Inhibition of HIV-1 infection by a unique short hairpin RNA to chemokine receptor 5 delivered into macrophages through hematopoietic progenitor cell transduction

ABSTRACT We demonstrate a novel approach for coexpression of a short hairpin RNA (shRNA) with an open reading frame which exploits transcriptional read-through of a minimal polyadenylation signal from a Pol II promoter. We first observed efficient inducible expression of enhanced green fluorescent protein along with an anti-rev shRNA. We took advantage of this observation to test coexpression of the transdominant negative mutant (humanized) of human immunodeficiency type 1 (HIV-1) Rev (huRevM10) along with an anti-rev shRNA via an HIV-1-inducible fusion promoter. The coexpression of the shRNA and transdominant protein resulted in potent, long-term inhibition of HIV-1 gene expression and suppression of shRNA-resistant mutants. This dual expression system has broad-based potential for other shRNA applications, such as cases where simultaneous knockdown of mutant and wild-type transcripts must be accompanied by replacement of the wild-type protein.

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Efficient Gene Transfer of HIV-1-Specific Short Hairpin RNA into Human Lymphocytic Cells Using Recombinant Adeno-associated Virus Vectors

Molecular Therapy ◽

10.1016/j.ymthe.2003.11.025 ◽

2004 ◽

Vol 9 (3) ◽

pp. 396-402 ◽

Cited By ~ 33

Author(s):

Daniel Boden ◽

Oliver Pusch ◽

Fred Lee ◽

Lynne Tucker ◽

Bharat Ramratnam

Keyword(s):

Gene Transfer ◽

Short Hairpin Rna ◽

Hairpin Rna ◽

Adeno Associated Virus ◽

Virus Vectors ◽

Efficient Gene ◽

Short Hairpin ◽

Hiv 1

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Nucleotide sequence homology requirements of HIV-1-specific short hairpin RNA

Nucleic Acids Research ◽

10.1093/nar/gkg876 ◽

2003 ◽

Vol 31 (22) ◽

pp. 6444-6449 ◽

Cited By ~ 50

Author(s):

O. Pusch

Keyword(s):

Nucleotide Sequence ◽

Sequence Homology ◽

Short Hairpin Rna ◽

Hairpin Rna ◽

Nucleotide Sequence Homology ◽

Short Hairpin ◽

Hiv 1

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Infection of hematopoietic progenitor cells by HIV-1 subtype C, and its association with anemia in southern Africa

Blood ◽

10.1182/blood-2007-04-086314 ◽

2007 ◽

Vol 110 (9) ◽

pp. 3143-3149 ◽

Cited By ~ 25

Author(s):

Andrew D. Redd ◽

Ava Avalos ◽

Max Essex

Keyword(s):

Progenitor Cells ◽

Southern Africa ◽

Progenitor Cell ◽

The United States ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor Cell ◽

Subtype C ◽

Hematopoietic Progenitor ◽

Subtype B ◽

Hiv 1

AbstractReports from southern Africa, an area in which human immunodeficiency virus type 1 (HIV-1) infection is caused almost exclusively by subtype C (HIV-1C), have shown increased rates of anemia in HIV-infected populations compared with similar acquired immunodeficiency syndrome (AIDS) patients in the United States, an area predominantly infected with subtype B (HIV-1B). Recent findings by our group demonstrated a direct association between HIV-1 infection and hematopoietic progenitor cell health in Botswana. Therefore, using a single-colony infection assay and quantitative proviral analysis, we examined whether HIV-1C could infect hematopoietic progenitor cells (HPCs) and whether this phenotype was associated with the higher rates of anemia found in southern Africa. The results show that a significant number of HIV-1C, but not HIV-1B, isolates can infect HPCs in vitro (P < .05). In addition, a portion of HIV-1C–positive Africans had infected progenitor cell populations in vivo, which was associated with higher rates of anemia in these patients (P < .05). This represents a difference in cell tropism between 2 geographically separate and distinct HIV-1 subtypes. The association of this hematotropic phenotype with higher rates of anemia should be considered when examining anti-HIV drug treatment regimens in HIV-1C–predominant areas, such as southern Africa.

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A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model

Blood ◽

10.1182/blood-2009-04-215855 ◽

2010 ◽

Vol 115 (8) ◽

pp. 1534-1544 ◽

Cited By ~ 105

Author(s):

Saki Shimizu ◽

Patrick Hong ◽

Balamurugan Arumugam ◽

Lauren Pokomo ◽

Joshua Boyer ◽

...

Keyword(s):

T Cell ◽

Mouse Model ◽

Lentiviral Vector ◽

Short Hairpin Rna ◽

Lymphoid Organs ◽

Great Promise ◽

Ccr5 Expression ◽

Hairpin Rna ◽

Short Hairpin ◽

Hiv 1

AbstractInhibiting the expression of the HIV-1 coreceptor CCR5 holds great promise for controlling HIV-1 infection in patients. Here we report stable knockdown of human CCR5 by a short hairpin RNA (shRNA) in a humanized bone marrow/liver/thymus (BLT) mouse model. We delivered a potent shRNA against CCR5 into human fetal liver-derived CD34+ hematopoietic progenitor/stem cells (HPSCs) by lentiviral vector transduction. We transplanted vector-transduced HPSCs solidified with Matrigel and a thymus segment under the mouse kidney capsule. Vector-transduced autologous CD34+ cells were subsequently injected in the irradiated mouse, intended to create systemic reconstitution. CCR5 expression was down-regulated in human T cells and monocytes/macrophages in systemic lymphoid tissues, including gut-associated lymphoid tissue, the major site of HIV-1 replication. The shRNA-mediated CCR5 knockdown had no apparent adverse effects on T-cell development as assessed by polyclonal T-cell receptor Vβ family development and naive/memory T-cell differentiation. CCR5 knockdown in the secondary transplanted mice suggested the potential of long-term hematopoietic reconstitution by the shRNA-transduced HPSCs. CCR5 tropic HIV-1 infection was effectively inhibited in mouse-derived human splenocytes ex vivo. These results demonstrate that lentiviral vector delivery of shRNA into human HPSCs could stably down-regulate CCR5 in systemic lymphoid organs in vivo.

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