Joseph S. Anderson

Bispecific Short Hairpin siRNA Constructs Targeted to CD4, CXCR4, and CCR5 Confer HIV-1 Resistance

Exploiting the phenomenon of RNA interference (RNAi), recent studies established the utility of monospecific small interfering RNAs (siRNAs) in suppressing HIV-1 infection. However, because of the high mutation rate of the HIV genome, there are considerable challenges in the design of fully efficacious gene therapeutic constructs. Therefore, approaches that simultaneously target different stages of the viral life cycle are desirable. In our current studies, we designed bispecific siRNA constructs against HIV-1 cell surface receptors to inhibit viral entry. Dual specific short hairpin siRNA constructs, containing an 8-nucleotide intervening spacer, targeted against either CXCR4 and CD4 or CCR5 and CXCR4 were synthesized by in vitro transcription. Cleavage of the bispecific constructs yielding monospecific siRNAs was shown to occur in cell extracts. Magi-CXCR4 and CCR5 cells transfected with bispecific siRNAs showed significant downregulation of their respective coreceptors, as determined by FACS analysis. This suggested that combinatorial constructs comprising multiple effector motifs were processed in transfected cells into their respective functional siRNAs. Transfected cells were challenged with either X4 (NL4-3) or R5-tropic (BaL-1) strains of HIV-1. Downregulation of the cell surface receptors coincided with resistance to in vitro viral challenge in both Magi cell lines and peripheral blood mononuclear cells (PBMCs). These results demonstrated the practical utility of short hairpin siRNA bispecific constructs synthesized as a single transcript. Because the short hairpin design will permit tandem assembly of multiple effector motifs, it is now possible to introduce promising multivalent siRNA constructs into retroviral and lentiviral vectors for in vivo gene therapeutic applications.

Safety and Efficacy of a Lentiviral Vector Containing Three Anti-HIV Genes—CCR5 Ribozyme, Tat-rev siRNA, and TAR Decoy—in SCID-hu Mouse–Derived T Cells

Gene therapeutic strategies show promise in controlling human immunodeficiency virus (HIV) infection and in restoring immunological function. A number of efficacious anti-HIV gene constructs have been described so far, including small interfering RNAs (siRNAs), RNA decoys, transdominant proteins, and ribozymes, each with a different mode of action. However, as HIV is prone to generating escape mutants, the use of a single anti-HIV construct would not be adequate to afford long range-viral protection. On this basis, a combination of highly potent anti-HIV genes-namely, a short hairpin siRNA (shRNA) targeting rev and tat, a transactivation response (TAR) decoy, and a CCR5 ribozyme-have been inserted into a third-generation lentiviral vector. Our recent in vitro studies with this construct, Triple-R, established its efficacy in both T-cell lines and CD34 cell-derived macrophages. In this study, we have evaluated this combinatorial vector in vivo. Vector-transduced CD34 cells were injected into severe combined immunodeficiency (SCID)-hu mouse thy/liv grafts to determine their capacity to give rise to T cells. Our results show that phenotypically normal transgenic T cells are generated that are able to resist HIV-1 infection when challenged in vitro. These important attributes of this combinatorial vector show its promise as an excellent candidate for use in human clinical trials.

HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector

BackgroundRNA interference (RNAi) mediated by small interfering RNAs (siRNAs) has proved to be a highly effective gene silencing mechanism with great potential for HIV/AIDS gene therapy. Previous work with siRNAs against cellular coreceptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance. Since monospecific siRNAs targeting individual coreceptors are inadequate in protecting against both T cell tropic (X4) and monocyte tropic (R5) viral strains simultaneously, bispecific constructs with dual specificity are required. For effective long range therapy, the bispecific constructs need to be stably transduced into HIV-1 target cells via integrating viral vectors.ResultsTo achieve this goal, lentiviral vectors incorporating both CXCR4 and CCR5 siRNAs of short hairpin design were constructed. The CXCR4 siRNA was driven by a U6 promoter whereas the CCR5 siRNA was driven by an H1 promoter. A CMV promoter driven EGFP reporter gene is also incorporated in the bispecific construct. High efficiency transduction into coreceptor expressing Magi and Ghost cell lines with a concomitant down regulation of respective coreceptors was achieved with lentiviral vectors. When the siRNA expressing transduced cells were challenged with X4 and R5 tropic HIV-1, they demonstrated marked viral resistance. HIV-1 resistance was also observed in bispecific lentiviral vector transduced primary PBMCs.ConclusionsBoth CXCR4 and CCR5 coreceptors could be simultaneously targeted for down regulation by a single combinatorial lentiviral vector incorporating respective anti-coreceptor siRNAs. Stable down regulation of both the coreceptors protects cells against infection by both X4 and R5 tropic HIV-1. Stable down regulation of cellular molecules that aid in HIV-1 infection will be an effective strategy for long range HIV gene therapy.

Potent Suppression of HIV Type 1 Infection By a Short Hairpin Anti-CXCR4 siRNA

The phenomenon of RNA interference (RNAi) sparked a new surge in the area of posttranscriptional gene silencing methodologies and their potential application for HIV-1 gene therapy. A potentially promising strategy is to exploit siRNAs to prevent viral entry at the cell surface by down-regulating essential cell surface HIV-1 coreceptors. In the present studies we targeted the CXCR4 coreceptor for disruption with siRNA to inhibit HIV-1 entry as a first step toward the ultimate goal of translating this to gene therapy for AIDS. A stem-loop hairpin structured anti-CXCR4 siRNA was designed and synthesized in vitro by transcription with T7 polymerase. Down-regulation of the coreceptor was assayed in U373-Magi-CXCR4 cells. FACS analysis showed marked down-regulation of CXCR4 on the cell surface and Western blot analysis confirmed the reduced levels of intracellular synthesis. When challenged with X4-tropic HIV-1 NL4-3, the siRNA-transfected cells exhibited marked viral resistance. Consistent with these results, siRNA-transfected primary lymphocytes also exhibited significant resistance to HIV-1 entry. These proof-of-concept studies demonstrated the efficacy of an siRNA targeted to an essential cellular coreceptor CXCR4 in protecting from HIV-1 infection. Delivery of this siRNA into hematopoietic stem cells via lentiviral vectors may have potential gene therapeutic applications.

CXCR4 and CCR5 shRNA transgenic CD34+ cell derived macrophages are functionally normal and resist HIV-1 infection

BackgroundStable simultaneous knock down of the HIV-1 coreceptors CCR5 and CXCR4 is a promising strategy to protect cells from both R5 macrophage tropic and X4 T cell tropic as well as dual tropic viral infections. The potency of shRNAs in targeted gene silencing qualifies them as powerful tools for long term HIV gene therapy. Our previous work with a bispecific lentiviral vector containing CXCR4 and CCR5 shRNAs showed efficacy in down regulating both coreceptors and conferring viral resistance to both X4 and R5-tropic strains of HIV-1 in cultured cell lines. To extend these results to a stem cell gene therapy setting, here we show transduction of primary CD34+ hematopoietic progenitor cells to derive normal end stage cells that are resistant to HIV-1 infection.ResultsThe bispecific XHR lentiviral vector harboring CXCR4 and CCR5 shRNA expression cassettes was efficient in transducing CD34+ cells. The transduced cells gave rise to morphologically normal transgenic macrophages when cultured in cytokine media. There was a marked down regulation of both coreceptors in the stably transduced macrophages which showed resistance to both R5 and X4 HIV-1 strains upon in vitro challenge. Since off target effects by some shRNAs may have adverse effects on transgenic cells, the stably transduced macrophages were further analyzed to determine if they are phenotypically and functionally normal. FACS evaluation showed normal levels of the characteristic surface markers CD14, CD4, MHC class II, and B7.1. Phagocytic functions were also normal. The transgenic macrophages demonstrated normal abilities in up-regulating the costimulatory molecule B7.1 upon LPS stimulation. Furthermore, IL-1 and TNFα cytokine secretion in response to LPS stimulation was also normal. Thus, the transgenic macrophages appear to be phenotypically and functionally normal.ConclusionThese studies have demonstrated for the first time that a bispecific lentiviral vector could be used to stably deliver shRNAs targeted to both CCR5 and CXCR4 coreceptors into CD34+ hematopoietic progenitor cells and derive transgenic macrophages. Transgenic macrophages with down regulated coreceptors were resistant to both R5 and X4 tropic HIV-1 infections. The differentiated cells were also phenotypically and functionally normal indicating no adverse effects of shRNAs on lineage specific differentiation of stem cells. It is now possible to construct gene therapeutic lentiviral vectors incorporating multiple shRNAs targeted to cellular molecules that aid in HIV-1 infection. Use of these vectors in a stem cell setting shows great promise for sustained HIV/AIDS gene therapy.

Generation of an HIV-1-Resistant Immune System with CD34+ Hematopoietic Stem Cells Transduced with a Triple-Combination Anti-HIV Lentiviral Vector

ABSTRACT HIV gene therapy has the potential to offer an alternative to the use of current small-molecule antiretroviral drugs as a treatment strategy for HIV-infected individuals. Therapies designed to administer HIV-resistant stem cells to an infected patient may also provide a functional cure, as observed in a bone marrow transplant performed with hematopoietic stem cells (HSCs) homozygous for the CCR5-Δ32-bp allele. In our current studies, preclinical evaluation of a combination anti-HIV lentiviral vector was performed, in vivo, in humanized NOD-RAG1−/− IL2rγ−/− knockout mice. This combination vector, which displays strong preintegration inhibition of HIV-1 infection in vitro, contains a human/rhesus macaque TRIM5α isoform, a CCR5 short hairpin RNA (shRNA), and a TAR decoy. Multilineage hematopoiesis from anti-HIV lentiviral vector-transduced human CD34+ HSCs was observed in the peripheral blood and in various lymphoid organs, including the thymus, spleen, and bone marrow, of engrafted mice. Anti-HIV vector-transduced CD34+ cells displayed normal development of immune cells, including T cells, B cells, and macrophages. The anti-HIV vector-transduced cells also displayed knockdown of cell surface CCR5 due to the expression of the CCR5 shRNA. After in vivo challenge with either an R5-tropic BaL-1 or X4-tropic NL4-3 strain of HIV-1, maintenance of human CD4+ cell levels and a selective survival advantage of anti-HIV gene-modified cells were observed in engrafted mice. The data provided from our study confirm the safety and efficacy of this combination anti-HIV lentiviral vector in a hematopoietic stem cell gene therapy setting for HIV and validates its potential application in future clinical trials.

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

The CCR5 co-receptor is necessary for cellular entry by R5 tropic viral strains involved in primary HIV infection, but is dispensable for normal human physiology. Owing to its crucial role in HIV-1 infection, the CCR5 co-receptor has been the subject of many therapeutic approaches, including gene therapy. siRNA targeting was shown to be effective in downregulating CCR5 expression and conferring significant protection against HIV-1 in susceptible cells. However, complete knockdown of CCR5 expression has not been achieved and thus remains an elusive goal. In these studies, we identified new CCR5 siRNAs capable of achieving complete knockdown of the co-receptor expression. Our transfection studies have shown that longer 28-mer short hairpin siRNAs are very effective in gene downregulation as assessed by fluorescence-activated cell sorting and transcript quantitation by quantitative real-time polymerase chain reaction. These siRNAs conferred strong antiviral protection during viral challenge. To obtain stable expression, highly potent siRNA expression cassettes were introduced into lentiviral vectors. Similar high levels of CCR5 downregulation were observed in stably transduced cells with concomitant viral protection in cultured cell lines. To translate these results to a stem cell gene therapy setting, CD34 hematopoietic progenitor cells were transduced with lentiviral vectors to derive transgenic macrophages. The transgenic cells also exhibited high levels of CCR5 downregulation and viral resistance. With regard to Pol-III promoter-mediated siRNA expression, higher efficacies were obtained with U6-driven CCR5 siRNAs. However, in contrast to previous reports, no apparent cytotoxicities were observed in transgenic cells containing U6-driven siRNA constructs. Thus the above anti-CCR5 siRNAs are among the most effective demonstrated to date and are very promising candidates for clinical applications.

Preintegration HIV-1 Inhibition by a Combination Lentiviral Vector Containing a Chimeric TRIM5α Protein, a CCR5 shRNA, and a TAR Decoy

Human immunodeficiency virus (HIV) gene therapy offers a promising alternative approach to current antiretroviral treatments to inhibit HIV-1 infection. Various stages of the HIV life cycle including pre-entry, preintegration, and postintegration can be targeted by gene therapy to block viral infection and replication. By combining multiple highly potent anti-HIV transgenes in a single gene therapy vector, HIV-1 resistance can be achieved in transduced cells while prohibiting the generation of escape mutants. Here, we describe a combination lentiviral vector that encodes three highly effective anti-HIV genes functioning at separate stages of the viral life cycle including a CCR5 short hairpin RNA (shRNA) (pre-entry), a human/rhesus macaque chimeric TRIM5α (postentry/preintegration), and a transactivation response element (TAR) decoy (postintegration). The major focus on designing this anti-HIV vector was to block productive infection of HIV-1 and to inhibit any formation of provirus that would maintain the viral reservoir. Upon viral challenge, potent preintegration inhibition of HIV-1 infection was achieved in combination vector-transduced cells in both cultured and primary CD34+ hematopoietic progenitor cell (HPC)-derived macrophages. The generation of escape mutants was also blocked as evaluated by long-term culture of challenged cells. The ability of this combination anti-HIV lentiviral vector to prevent HIV-1 infection, in vitro, warrants further evaluation of its in vivo efficacy.Human immunodeficiency virus (HIV) gene therapy offers a promising alternative approach to current antiretroviral treatments to inhibit HIV-1 infection. Various stages of the HIV life cycle including pre-entry, preintegration, and postintegration can be targeted by gene therapy to block viral infection and replication. By combining multiple highly potent anti-HIV transgenes in a single gene therapy vector, HIV-1 resistance can be achieved in transduced cells while prohibiting the generation of escape mutants. Here, we describe a combination lentiviral vector that encodes three highly effective anti-HIV genes functioning at separate stages of the viral life cycle including a CCR5 short hairpin RNA (shRNA) (pre-entry), a human/rhesus macaque chimeric TRIM5 alpha (postentry/preintegration), and a transactivation response element (TAR) decoy (postintegration). The major focus on designing this anti-HIV vector was to block productive infection of HIV-1 and to inhibit any formation of provirus that would maintain the viral reservoir. Upon viral challenge, potent preintegration inhibition of HIV-1 infection was achieved in combination vector-transduced cells in both cultured and primary CD34(+) hematopoietic progenitor cell (HPC)-derived macrophages. The generation of escape mutants was also blocked as evaluated by long-term culture of challenged cells. The ability of this combination anti-HIV lentiviral vector to prevent HIV-1 infection, in vitro, warrants further evaluation of its in vivo efficacy.

Generation of HIV-1 Resistant and Functional Macrophages From Hematopoietic Stem Cell–derived Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. By developing iPSCs to treat HIV, there is the potential for generating a continuous supply of therapeutic cells for transplantation into HIV-infected patients. In this study, we have used human hematopoietic stem cells (HSCs) to generate anti-HIV gene expressing iPSCs for HIV gene therapy. HSCs were dedifferentiated into continuously growing iPSC lines with four reprogramming factors and a combination anti-HIV lentiviral vector containing a CCR5 short hairpin RNA (shRNA) and a human/rhesus chimeric TRIM5α gene. Upon directed differentiation of the anti-HIV iPSCs toward the hematopoietic lineage, a robust quantity of colony-forming CD133(+) HSCs were obtained. These cells were further differentiated into functional end-stage macrophages which displayed a normal phenotypic profile. Upon viral challenge, the anti-HIV iPSC-derived macrophages exhibited strong protection from HIV-1 infection. Here, we demonstrate the ability of iPSCs to develop into HIV-1 resistant immune cells and highlight the potential use of iPSCs for HIV gene and cellular therapies.

Human Immunodeficiency Virus Type 1 Restriction by Human–Rhesus Chimeric Tripartite Motif 5α (TRIM 5α) in CD34+ Cell-Derived Macrophages In Vitro and in T Cells In Vivo in Severe Combined Immunodeficient (SCID-hu) Mice Transplanted with Human Fetal Tissue

Species-specific innate resistance against viral infections offers novel avenues for antiviral therapeutics. The retroviral restriction factor TRIM5alpha (tripartite motif 5alpha protein) has been shown to potently restrict human immunodeficiency virus (HIV)-1 infection in otherwise susceptible cell lines and CD34(+) cell-derived macrophages. A 13-amino acid patch in the C-terminal B30.2 (SPRY) domain of rhesus macaque TRIM5alpha has been shown to be involved in HIV-1 capsid recognition and is critical for viral inhibition. A chimeric human-rhesus TRIM5alpha (TRIM5alpha-HRH) was generated by replacing an 11-amino acid patch in the human isoform with the rhesus 13-amino acid patch. Here we show that lentiviral vector expression of this human-rhesus chimera in HIV-1-permissive MAGI-CXCR4 cells conferred resistance as well as a selective survival advantage on HIV-1 challenge. To apply these findings in a stem cell gene therapy setting, TRIM5alpha-HRH was expressed in CD34(+) cell-derived macrophages in vitro and in SCID-hu mouse-derived thymocytes in vivo. On viral challenge, transgenic macrophages and thymocytes were highly resistant to HIV-1 compared with control cells. Normal development of TRIM5alpha-HRH-expressing macrophages and in vivo-derived T cells was also observed by phenotypic flow cytometric analysis. These results demonstrate the efficacy of TRIM5alpha-HRH in a stem cell setting and its further advancement for use in gene therapy applications.