Axl receptor tyrosine kinase is a regulator of apolipoprotein E.

Alzheimer's disease (AD), the leading cause of dementia, is a chronic neurodegenerative disease. Apolipoprotein E (apoE), which carries lipids in the brain in the form of lipoproteins, plays an undisputed role in AD pathophysiology. A high-throughput phenotypic screen was conducted using a CCF-STTG1 human astrocytoma cell line to identify small molecules that could upregulate apoE secretion. AZ7235, a previously discovered Axl kinase inhibitor, was identified to have robust apoE activity in brain microglia, astrocytes and pericytes. AZ7235 also increased expression of ATP-binding cassette protein A1 (ABCA1), which is involved in the lipidation and secretion of apoE. Moreover, AZ7235 did not exhibit Liver-X-Receptor (LXR) activity and stimulated apoE and ABCA1 expression in the absence of LXR. Target validation studies using AXL-/- CCF-STTG1 cells showed that Axl is required to mediate AZ7235 upregulation of apoE and ABCA1. Intriguingly, apoE expression and secretion was significantly attenuated in AXL-deficient CCF-STTG1 cells and reconstitution of Axl or kinase-dead Axl significantly restored apoE baseline levels, demonstrating that Axl also plays a role in maintaining apoE homeostasis in astrocytes independent of its kinase activity. Lastly, these effects may require human apoE regulatory sequences, as AZ7235 exhibited little stimulatory activity toward apoE and ABCA1 in primary murine glia derived from neonatal human APOE3 targeted-replacement mice. Collectively, we identified a small molecule that exhibits robust apoE and ABCA1 activity independent of the LXR pathway in human cells and elucidated a novel relationship between Axl and apoE homeostasis in human astrocytes.

Viral Apoptosis Evasion via the MAPK Pathway by Use of a Host Long Noncoding RNA.

An emerging realization of infectious disease is that pathogens can cause a high incidence of genetic instability within the host as a result of infection-induced DNA lesions. These often lead to classical hallmarks of cancer, one of which is the ability to evade apoptosis despite the presence of numerous genetic mutations that should be otherwise lethal. The Human Immunodeficiency Virus type 1 (HIV-1) is one such pathogen as it induces apoptosis in CD4+ T cells but is largely non-cytopathic in macrophages. As a consequence there is long-term dissemination of the pathogen specifically by these infected yet surviving host cells. Apoptosis is triggered by double-strand breaks (DSBs), such as those induced by integrating retroviruses like HIV-1, and is coordinated by the p53-regulated long noncoding RNA lincRNA-p21. As is typical for a long noncoding RNA, lincRNA-p21 mediates its activities in a complex with one of its two protein binding partners, namely HuR and hnRNP-K. In this work, we monitor the cellular response to infection to determine how HIV-1 induces DSBs in macrophages yet evades apoptosis in these cells. We show that the virus does so by securing the pro-survival MAP2K1/ERK2 cascade early upon entry, in a gp120-dependent manner, to orchestrate a complex dysregulation of lincRNA-p21. By sequestering the lincRNA-p21 partner HuR in the nucleus, HIV-1 enables lincRNA-p21 degradation. Simultaneously, the virus permits transcription of pro-survival genes by sequestering lincRNA-p21's other protein partner hnRNP-K in the cytoplasm via the MAP2K1/ERK2 pathway. Of particular note, this MAP2K1/ERK2 pro-survival cascade is switched off during T cell maturation and is thus unavailable for similar viral manipulation in mature CD4+ T cells. We show that the introduction of MAP2K1, ERK2, or HDM2 inhibitors in HIV-infected macrophages results in apoptosis, providing strong evidence that the viral-mediated apoptotic block can be released, specifically by restoring the nuclear interaction of lincRNA-p21 and its apoptosis protein partner hnRNP-K. Together, these results reveal a unique example of pathogenic control over mammalian apoptosis and DNA damage via a host long noncoding RNA, and present MAP2K1/ERK2 inhibitors as a novel therapeutic intervention strategy for HIV-1 infection in macrophages.

Small molecule inducers of ABCA1 and apoE that act through indirect activation of the LXR pathway.

apoE is the primary lipid carrier within the CNS and the strongest genetic risk factor for late onset Alzheimer's disease (AD). apoE is primarily lipidated via ABCA1, and both are under transcriptional regulation by the nuclear liver X receptor (LXR). Considerable evidence from genetic (using ABCA1 overexpression) and pharmacological (using synthetic LXR agonists) studies in AD mouse models suggests that increased levels of lipidated apoE can improve cognitive performance and, in some strains, can reduce amyloid burden. However, direct synthetic LXR ligands have hepatotoxic side effects that limit their clinical use. Here, we describe a set of small molecules, previously annotated as antagonists of the purinergic receptor, P2X7, which enhance ABCA1 expression and activity as well as apoE secretion, and are not direct LXR ligands. Furthermore, P2X7 is not required for these molecules to induce ABCA1 upregulation and apoE secretion, demonstrating that the ABCA1 and apoE effects are mechanistically independent of P2X7 inhibition. Hence, we have identified novel dual activity compounds that upregulate ABCA1 across multiple CNS cell types, including human astrocytes, pericytes, and microglia, through an indirect LXR mechanism and that also independently inhibit P2X7 receptor activity.

Non-coding RNAs and HIV: viral manipulation of host dark matter to shape the cellular environment.

On October 28th 1943 Winston Churchill said "we shape our buildings, and afterward our buildings shape us" (Humes, 1994). Churchill was pondering how and when to rebuild the British House of Commons, which had been destroyed by enemy bombs on May 10th 1941. The old House had been small and insufficient to hold all its members, but was restored to its original form in 1950 in order to recapture the "convenience and dignity" that the building had shaped into its parliamentary members. The circular loop whereby buildings or dwellings are shaped and go on to shape those that reside in them is also true of pathogens and their hosts. As obligate parasites, pathogens need to alter their cellular host environments to ensure survival. Typically pathogens modify cellular transcription profiles and in doing so, the pathogen in turn is affected, thereby closing the loop. As key orchestrators of gene expression, non-coding RNAs provide a vast and extremely precise set of tools for pathogens to target in order to shape the cellular environment. This review will focus on host non-coding RNAs that are manipulated by the infamous intracellular pathogen, the human immunodeficiency virus (HIV). We will briefly describe both short and long host non-coding RNAs and discuss how HIV gains control of these factors to ensure widespread dissemination throughout the host as well as the establishment of lifelong, chronic infection.

Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens.

Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta-analysis of findings from genome-wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.

A discussion of molecular biology methods for protein engineering.

A number of molecular biology techniques are available to generate variants from a particular start gene for eventual protein expression. We discuss the basic principles of these methods in a repertoire that may be used to achieve the elemental steps in protein engineering. These include site-directed, deletion and insertion mutagenesis. We provide detailed case studies, drawn from our own experiences, packaged together with conceptual discussions and include an analysis of the techniques presented with regards to their uses in protein engineering.

Inhibition of hepatitis B virus replication in vivo using lipoplexes containing altritol-modified antiviral siRNAs.

Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the world's population and carriers of the virus are at risk for complicating hepatocellular carcinoma. Current treatment options have limited efficacy and chronic HBV infection is likely to remain a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realized. Limiting off target effects, improvement of delivery efficiency, dose regulation and preventing reactivation of viral replication are some of the hurdles that need to be overcome. To address this, we assessed the usefulness of the recently described class of altritol-containing synthetic siRNAs (ANA siRNAs), which were administered as lipoplexes and tested in vivo in a stringent HBV transgenic mouse model. Our observations show that ANA siRNAs are capable of silencing of HBV replication in vivo. Importantly, non specific immunostimulation was observed with unmodified siRNAs and this undesirable effect was significantly attenuated by ANA modification. Inhibition of HBV replication of approximately 50% was achieved without evidence for induction of toxicity. These results augur well for future application of ANA siRNA therapeutic lipoplexes.

RNA interference-based gene expression strategies aimed at sustained therapeutic inhibition of HIV.

The naturally-occurring RNA interference (RNAi) pathway represents a powerful tool for the sequence-specific post-transcriptional silencing of gene expression. By exploiting the endogenous mammalian RNAi pathway, several expression-based strategies have been developed to inhibit human immunodeficiency virus (HIV) gene expression and replication. This approach potentially has utility as a protective 'therapeutic vaccine' of virus-susceptible lymphocytes. In this review we discuss new developments aimed at improving efficacy and delivery of novel RNAi-based gene expression antiviral strategies. Particular attention is given to advances in combinatorial gene expression systems that prevent the emergence of RNAi-resistant virus by simultaneously targeting multiple HIV targets. Potential usefulness of silencing host factors that are required for viral replication is also discussed. These approaches form the basis for a number of promising ongoing and future clinical trials aimed at providing an effective, safe and prolonged single-intervention therapy for HIV/AIDS.

The efficacy of generating three independent anti-HIV-1 siRNAs from a single U6 RNA Pol III-expressed long hairpin RNA.

RNA Interference (RNAi) effectors have been used to inhibit rogue RNAs in mammalian cells. However, rapidly evolving sequences such as the human immunodeficiency virus type 1 (HIV-1) require multiple targeting approaches to prevent the emergence of escape variants. Expressed long hairpin RNAs (lhRNAs) have recently been used as a strategy to produce multiple short interfering RNAs (siRNAs) targeted to highly variant sequences. We aimed to characterize the ability of expressed lhRNAs to generate independent siRNAs that silence three non-contiguous HIV-1 sites by designing lhRNAs comprising different combinations of siRNA-encoding sequences. All lhRNAs were capable of silencing individual target sequences. However, silencing efficiency together with concentrations of individual lhRNA-derived siRNAs diminished from the stem base (first position) towards the loop side of the hairpin. Silencing efficacy against HIV-1 was primarily mediated by siRNA sequences located at the base of the stem. Improvements could be made to first and second position siRNAs by adjusting spacing arrangements at their junction, but silencing of third position siRNAs remained largely ineffective. Although lhRNAs offer advantages for combinatorial RNAi, we show that good silencing efficacy across the span of the lhRNA duplex is difficult to achieve with sequences that encode more than two adjacent independent siRNAs.

An RNA targeted to the HIV-1 LTR promoter modulates indiscriminate off-target gene activation.

Transcriptional gene silencing (TGS) can be achieved by small RNAs targeted to upstream promoter regions. Previously we characterized siRNAs targeted to the HIV-1 long terminal repeat (LTR) promoter at site 247, and found that a 21-base antisense strand of siRNA-247 (LTR-247as) suppressed LTR-mediated expression. To characterize the specificity of LTR-247as, vectors expressing antisense RNAs targeted to a region spanning 50 bases up- and downstream of the 247 target site were generated. LTR-247as+7, a approximately 22 base antisense RNA that is shifted by only seven bases upstream of LTR-247as, showed a significant increase in LTR-driven reporter gene expression that was independent of cell type and active chromatin methyl-marks. Promoter-targeting siRNAs have been recently shown to induce gene activation. However, here we demonstrate gene activation via a sequence-specific off-target effect. Microarray analysis of LTR-247as+7-treated cultures resulted in the deregulation of approximately 185 genes. A gene of unknown function, C10orf76, was responsive to inhibition by LTR-247as+7 and the loss of C10orf76 resulted in the upregulation of several genes that were activated by LTR-247as+7. These data suggest caution when using short antisense RNAs or siRNAs designed to target promoter sequences, since promoter-targeted RNAs may have unintended inhibitory effects against factors with suppressive gene activity.

The inhibitory efficacy of RNA POL III-expressed long hairpin RNAs targeted to untranslated regions of the HIV-1 5' long terminal repeat.

Human immunodeficiency virus type 1 (HIV-1) is a lentivirus that causes persistent infection resulting in the demise of immune regulatory cells, and ensuing diseases associated with acquired immune deficiency syndrome (AIDS). Although current therapeutic modalities have had a significant impact on mortality rates, novel therapies are constantly needed to prevent the emergence of resistant viral variants that escape the effects of antivirals. RNA Interference (RNAi) is a promising therapeutic modality for the inhibition of HIV-1 RNAs. Traditionally, RNAi effector sequences include expressed short hairpin RNAs (shRNAs) or short interfering RNAs (siRNAs). Recently, expressed long hairpin RNAs (lhRNAs) have been used with the aim of generating multiple independent siRNAs, which simultaneously target different susceptible sites on HIV-1. Here, modified lhRNAs expressed from U6 RNA Pol III promoters were targeted to sites within the first transcribed sequences of the HIV-1 5' long terminal repeat (LTR) region. Both Tat-dependent and independent suppressive efficacy was demonstrated against subtype B and C reporter sequences; however, lhRNAs complementary to the TAR stem-loop were refractory to silencing. None of the lhRNAs induced an unwanted interferon response as measured by interferon beta levels. Silencing by the lhRNAs was not equal across the extent of its cognate sequence, with the greatest efficacy observed for sequences located at the base of the stem. Nevertheless, direct antireplicative activity was seen when targeting lhRNAs to a subtype B HIV clone pNL4-3 Luc and a subtype C wild-type HIV-1 strain, FV5. These data highlight distinct target loci within the 5' LTR of HIV-1 that are susceptible to lhRNA targeting, and may prove to have an important advantage over other RNAi target sites within HIV-1. Although lhRNAs themselves require further manipulation to improve their overall efficacy in generating multiple functioning siRNAs, they may prove useful in any combinatorial-based approach to treating HIV-1 infection.

Specific inhibition of HBV replication in vitro and in vivo with expressed long hairpin RNA.

Activating RNA interference to achieve specific gene silencing has shown promise for the development of RNA-based treatment of chronic hepatitis B virus (HBV) infection. To further this approach, we assessed the efficacy of expressed long hairpin RNAs (lhRNAs) that target the conserved HBx open reading frame of HBV. As substrates for Dicer, lhRNAs have the potential to generate multiple short interfering RNAs (siRNAs) to enable simultaneous targeting of different sites. Two U6 Pol III vectors were constructed that encode anti-HBV lhRNAs with a 62 base pair stem sequence containing multiple G:U pairings. Assessment in transfected cultured cells and also in vivo using the murine hydrodynamic injection model showed that one of the lhRNA vectors (lhRNA 1) diminished markers of virus replication by 70-90% without evidence of interferon response induction. Greatest silencing efficacy was observed for targets that are complementary to sequences located at the base of the hairpin stem and this correlated with a higher concentration of siRNAs derived from this region of the lhRNA. Although lhRNA 1 has the advantage of targeting a greater viral sequence, incomplete cellular processing may result in unequal silencing across the span of the viral target RNA.

In vitro analysis of rates and spectra of mutations in a polymorphic region of the Rv0746 PE_PGRS gene of Mycobacterium tuberculosis.

An assay modeled on a known polymorphism in the PE_PGRS9 gene of Mycobacterium tuberculosis was designed to assess the mutability of a sequence containing interspersed PGRS repeats. Application of the assay in Mycobacterium smegmatis revealed sequence plasticity: in addition to recapitulating the mutation on which it was based, other mutations likely mediated by replication slippage between PGRS repeats were detected. However, the mutation rates argued against marked hypermutability of such sequences in mycobacteria.