpg)