MicroRNAs and long non-coding RNAs during transcriptional regulation and latency of HIV and HTLV.

Human immunodeficiency virus (HIV) and human T cell leukemia virus (HTLV) have replicative and latent stages of infection. The status of the viruses is dependent on the cells that harbour them and on different events that change the transcriptional and post-transcriptional events. Non-coding (nc)RNAs are key factors in the regulation of retrovirus replication cycles. Notably, micro (mi)RNAs and long non-coding (lnc)RNAs are important regulators that can induce switches between active transcription-replication and latency of retroviruses and have important impacts on their pathogenesis. Here, we review the functions of miRNAs and lncRNAs in the context of HIV and HTLV. We describe how specific miRNAs and lncRNAs are involved in the regulation of the viruses' transcription, post-transcriptional regulation and latency. We further discuss treatment strategies using ncRNAs for HIV and HTLV long remission, reactivation or possible cure.

[Gene therapy to cure HIV infection]. / Thérapie génique pour guérir l'infection par le VIH.

To date, the only intervention that has cured HIV infection has been bone marrow transplants from HIV-resistant donors to HIV-infected recipients. This approach has been used to both cure hematological malignancies and HIV infection, but it cannot be widely adopted due to the high risk of mortality associated with cell transplants between individuals. To overcome this limitation, several approaches have been developed to generate HIV resistance using gene therapy in an infected individual's own cells. With the growing arsenal of effective methods to generate HIV-resistant cells, a safe and effective combination gene therapy approach to cure HIV infection is fast approaching. Here, we review several gene therapy-based methods to generate HIV-resistant cells including the expression of antiviral genes, genome editing, and transcriptional gene silencing. Their varied mechanisms, advantages, and disadvantages are discussed, and perspectives are provided for how they may be combined to design an effective gene therapy for HIV.

Gene therapy to cure HIV infection.

To date, the only intervention that has cured HIV infection has been bone marrow transplants from HIV-resistant donors to HIV-infected recipients. This approach has been used to both cure hematological malignancies and HIV infection, but it cannot be widely adopted due to the high risk of mortality associated with cell transplants between individuals. To overcome this limitation, several approaches have been developed to generate HIV resistance using gene therapy in an infected individual's own cells. With the growing arsenal of effective methods to generate HIV-resistant cells, a safe and effective combination gene therapy approach to cure HIV infection is fast approaching. Here, we review several gene therapy-based methods to generate HIV-resistant cells including the expression of antiviral genes, genome editing, and transcriptional gene silencing. Their varied mechanisms, advantages, and disadvantages are discussed, and perspectives are provided for how they may be combined to design an effective gene therapy for HIV.

Effective inhibition of HIV-1 production by short hairpin RNAs and small interfering RNAs targeting a highly conserved site in HIV-1 Gag RNA is optimized by evaluating alternative length formats.

We have previously identified a target site in HIV-1 RNA that was particularly accessible to a ribozyme and a short hairpin RNA (shRNA). To design small interfering RNAs (siRNAs) targeting this site, we evaluated the effects of siRNAs with different lengths on HIV-1 production. The potency and efficacy of these siRNAs were dependent on the length of their intended sense strand with trends for symmetrical and asymmetrical formats that were similar. Although a typical canonical format with a 21-nucleotide (nt) sense strand was effective at inhibiting HIV-1 production, Dicer substrate siRNAs (dsiRNAs) with the longest lengths (27 to 29 nucleotides) were the most effective. Induction of double-stranded RNA immune responses and effects on cell viability were not detected in cells transfected with different siRNAs, suggesting that the differences observed were not related to indirect effects on HIV-1 production. For the corresponding shRNA designs, a different trend in potency and efficacy against HIV-1 production was observed, with the most effective shRNAs having stem lengths from 20 to 27 bp. Our results highlight the importance of evaluating different designs to identify the best siRNA and shRNA formats for any particular target site and provide a set of highly effective molecules for further development as drug and gene therapies for HIV-1 infection.

HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs.

Several proteins and RNAs expressed by mammalian viruses have been reported to interfere with RNA interference (RNAi) activity. We investigated the ability of the HIV-1-encoded RNA elements Trans-Activation Response (TAR) and Rev-Response Element (RRE) to alter RNAi. MicroRNA let7-based assays showed that RRE is a potent suppressor of RNAi activity, while TAR displayed moderate RNAi suppression. We demonstrate that RRE binds to TAR-RNA Binding Protein (TRBP), an essential component of the RNA Induced Silencing Complex (RISC). The binding of TAR and RRE to TRBP displaces small interfering (si)RNAs from binding to TRBP. Several stem-deleted RRE mutants lost their ability to suppress RNAi activity, which correlated with a reduced ability to compete with siRNA-TRBP binding. A lentiviral vector expressing TAR and RRE restricted RNAi, but RNAi was restored when Rev or GagPol were coexpressed. Adenoviruses are restricted by RNAi and encode their own suppressors of RNAi, the Virus-Associated (VA) RNA elements. RRE enhanced the replication of wild-type and VA-deficient adenovirus. Our work describes RRE as a novel suppressor of RNAi that acts by competing with siRNAs rather than by disrupting the RISC. This function is masked in lentiviral vectors co-expressed with viral proteins and thus will not affect their use in gene therapy. The potent RNAi suppressive effects of RRE identified in this study could be used to enhance the expression of RNAi restricted viruses used in oncolysis such as adenoviruses.

HIV and Ribozymes.

Ribozymes are structured RNA molecules that act as catalysts in different biological reactions. From simple genome cleaving activities in satellite RNAs to more complex functions in cellular protein synthesis and gene regulation, ribozymes play important roles in all forms of life. Several naturally existing ribozymes have been modified for use as therapeutics in different conditions, with HIV-1 infection being one of the most studied. This chapter summarizes data from different preclinical and clinical studies conducted to evaluate the potential of ribozymes to be used in HIV-1 therapies. The different ribozyme motifs that have been modified, as well as their target sites and expression strategies, are described. RNA conjugations used to enhance the antiviral effect of ribozymes are also presented and the results from clinical trials conducted to date are summarized. Studies on anti-HIV-1 ribozymes have provided valuable information on the optimal expression strategies and clinical protocols for RNA gene therapy and remain competitive candidates for future therapy.

The discovery and development of RNA-based therapies for treatment of HIV-1 infection.

INTRODUCTION: Long-term control of HIV-1 infection can potentially be achieved using autologous stem cell transplants with gene-modified cells. Non-coding RNAs represent a diverse class of therapeutic agents including ribozymes, RNA aptamers and decoys, small interfering RNAs, short hairpin RNAs, and U1 interference RNAs that can be designed to inhibit HIV-1 replication. They have been engineered for delivery as drugs to complement current HIV-1 therapies and as gene therapies for a potential HIV-1 functional cure. AREAS COVERED: This review surveys the past three decades of development of these RNA technologies with a focus on their efficacy and safety for treating HIV-1 infections. We describe the mechanisms of each RNA-based agent, targets they have been developed against, efforts to enhance their stability and efficacy, and we evaluate their performance in past and ongoing preclinical and clinical trials. EXPERT OPINION: RNA-based technologies are among the top candidates for gene therapies where they can be stably expressed for long-term suppression of HIV-1. Advances in both gene and drug delivery strategies and improvements to non-coding RNA stability and antiviral properties will cooperatively drive forward progress in improving drug therapy and engineering HIV-1 resistant cells.

CT53518, a novel selective FLT3 antagonist for the treatment of acute myelogenous leukemia (AML).

Up to 30% of acute myelogenous leukemia (AML) patients harbor an activating internal tandem duplication (ITD) within the juxtamembrane domain of the FLT3 receptor, suggesting that it may be a target for kinase inhibitor therapy. For this purpose we have developed CT53518, a potent antagonist that inhibits FLT3, platelet-derived growth factor receptor (PDGFR), and c-Kit (IC(50) approximately 200 nM), while other tyrosine or serine/threonine kinases were not significantly inhibited. In Ba/F3 cells expressing different FLT3-ITD mutants, CT53518 inhibited IL-3-independent cell growth and FLT3-ITD autophosphorylation with an IC(50) of 10-100 nM. In human FLT3-ITD-positive AML cell lines, CT53518 induced apoptosis and inhibited FLT3-ITD phosphorylation, cellular proliferation, and signaling through the MAP kinase and PI3 kinase pathways. Therapeutic efficacy of CT53518 was demonstrated both in a nude mouse model and in a murine bone marrow transplant model of FLT3-ITD-induced disease.

In vitro and in vivo cleavage of HIV-1 RNA by new SOFA-HDV ribozymes and their potential to inhibit viral replication.

RNA-based compounds are promising agents to inactivate viruses. New specific hepatitis delta virus (HDV)-derived ribozymes are natural molecules that can be engineered to specifically target a viral RNA. We have designed specific on-off adaptor (SOFA)-HDV ribozymes targeting the tat and rev sequences of the human immunodeficiency virus type 1 (HIV-1) RNA. We show that the SOFA-HDV ribozymes cleave their RNA target in vitro. They inhibit the Tat-mediated transactivation of HIV-1 from 62% to 86% in different assays. In vivo, the amount of HIV RNA was decreased by 60 and 86% with two distinct ribozymes, which indicates that the inhibition of HIV production is directly correlated to the decline in spliced and unspliced viral RNAs. These SOFAHDV- ribozymes inhibited the expression and the viral production of four HIV-1 strains, indicating an extended potential to act on multiple HIV variants. In HEK 293T and HeLa cells transfected with pNL4-3 and the SOFA-HDV-ribozymes, the reduced RNA levels consequently decreased the Gag protein expression in the cell and virus production in the supernatant. When transfected before HIV-1 infection, the ribozymes prevented the incoming virus from being expressed. The ribozymes inhibited HIV production up to 90% when transfected in combination with the HIV protease inhibitor Atazanavir. Our results strongly suggest that SOFA-HDV ribozymes have a great potential to target HIV-1 and to be used as therapeutic agents in combination therapy.

Cloning and Detection of Aptamer-Ribozyme Conjugations.

RNA aptamers can be used to target proteins or nucleic acids for therapeutic purposes and are candidates for RNA-mediated gene therapy. Like other small therapeutic RNAs, they can be expressed in cells from DNA templates that include a cellular promoter upstream of the RNA coding sequence. Secondary structures flanking aptamers can be used to enhance the activity or stability of these molecules. Notably, flanking self-cleaving ribozymes to remove extraneous nucleotides included during transcription as well as flanking hairpins to improve RNA stability have been used to increase the effect of therapeutic aptamers. Here we describe the cloning procedure of aptamers containing different flanking secondary structures and methods to compare their expression levels by a northern blot protocol optimized for the detection of small RNA molecules.

Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promoters.

The expression of short hairpin RNAs (shRNAs) in cells has many potential therapeutic applications, including as a functional cure for HIV. The RNA polymerase III promoters H1, 7SK, and U6 have all been used to express shRNAs. However, there have been no direct and simultaneous comparisons of shRNA potency, expression level, and transcriptional profile between the promoters. We show that the 7SK and U6 promoters result in higher shRNA levels and potency compared to the H1 promoter but that in transduced T lymphocytes, higher expression levels can also lead to growth defects. We present evidence that Dicer cleavage of shRNAs is measured from the first base pair in the shRNA stem, rather than from the 5' end as previously shown for structurally related microRNAs. As a result, guide-strand identity was unaffected by variations in 5' transcription start sites among the different promoters, making expression levels the main determinant of shRNA potency. While all promoters generated shRNAs with variable start sites, the U6 promoter was the most accurate in using its intended +1 position. Our results have implications for the development of therapeutic small RNAs for gene therapy and for our understanding of how shRNAs are processed in cells.

A pharmacokinetic study of plerixafor in subjects with varying degrees of renal impairment.

Plerixafor is a selective antagonist of CXCR4 used for mobilization of hematopoietic stem cells (HSCs) for autologous stem cell transplantation (SCT) in patients with multiple myeloma (MM) and non-Hodgkin lymphoma (NHL). This Phase 1 open-label study in healthy subjects was conducted to evaluate the pharmacokinetic characteristics of plerixafor in subjects with renal impairment. All subjects received a single 0.24 mg/kg subcutaneous dose of plerixafor. Subjects were stratified into 4 cohorts based on creatinine clearance determined from a 24-hour urine collection: control (>90 mL/min), mild renal impairment (51-80 mL/min), moderate renal impairment (31-50 mL/min), and severe renal impairment (<31 mL/min, not requiring dialysis). Eleven female subjects (48%) and 12 male subjects (52%), ranging in age from 35 to 73 years, were enrolled. Plerixafor clearance was reduced in subjects with renal impairment and was positively correlated with creatinine clearance. The mean area under the concentration- versus-time curve from time 0 to 24 hours postdose of plerixafor in subjects with mild, moderate, and severe renal impairment was 7%, 32%, and 39% higher, respectively, than that in subjects with normal renal function. Renal impairment had no effect on maximal plasma concentrations. The safety profile was similar among subjects with renal impairment and controls. No renal impairment-related trends in the incidence of adverse events were apparent. A plerixaflor dose reduction to 160 microg/kg in patients with a creatinine clearance value

Characterization of the TRBP domain required for dicer interaction and function in RNA interference.

BACKGROUND: Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain. RESULTS: We show that the TRBP binding site in Dicer is a 165 amino acid (aa) region located between the ATPase and the helicase domains. The binding site in TRBP is a 69 aa domain, called C4, located at the C-terminal end of TRBP. The TRBP1 and TRBP2 isoforms, but not TRBPs lacking the C4 site (TRBPsDeltaC4), co-immunoprecipitated with Dicer. The C4 domain is therefore necessary to bind Dicer, irrespective of the presence of RNA. Immunofluorescence shows that while full-length TRBPs colocalize with Dicer, TRBPsDeltaC4 do not. tarbp2-/- cells, which do not express TRBP, do not support RNA interference (RNAi) mediated by short hairpin or micro RNAs against EGFP. Both TRBPs, but not TRBPsDeltaC4, were able to rescue RNAi function. In human cells with low RNAi activity, addition of TRBP1 or 2, but not TRBPsDeltaC4, rescued RNAi function. CONCLUSION: The mapping of the interaction sites between TRBP and Dicer show unique domains that are required for their binding. Since TRBPsDeltaC4 do not interact or colocalize with Dicer, we suggest that TRBP and Dicer, both dsRBPs, do not interact through bound dsRNA. TRBPs, but not TRBPsDeltaC4, rescue RNAi activity in RNAi-compromised cells, indicating that the binding of Dicer to TRBP is critical for RNAi function.

Anthranilamide-based N,N-dialkylbenzamidines as potent and orally bioavailable factor Xa inhibitors: P4 SAR.

Anthranilamide-based benzamidine compound 4 and its N-substituted analogs were designed and examined as factor Xa inhibitors using substituted benzamidines as unconventional S4 binding element. A group of N,N-dialkylbenzamidines (11, 17 and 24) have been discovered as potent factor Xa inhibitors with strong anticoagulant activity and promising oral PK profiles.

Discovery of betrixaban (PRT054021), N-(5-chloropyridin-2-yl)-2-(4-(N,N-dimethylcarbamimidoyl)benzamido)-5-methoxybenzamide, a highly potent, selective, and orally efficacious factor Xa inhibitor.

Systematic SAR studies of in vitro factor Xa inhibitory activity around compound 1 were performed by modifying each of the three phenyl rings. A class of highly potent, selective, efficacious and orally bioavailable direct factor Xa inhibitors was discovered. These compounds were screened in hERG binding assays to examine the effects of substitution groups on the hERG channel affinity. From the leading compounds, betrixaban (compound 11, PRT054021) has been selected as the clinical candidate for development.

Design, synthesis and discovery of 1-(2-(6-Chloro-3-methylsulfonyl)-naphthyl)-1H-pyrazole-5-carboxylamides as highly potent factor Xa inhibitors.

Based upon the biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamides reported in our previous communications, we designed and discovered 2-(6-chloro-3-methylsulfonyl)-naphthyl as an optimal factor Xa S1 binding element. Employing a key Diels-Alder reaction of 1,4-dihydro-2,3-benzoxathiin-3-oxide with maleic anhydride and a key Cu(I)-mediated methylsulfonylation, we prepared two biphenyl 1-(2-(6-chloro-3-methylsulfonyl)-naphthyl)-1H-pyrazole-5-carboxylamides as highly potent factor Xa inhibitors with K(i) values of 0.065 nM and 0.045 nM respectively, and demonstrated the synergistically enhanced binding interaction in the factor Xa S1 site.