Successful treatment of human immunodeficiency virus infection may ultimately require targeting of hematopoietic stem cells. Here we used retroviral vectors carrying the ribozyme gene to transduce CD34+ cells from human fetal cord blood. Transduction and ribozyme expression had no apparent adverse effect on cell differentiation and/or proliferation. The macrophage-like cells, differentiated from the stem/progenitor cells in vitro, expressed the ribozyme gene and resisted infection by a macrophage tropic human immunodeficiency virus type 1. These results suggest the feasibility of stem cell gene therapy for human immunodeficiency virus-infected patients.Moloney murine leukemia virus long-terminal repeat (LTR) promoter was inefficient or completely turned off in the transduced stem cells upon maturation. In this study, we largely followed the previous protocols for enrichment of CD34+ cells and for retroviral transduction (7). We report here studies using retrovirus vectors carrying the ribozyme gene driven by two different polymerase III promoters to transduce CD34+ hematopoietic stem/progenitor cells isolated from fetal cord blood and then challenging the progeny cells with a macrophage tropic strain of HIV-1. The experimental results show the in vitro efficacy of using stem/progenitor cells as the target for gene therapy for the treatment of HIV infection.When the concept of "intracellular immunization" was first introduced as a gene therapy approach for the treatment of human immunodeficiency virus (HIV)/AIDS in 1988 (1), it was proposed that gene transfer into hematopoietic stem cells would be a preferred strategy to achieve sustained immune reconstitution. Stem cells possess both the capacity for selfrenewal and the ability to give rise to all hematopoietic cell lineages, including brain macrophage and microglial cells, which are also targets for HIV infection. Autologous or allogeneic transplantation with transduced stem cells might thus allow the permanent repopulation of all hematopoietic cell lineages of the immune system with "intracellularly immunized" cells (2). We have previously reported that a hairpin ribozyme designed to cleave HIV-1 RNA in the 5' leader sequence suppressed virus expression in HeLa cells cotransfected with proviral DNAs (3, 4). More recently, we showed that human T-cell lines (5) and primary T cells (6) transduced with retroviral vectors containing this ribozyme were resistant to challenge with diverse strains of HIV, including several uncloned clinical isolates.Transduction and expression of transgenes in bone marrow cells were hampered by the paucity of stem cells in normal bone marrow and by the inefficiency of most methods of gene transfer. Ho and colleagues (7) used immunomagnetic beads to enrich for CD34+ cells from bone marrow, mobilized peripheral blood, and fetal cord blood. A purity of 85%-95% of CD34+ cells was routinely obtained by using this procedure (7). Moreover, the isolated CD34+ cells were highly susceptible to retroviral vector-mediated gene transfer: u...
CorrectionsMICROBIOLOGY. For the article ''Identification of eIF2B␥ and eIF2␥ as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach''
We reported previously that human CD4+ T cell lines stably expressing a hairpin ribozyme targeted to the human immunodeficiency virus type 1 (HIV-1) U5 leader sequence were resistant to challenge with diverse HIV-1 viral clones and clinical isolates (Yamada et al., 1994). To simulate more closely the in vivo infection process for investigations of anti-HIV-1 ribozyme gene therapy, we developed a system to transfer this ribozyme gene into freshly isolated human peripheral blood lymphocytes (PBLs) using a murine retrovirus vector. Following transduction and G418 selection, human PBLs from multiple donors expressed the ribozyme and resisted challenge by HIV-1 viral clones and clinical isolates, while control vector-transduced PBLs remained fully permissive for HIV-1 infection. No inhibition of an HIV-2 clone lacking the target was seen in ribozyme-expressing PBLs. Ribozyme expression had no effect on viability or proliferation kinetics of the primary lymphocytes. This study is the first demonstration in primary human T cells of resistance to HIV-1 infection conferred by gene transfer. A human clinical trial is in development to test further the safety and efficacy of this ribozyme in PBLs of HIV-1-infected patients in vivo.
A small lipid-containing bacteriophage PRD1 specifies its own DNA polymerase that utilizes terminal protein as a primer for DNA synthesis. The PRD1 DNA polymerase gene has been sequenced, and its amino acid sequence has been deduced. This protein-primed DNA polymerase consists of 553 amino acid residues with a calculated molecular weight of 63,300. Thus, it appears to be the smallest DNA polymerase ever isolated from prokaryotic cells. Comparison of the PRD1 DNA polymerase sequence with other DNA polymerase sequences that have been published yielded segmental but sighificant homologies. These results strongly suggest that many pi-okaryotic and eukaryotic DNA polymerase genes, regardless of size, have evolved from a common ancestral gene. The results further indicate that those DNA polymerases that use either an RNA or protein primer are related. We propose to classify DNA polymerases on the basis of their evolutionary relatedness.sensitive to the drug aphidicolin, which is a specific inhibitor of eukaryotic DNA polymerases a and A (10, 16).In this communication, we report the nucleotide sequence and the deduced amino acid sequence of PRD1 DNA polymerase.* We have compared the amino acid sequence of this polymerase with those of other DNA polymerases and found that PRD1 DNA polymerase, the smallest known DNA polymerase possessing a proofreading function, has partial homology with many other DNA polymerases including phage T4 DNA polymerase. These results together with those of others (17-22) suggest strongly that many prokaryotic and eukaryotic DNA polymerase genes, regardless of size, have evolved from a common ancestral gene. The results also suggest that DNA polymerases that use either an RNA or protein primer have arisen from a common ancestral progenitor.
We have constructed a hairpin ribozyme targeted to cleave a conserved sequence in the HIV-1 pol gene. The ribozyme was modified to include a structure-stabilizing tetraloop. In vitro studies revealed a cleavage efficiency unprecedented for hairpin ribozymes (Kcat/Km = 75 min-1 microM-1). Stable retroviral vector transduction of this ribozyme gene in T-cell lines resulted in long-term ribozyme expression. As compared to control vector transduced T-cells, the pol ribozyme-transduced cells exhibited significant inhibition of different strains of HIV-1 virus production; this protection was greater when ribozyme expression was driven from an internal pol III promoter (adenovirus VA1) than when driven by a pol II promoter (the MMLV LTR). These results further demonstrate the potential of hairpin ribozymes as anti-HIV gene therapy agents and suggest possibilities for employing combinations of independently targeted hairpin ribozymes.
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