Characterization of Anti-CCR5 Ribozyme-Transduced CD34+ Hematopoietic Progenitor Cells in Vitro and in a SCID-hu Mouse Model in Vivo

Abstract

The cellular entry of HIV is mediated by the specific interaction of viral envelope glycoproteins with the cell-surface marker CD4 and a chemokine receptor (CCR5 or CXCR4). Individuals with a 32-base-pair (bp) deletion in the CCR5 coding region, which results in a truncated peptide, show resistance to HIV-1 infection. This suggests that the downregulation of CCR5 expression on target cells may prevent HIV infection. Therefore, ribozymes that inhibit the CCR5 expression offer a novel approach for anti-HIV gene therapy. To assess the effect of an anti-CCR5 ribozyme (R5Rbz) on macrophage differentiation, CD34+ hematopoietic progenitor cells were transduced with a retroviral vector carrying RSRbz and allowed to differentiate in the presence of appropriate cytokines. R5Rbz-transduced CD34+ cells differentiated normally into mature macrophages that carried CD14 and CD4 surface markers, expressed the anti-CCR5 ribozyme, and showed significant resistance to viral infection upon challenge with the HIV-1 BaL strain. Using an in vivo thymopoiesis model, the effect of RSRbz on stem cell differentiation into thymocytes was evaluated by reconstituting SCID-hu mice thymic grafts with ribozyme-transduced CD34+ cells. FACS analysis of cell biopsies at 4 and 6 weeks postengraftment for HLA, CD4, and CD8 markers showed comparable levels of reconstitution and similar percentages of subpopulations of thymocytes between grafts receiving R5Rbz-transduced and control CD34+ cells. RT-PCR assays demonstrated the expression of the anti-CCR5 ribozyme in CD4+, CD8+, and CD4+/CD8+ thymocyte subsets derived from RSRbz-transduced CD34+ cells. These results indicate that anti-CCR5 ribozyme can be introduced into hematopoietic stem cells without adverse effects on their subsequent lineage-specific differentiation and maturation. The expression of anti-CCR5 ribozymes in HIV-1 target cells offers a novel gene therapy strategy to control HIV infection.

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

“…40,41,[51][52][53] An alternative therapeutic method for disrupting HIV entry is the use of gene delivery to provide a genetic means to alter expression or block the function of chemokine receptors, for example, intrabodies, ribozymes, intrakines, zinc-fingers or RNAi. [13][14][15][16]54 Our approach to disrupting CCR5 expression on the cell surface relies on cellular expression of a humanized intrabody targeting CCR5. 16 The CCR5 intrabody recog- …”

“…[13][14][15][16] However, these viral entry disruption strategies have shown varying levels of protection when the viral challenge dose is increased, thus providing the caution that therapeutic efficacy may be breached by high viral loads. [17][18][19] Recent studies have indicated that HIV-1 may be more effectively transmitted during antigen presentation between CD4 + T cells and HIV-1 exposed or infected dendritic cells (DCs), termed an infectious immunological synapse, thus providing a means for increased local viral levels.…”

Inhibition of HIV-1 replication by simian restriction factors, TRIM5α and APOBEC3G

“…So far, intracellular antibodies against viral proteins, 1 anti-human immunodeficiency virus Type 1 (HIV-1) ribozymes, [2][3][4][5] RNA decoys, 6 transdominant viral genes, 7,8 antisense and short interfering RNA (siRNA) against viral and essential cellular proteins for HIV-1 replication [9][10][11][12][13][14] have been used as therapeutic constructs. However, the inhibitory effects on HIV-1 replication by anti-HIV-1 ribozymes, decoys or antisense constructs are generally mild.…”

Complete knockdown of CCR5 by lentiviral vector-expressed siRNAs and protection of transgenic macrophages against HIV-1 infection

“…So far, a number of promising intracellular immunization strategies have been employed using different molecules that act by a variety of mechanisms such as ribozymes, antisense constructs and siRNAs. [3][4][5][6][7][8] Of these, siRNA-based constructs have received considerable attention due to their target specificity and improved potency. siRNAs use the endogenous RNAi pathway for target recognition and gene silencing.…”

“…[4][5][6][7]20,[22][23][24][25][26][27]29 Both synthetic siRNAs delivered via transfection and endogenously expressed siRNAs via viral vectors have been employed. Although marked downregulation of CCR5 expression has been observed, complete silencing of coreceptor expression has yet to be achieved.…”