Engineering HIV-1-Resistant T-Cells from Short-Hairpin RNA-Expressing Hematopoietic Stem/Progenitor Cells in Humanized BLT Mice

Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT) mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC) transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA). However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR). Here, we report that human CD4⁺ T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4⁺ T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4⁺ T-cells ex vivo. Furthermore, levels of gene-marked CD4⁺ T-cells in peripheral blood increased despite systemic infection with either CXCR4- or CCR5- tropic HIV-1 in vivo. These results demonstrate that transplantation of HSPCs engineered with our combination shRNA vector may be a potential therapy against HIV disease.     

Mechanism of U-Insertion RNA Editing in Trypanosome Mitochondria: Characterization of RET2 Functional Domains by Mutational Analysis

3′-Terminal uridylyl transferases (TUTases) selectively bind uridine 5′-triphosphate (UTP) and catalyze the addition of uridine 5′-monophosphate to the 3′-hydroxyl of RNA substrates in a template-independent manner. RNA editing TUTase 1 and RNA editing TUTase 2 (RET2) play central roles in uridine insertion/deletion RNA editing, which is an essential part of mitochondrial RNA processing in trypanosomes. Although the conserved N-terminal (catalytic) domain and C-terminal (nucleotide base recognition) domain are readily distinguished in all known TUTases, nucleotide specificity, RNA substrate preference, processivity, quaternary structures, and auxiliary domains vary significantly among enzymes of divergent biological functions. RET2 acts as a subunit of the RNA editing core complex to carry out guide-RNA-dependent U-insertion into mitochondrial mRNA. By correlating mutational effects on RET2 activity as recombinant protein and as RNA editing core complex subunit with RNAi-based knock-in phenotypes, we have assessed the UTP and RNA binding sites in RET2. Here we demonstrate functional conservation of key UTP-binding and metal-ion-coordinating residues and identify amino acids involved in RNA substrate recognition. Invariant arginine residues 144 and 435 positioned in the vicinity of the UTP binding site are critical for RET2 activity on single-stranded and double-stranded RNAs, as well as function in vivo. Recognition of a double-stranded RNA, which resembles a guide RNA/mRNA duplex, is further facilitated by multipoint contacts across the RET2-specific middle domain.     

Mechanism of U Insertion RNA Editing in Trypanosome Mitochondria: The Bimodal TUTase Activity of the Core Complex

Expression of the trypanosomal mitochondrial genome requires the insertion and deletion of uridylyl residues at specific sites in pre-mRNAs. RET2 terminal uridylyl transferase is an integral component of the RNA editing core complex (RECC) and is responsible for the guide-RNA-dependent U insertion reaction. By analyzing RNA-interference-based knock-in Trypanosoma brucei cell lines, purified editing complex, and individual protein, we have investigated RET2's association with the RECC. In addition, the U insertion activity exhibited by RET2 as an RECC subunit was compared with characteristics of the monomeric protein. We show that interaction of RET2 with RECC is accomplished via a protein-protein contact between its middle domain and a structural subunit, MP81. The recombinant RET2 catalyzes a faithful editing on gapped (precleaved) double-stranded RNA substrates, and this reaction requires an internal monophosphate group at the 5′ end of the mRNA 3′ cleavage fragment. However, RET2 processivity is limited to insertion of three Us. Incorporation into the RECC voids the internal phosphate requirement and allows filling of longer gaps similar to those observed in vivo. Remarkably, monomeric and RECC-embedded enzymes display a similar bimodal activity: the distributive insertion of a single uracil is followed by a processive extension limited by the number of guiding nucleotides. Based on the RNA substrate specificity of RET2 and the purine-rich nature of U insertion sites, we propose that the distributive + 1 insertion creates a substrate for the processive gap-filling reaction. Upon base-pairing of the + 1 extended 5′ cleavage fragment with a guiding nucleotide, this substrate is recognized by RET2 in a different mode compared to the product of the initial nucleolytic cleavage. Therefore, RET2 distinguishes base pairs in gapped RNA substrates which may constitute an additional checkpoint contributing to overall fidelity of the editing process.     

Lentiviral vector retargeting to P-glycoprotein on metastatic melanoma through intravenous injection

Targeted gene transduction to specific tissues and organs through intravenous injection would be the ultimate preferred method of gene delivery. Here, we report successful targeting in a living animal through intravenous injection of a lentiviral vector pseudotyped with a modified chimeric Sindbis virus envelope (termed m168). m168 pseudotypes have high titer and high targeting specificity and, unlike other retroviral pseudotypes, have low nonspecific infectivity in liver and spleen. A mouse cancer model of metastatic melanoma was used to test intravenous targeting with m168. Human P-glycoprotein was ectopically expressed on the surface of melanoma cells and targeted by the m168 pseudotyped lentiviral vector conjugated with antibody specific for P-glycoprotein. m168 pseudotypes successfully targeted metastatic melanoma cells growing in the lung after systemic administration by tail vein injection. Further development of this targeting technology should result in applications not only for cancers but also for genetic, infectious and immune diseases.