RESUMO
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The majority of CFTR mutations result in impaired chloride channel function as only a fraction of the mutated CFTR reaches the plasma membrane. The development of a therapeutic approach that facilitates increased cell-surface expression of CFTR could prove clinically relevant. Here, we evaluate and contrast two molecular approaches to activate CFTR expression. We find that an RNA-guided nuclease null Cas9 (dCas9) fused with a tripartite activator, VP64-p65-Rta can activate endogenous CFTR in cultured human nasal epithelial cells from CF patients. We also find that targeting BGas, a long non-coding RNA involved in transcriptionally modulating CFTR expression with a gapmer, induced both strong knockdown of BGas and concordant activation of CFTR. Notably, the gapmer can be delivered to target cells when generated as electrostatic particles with recombinant HIV-Tat cell penetrating peptide (CPP), when packaged into exosomes, or when loaded into lipid nanoparticles (LNPs). Treatment of patient-derived human nasal epithelial cells containing F508del with gapmer-CPP, gapmer-exosomes, or LNPs resulted in increased expression and function of CFTR. Collectively, these observations suggest that CRISPR/dCas-VPR (CRISPR) and BGas-gapmer approaches can target and specifically activate CFTR.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/genética , Terapia de Alvo Molecular/métodos , Mucosa Nasal/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/genética , Fibrose Cística/metabolismo , Fibrose Cística/terapia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Nanopartículas/química , Mucosa Nasal/citologia , RNA Guia de Cinetoplastídeos/farmacologia , RNA Longo não Codificante/genética , Ativação Transcricional , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Cystic fibrosis (CF) is a life-shortening genetic disease. The root cause of CF is heritable recessive mutations that affect the cystic fibrosis transmembrance conductance regulator (CFTR) gene and the subsequent expression and activity of encoded ion channels at the cell surface. We show that CFTR is regulated transcriptionally by the actions of a novel long noncoding RNA (lncRNA), designated as BGas, that emanates from intron 11 of the CFTR gene and is expressed in the antisense orientation relative to the protein coding sense strand. We find that BGas functions in concert with several proteins including HMGA1, HMGB1, and WIBG to modulate the local chromatin and DNA architecture of intron 11 of the CFTR gene and thereby affects transcription. Suppression of BGas or its associated proteins results in a gain of both CFTR expression and chloride ion function. The observations described here highlight a previously underappreciated mechanism of transcriptional control and suggest that BGas may serve as a therapeutic target for specifically activating expression of CFTR.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Regulação da Expressão Gênica , RNA Antissenso/genética , RNA Longo não Codificante , Fibrose Cística/metabolismo , Proteínas de Ligação a DNA/metabolismo , Loci Gênicos , Humanos , Modelos Biológicos , Ligação ProteicaRESUMO
HIV-1 provirus integration results in a persistent latently infected reservoir that is recalcitrant to combined antiretroviral therapy (cART) with lifelong treatment being the only option. The "shock and kill" strategy aims to eradicate latent HIV by reactivating proviral gene expression in the context of cART treatment. Gene-specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising single guide RNAs (sgRNAs) with a nuclease-deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the long terminal repeat promoter of HIV-1 and identified a "hotspot" for activation within the viral enhancer sequence. Activating sgRNAs transcriptionally modulated the latent proviral genome across multiple different in vitro latency cell models including T cells comprising a clonally integrated mCherry-IRES-Tat (LChIT) latency system. We detected consistent and effective activation of latent virus mediated by activator sgRNAs, whereas latency reversal agents produced variable activation responses. Transcriptomic analysis revealed dCas9-VP64/sgRNAs to be highly specific, while the well-characterized chemical activator TNFα induced widespread gene dysregulation. CRISPR-mediated gene activation represents a novel system which provides enhanced efficiency and specificity in a targeted latency reactivation strategy and represents a promising approach to a "functional cure" of HIV/AIDS.
Assuntos
Sistemas CRISPR-Cas , HIV-1/fisiologia , Complexos Multiproteicos/metabolismo , Ativação Viral , Latência Viral , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Regulação Viral da Expressão Gênica , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/genética , Humanos , NF-kappa B/metabolismo , Motivos de Nucleotídeos , Ligação Proteica , RNA Guia de Cinetoplastídeos/genética , Ativação TranscricionalRESUMO
Amplification or overexpression of neuronal MYC (MYCN) is associated with poor prognosis of human neuroblastoma. Three isoforms of the MYCN protein have been described as well as a protein encoded by an antisense transcript (MYCNOS) that originates from the opposite strand at the MYCN locus. Recent findings suggest that some antisense long non-coding RNAs (lncRNAs) can play a role in epigenetically regulating gene expression. Here we report that MYCNOS transcripts function as a modulator of the MYCN locus, affecting MYCN promoter usage and recruiting various proteins, including the Ras GTPase-activating protein-binding protein G3BP1, to the upstream MYCN promoter. Overexpression of MYCNOS results in a reduction of upstream MYCN promoter usage and increased MYCN expression, suggesting that the protein-coding MYCNOS also functions as a regulator of MYCN ultimately controlling MYCN transcriptional variants. The observations presented here demonstrate that protein-coding transcripts can regulate gene transcription and can tether regulatory proteins to target loci.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Regiões Promotoras Genéticas/genética , RNA Antissenso/genética , Azacitidina/farmacologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , DNA Helicases , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Modelos Genéticos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proteínas de Ligação a Poli-ADP-Ribose , Isoformas de Proteínas/genética , Inibidores da Síntese de Proteínas/farmacologia , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The abundance of long noncoding RNAs (lncRNAs) and their wide range of functional roles in human cells are fast becoming realized. Importantly, lncRNAs have been identified as epigenetic modulators and consequently play a pivotal role in the regulation of gene expression. A human immunodeficiency virus-encoded antisense RNA transcript has recently been reported and we sought to characterize this RNA and determine its potential role in viral transcription regulation. The intrinsic properties of this human immunodeficiency virus-expressed lncRNA were characterized and the data presented here suggest that it functions as an epigenetic brake to modulate viral transcription. Suppression of this long antisense transcript with small single-stranded antisense RNAs resulted in the activation of viral gene expression. This lncRNA was found to localize to the 5' long-term repeats (LTR) and to usurp components of endogenous cellular pathways that are involved in lncRNA directed epigenetic gene silencing. Collectively, we find that this viral expressed antisense lncRNA is involved in modulating human immunodeficiency virus gene expression and that this regulatory effect is due to an alteration in the epigenetic landscape at the viral promoter.
Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , HIV-1/fisiologia , RNA Antissenso/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Viral/genética , Azacitidina/farmacologia , Epigênese Genética , Células HEK293 , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Ácidos Hidroxâmicos/farmacologia , Células Jurkat , Regiões Promotoras GenéticasRESUMO
The Human Immunodeficiency Virus (HIV) belongs to the subfamily of lentiviruses that are characterized by long incubation periods and chronic, persistent infection. The virus integrates into the genome of infected CD4+ cells and, in a subpopulation of cells, adopts a transcriptionally silent state, a process referred to a viral latency. This property makes it exceedingly difficult to therapeutically target the virus and eradicate infection. If left untreated, the inexorable demise of the infected individual's immune system ensues, a causal result of Acquired Immunodeficiency Syndrome (AIDS). Latently infected cells provide a reservoir that maintains viral infection indefinitely. In this chapter we explore the role of noncoding RNAs in HIV infection and in the establishment and maintenance of viral latency. Both short and long noncoding RNAs are endogenous modulators of epigenetic regulation in human cells and play an active role in gene expression. Lastly, we explore therapeutic modalities based on expressed RNAs that are capable of countering infection, transcriptionally regulating the virus, and suppressing or activating the latent state.
Assuntos
Terapia Genética/métodos , Infecções por HIV/terapia , HIV-1/fisiologia , RNA não Traduzido/genética , Latência Viral , Animais , Doença Crônica , Infecções por HIV/genética , HIV-1/genética , Humanos , Terapia de Alvo Molecular/métodos , Interferência de RNA/fisiologia , Transcrição Gênica , Latência Viral/genéticaRESUMO
Mirtrons, short hairpin pre-microRNA (miRNA) mimics directly produced by intronic splicing, have recently been identified and experimentally confirmed in invertebrates. While there is evidence to suggest several mammalian miRNAs have mirtron origins, this has yet to be experimentally demonstrated. Here, we characterize the biogenesis of mammalian mirtrons by ectopic expression of splicing-dependent mirtron precursors. The putative mirtrons hsa-miR-877, hsa-miR-1226 and mmu-miR-1224 were designed as introns within eGFP. Correct splicing and function of these sequences as introns was shown through eGFP fluorescence and RT-PCR, while all mirtrons suppressed perfectly complementary luciferase reporter targets to levels similar to that of corresponding independently expressed pre-miRNA controls. Splicing-deficient mutants and disruption of key steps in miRNA biogenesis demonstrated that mirtron-mediated gene knockdown was splicing-dependent, Drosha-independent and had variable dependence on RNAi pathway elements following pre-miRNA formation. The silencing effect of hsa-miR-877 was further demonstrated to be mediated by the generation of short anti-sense RNA species expressed with low abundance. Finally, the mammalian mirtron hsa-miR-877 was shown to reduce mRNA levels of an endogenous transcript containing hsa-miR-877 target sites in neuronal SH-SY5Y cells. This work confirms the mirtron origins of three mammalian miRNAs and suggests that they are a functional class of splicing-dependent miRNAs which are physiologically active.
Assuntos
MicroRNAs/metabolismo , Interferência de RNA , Precursores de RNA/metabolismo , Animais , Linhagem Celular , Humanos , Íntrons , Camundongos , Neurônios/metabolismo , Processamento Pós-Transcricional do RNA , Splicing de RNA , Ribonuclease III/fisiologiaRESUMO
Several different approaches exist to generate expressed RNA interference (RNAi) precursors for multiple target inhibition, a strategy referred to as combinatorial (co)RNAi. One such approach makes use of RNA Pol III-expressed long hairpin RNAs (lhRNAs), which are processed by Dicer to generate multiple unique short interfering siRNA effectors. However, because of inefficient intracellular Dicer processing, lhRNA duplexes have been limited to generating two independent effective siRNA species. In this study, we describe a novel strategy whereby four separate anti-HIV siRNAs were generated from a single RNA Pol III-expressed transcript. Two optimized lhRNAs, each comprising two active anti-HIV siRNAs, were placed in tandem to form a double long hairpin (dlhRNA) expression cassette, which encodes four unique and effective siRNA sequences. Processing of the 3' position lhRNA was more variable but effective multiple processing was possible by manipulating the order of the siRNA-encoding sequences. Importantly, unlike shRNAs, Pol III-expressed dlhRNAs did not compete with endogenous and exogenous microRNAs to disrupt the RNAi pathway. The versatility of expressed lhRNAs is greatly expanded and we provide a mechanism for generating transcripts with modular lhRNAs motifs that contribute to improved coRNAi.
Assuntos
Fármacos Anti-HIV/metabolismo , Interferência de RNA , RNA Polimerase III/metabolismo , Precursores de RNA/metabolismo , RNA Interferente Pequeno/metabolismo , Fármacos Anti-HIV/química , Linhagem Celular , Vetores Genéticos , Humanos , Interferons/metabolismo , Precursores de RNA/química , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Nuclear Pequeno/genéticaRESUMO
Gene-based therapies represent a promising therapeutic paradigm for the treatment of HIV-1, as they have the potential to maintain sustained viral inhibition with reduced treatment interventions. Such an option may represent a long-term treatment alternative to highly active antiretroviral therapy. Methods: We previously described a therapeutic approach, referred to as transcriptional gene silencing (TGS), whereby small noncoding RNAs directly inhibit the transcriptional activity of HIV-1 by targeting sites within the viral promoter, specifically the 5' long terminal repeat (LTR). TGS differs from traditional RNA interference (RNAi) in that it is characterized by concomitant silent-state epigenetic marks on histones and DNA. To deliver TGS-inducing RNAs, we developed functional RNA conjugates based on the previously reported dual function of the gp120 (A-1) aptamer conjugated to 27-mer Dicer-substrate anti-HIV-1 siRNA (dsiRNA), LTR-362. Results: We demonstrate here that high levels of processed guide RNAs localize to the nucleus in infected T lymphoblastoid CEM cell line and primary human CD4+ T-cells. Treatment of the aptamer-siRNA conjugates induced TGS with an ~10-fold suppression of viral p24 levels as measured at day 12 post infection. To explore the silencing efficacy of aptamer-siRNA conjugates in vivo, HIV-1-infected humanized NOD/SCID/IL2 rγnull mice (hu-NSG) were treated with the aptamer-siRNA conjugates. Systemic delivery of the A-1-stick-LTR-362 27-mer siRNA conjugates suppressed HIV-1 infection and protected CD4+ T cell levels in viremia hu-NSG mice. Principle conclusions: Collectively these data suggest that the gp120 aptamer-dsiRNA conjugate design is suitable for systemic delivery of small RNAs that can be used to suppress HIV-1.
Assuntos
Aptâmeros de Nucleotídeos/genética , RNA Helicases DEAD-box/genética , Regulação Viral da Expressão Gênica , Inativação Gênica , Infecções por HIV/terapia , HIV-1/genética , RNA Viral/genética , Ribonuclease III/genética , Animais , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular Tumoral , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/metabolismo , Modelos Animais de Doenças , Terapia Genética/métodos , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , Repetição Terminal Longa de HIV , HIV-1/crescimento & desenvolvimento , HIV-1/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Conformação de Ácido Nucleico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/antagonistas & inibidores , RNA Viral/metabolismo , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/metabolismo , Transcrição GênicaRESUMO
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.
Assuntos
Repetição Terminal Longa de HIV , HIV-1/genética , HIV-1/fisiologia , Interferência de RNA , RNA Antissenso/genética , Sequência de Bases , Linhagem Celular , Humanos , Interferon beta/biossíntese , Dados de Sequência Molecular , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , RNA Antissenso/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transfecção , Regiões não TraduzidasRESUMO
The discovery of long non-coding RNAs (lncRNAs) and the elucidation of the mechanisms by which they affect different disease states are providing researchers with a better understanding of a wide array of disease pathways. Moreover, lncRNAs are presenting themselves as both unique diagnostic biomarkers as well as novel targets against which to develop new therapeutics. Here we will explore the intricate network of non-coding RNAs associated with infection by the human immunodeficiency virus (HIV). Non-coding RNAs derived from both the human host as well as those from HIV itself are emerging as important regulatory elements. We discuss here the various mechanisms through which both small and long non-coding RNAs impact viral replication, pathogenesis and disease progression. Given the lack of an effective vaccine or cure for HIV and the scale of the current pandemic, a deeper understanding of the complex interplay between non-coding RNAs and HIV will support the development of innovative strategies for the treatment of HIV/acquired immunodeficiency disease (AIDS).
Assuntos
Infecções por HIV/metabolismo , HIV-1/metabolismo , RNA Longo não Codificante/metabolismo , RNA Viral/metabolismo , Animais , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Interações Hospedeiro-Patógeno , Humanos , RNA Longo não Codificante/genética , RNA Viral/genéticaRESUMO
INTRODUCTION: The use of antiretroviral therapy has led to a significant decrease in morbidity and mortality in HIV-infected individuals. Nevertheless, gene-based therapies represent a promising therapeutic paradigm for HIV-1, as they have the potential for sustained viral inhibition and reduced treatment interventions. One new method amendable to a gene-based therapy is the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) gene editing system. AREAS COVERED: CRISPR/Cas9 can be engineered to successfully modulate an array of disease-causing genetic elements. We discuss the diverse roles that CRISPR/Cas9 may play in targeting HIV and eradicating infection. The Cas9 nuclease coupled with one or more small guide RNAs can target the provirus to mediate excision of the integrated viral genome. Moreover, a modified nuclease-deficient Cas9 fused to transcription activation domains may induce targeted activation of proviral gene expression allowing for the purging of the latent reservoirs. These technologies can also be exploited to target host dependency factors such as the co-receptor CCR5, thus preventing cellular entry of the virus. EXPERT OPINION: The diversity of the CRISPR/Cas9 technologies offers great promise for targeting different stages of the viral life cycle, and have the capacity for mediating an effective and sustained genetic therapy against HIV.
Assuntos
Proteínas Associadas a CRISPR/uso terapêutico , Terapia Genética/tendências , Infecções por HIV/genética , Infecções por HIV/terapia , Animais , Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Terapia Genética/métodos , HIV-1/genética , HumanosRESUMO
The RNA interference (RNAi) pathway has in recent years been exploited for the development of novel antiviral therapies. The emergence of viral escape mutants, however, is a major impediment to the use of RNAi effectors to treat highly mutable viruses such as HIV-1. A combinatorial approach is therefore required for long-term inhibition of gene expression. RNA Pol III-driven long hairpin RNA (lhRNA) duplexes can be cleaved several times by Dicer, yielding multiple functional siRNAs from a single construct. Here we describe a method for the generation of ectopically expressed U6-lhRNAs encoding three separate siRNA sequences targeting unique sites in HIV-1. This methodological overview explains some crucial aspects of lhRNA design and cloning as well as facile experiments to determine their efficacy in cell culture.
Assuntos
DNA Polimerase III/metabolismo , Técnicas de Transferência de Genes , HIV-1/genética , Conformação de Ácido Nucleico , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/metabolismo , Sequência de Bases , Northern Blotting , Linhagem Celular , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Humanos , Dados de Sequência Molecular , Plasmídeos/genética , Reação em Cadeia da Polimerase , RNA Nuclear Pequeno/genéticaRESUMO
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.
Assuntos
Expressão Gênica , HIV/genética , Interferência de RNA , Fármacos Anti-HIV/uso terapêutico , RNA Viral/genéticaRESUMO
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.