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Gene therapy for severe hemophilia B is advancing and offers sustained disease amelioration with a single treatment. We have reported the efficacy and safety of AMT-060, an investigational gene therapy comprising an adeno-associated virus serotype 5 capsid encapsidating the codon-optimized wild-type human factor IX (WT hFIX) gene with a liver-specific promoter, in patients with severe hemophilia B. Treatment with 2 × 1013 gc/kg AMT-060 showed sustained and durable FIX activity of 3%–13% and a substantial reduction in spontaneous bleeds without T cell-mediated hepatoxicity. To achieve higher FIX activity, we modified AMT-060 to encode the R338L “Padua” FIX variant that has increased specific activity (AMT-061). We report the safety and increased FIX activity of AMT-061 in non-human primates. Animals (n = 3/group) received intravenous AMT-060 (5 × 1012 gc/kg), AMT-061 (ranging from 5 × 1011 to 9 × 1013 gc/kg), or vehicle. Human FIX protein expression, FIX activity, and coagulation markers including D-dimer and thrombin-antithrombin complexes were measured. At equal doses, AMT-060 and AMT-061 resulted in similar human FIX protein expression, but FIX activity was 6.5-fold enhanced using AMT-061. Both vectors show similar safety and transduction profiles. Thus, AMT-061 holds great promise as a more potent FIX replacement gene therapy with a favorable safety profile. Keywords: AAV, AAV5, AMT-060, AMT-061, adeno-associated virus, factor IX, gene therapy, hemophilia B, non-human primate

Short hairpin RNAs (shRNAs) are widely used for gene knockdown by inducing the RNA interference (RNAi) mechanism, both for research and therapeutic purposes. The shRNA precursor is processed by the RNase III-like enzyme Dicer into biologically active small interfering RNA (siRNA). This effector molecule subsequently targets a complementary mRNA for destruction via the Argonaute 2 (AGO2) complex. The cellular role of Dicer concerns the processing of pre-miRNAs into mature microRNA (miRNA). Recently, a non-canonical pathway was reported for the biogenesis of miR-451, which bypasses Dicer and is processed instead by the slicer activity of AGO2, followed by the regular AGO2-mediated mRNA targeting step. Interestingly, shRNA designs that are characterized by a relatively short basepaired stem also bypass Dicer to be processed by AGO2. We named this design AgoshRNA as these molecules depend on AGO2 both for processing and silencing activity. In this study, we investigated diverse mechanistic aspects of this new class of AgoshRNA molecules. We probed the requirements for AGO2-mediated processing of AgoshRNAs by modification of the proposed cleavage site in the hairpin. We demonstrate by deep sequencing that AGO2-processed AgoshRNAs produce RNA effector molecules with more discrete ends than the products of the regular shRNA design. Furthermore, we tested whether trimming and tailing occurs upon AGO2-mediated processing of AgoshRNAs, similar to what has been described for miR-451. Finally, we tested the prediction that AgoshRNA activity, unlike that of regular shRNAs, is maintained in Dicer-deficient cell types. These mechanistic insights could aid in the design of optimised AgoshRNA tools and therapeutics.

RNA interference (RNAi) is highly effective in inhibiting human immunodeficiency virus type 1 (HIV-1) replication by the expression of antiviral short hairpin RNA (shRNA) in stably transduced T-cell lines. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs against highly conserved regions of the HIV-1 RNA genome. The future in vivo application of such a gene therapy protocol will reach only a fraction of the T cells, such that HIV-1 replication will continue in the unmodified T cells, thereby possibly frustrating the therapy by generation of HIV-1 variants that escape from the inhibition imposed by the protected cells. We studied virus inhibition and evolution in pure cultures of shRNA-expressing cells versus mixed cell cultures of protected and unprotected T cells. The addition of the unprotected T cells indeed seems to accelerate HIV-1 evolution and escape from a single shRNA inhibitor. However, expression of three antiviral shRNAs from a single lentiviral vector prevents virus escape even in the presence of unprotected cells. These results support the idea to validate the therapeutic potential of this anti-HIV approach in appropriate in vivo models.

Short hairpin RNAs (shRNAs) are widely used to induce RNA interference (RNAi). We tested a variety of shRNAs that differed in stem length and terminal loop size and revealed strikingly different RNAi activities and shRNA-processing patterns. Interestingly, we identified a specific shRNA design that uses an alternative Dicer-independent processing pathway. Detailed analyses indicated that a short shRNA stem length is critical for avoiding Dicer processing and activation of the alternative processing route, in which the shRNA is incorporated into RISC and processed by the AGO2-mediated slicer activity. Such alternatively processed shRNAs (AgoshRNAs) yield only a single RNA strand that effectively induces RNAi, whereas conventional shRNA processing results in an siRNA duplex of which both strands can trigger RNAi. Both the processing and subsequent RNAi activity of these AgoshRNAs are thus mediated by the RISC-component AGO2. These results have important implications for the future design of more specific RNAi therapeutics.

The discovery of RNA interference (RNAi), an evolutionary conserved gene silencing mechanism that is triggered by double stranded RNA, has led to tremendous efforts to use this technology for basic research and new RNA therapeutics. RNAi can be induced via transfection of synthetic small interfering RNAs (siRNAs), which results in a transient knockdown of the targeted mRNA. For stable gene silencing, short hairpin RNA (shRNA) or microRNA (miRNA) constructs have been developed. In mammals and humans, the natural RNAi pathway is triggered via endogenously expressed miRNAs. The use of modified miRNA expression cassettes to elucidate fundamental biological questions or to develop therapeutic strategies has received much attention. Viral vectors are particularly useful for the delivery of miRNA genes to specific target cells. To date, many viral vectors have been developed, each with distinct characteristics that make one vector more suitable for a certain purpose than others. This review covers the recent progress in miRNA-based gene-silencing approaches that use viral vectors, with a focus on their unique properties, respective limitations and possible solutions. Furthermore, we discuss a related topic that involves the insertion of miRNA-target sequences in viral vector systems to restrict their cellular range of gene expression. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.

Gene silencing by RNA interference (RNAi) can be achieved by intracellular expression of a short hairpin RNA (shRNA) that is processed into the effective small interfering RNA (siRNA) inhibitor by the RNAi machinery. Previous studies indicate that shRNA molecules do not always reflect the activity of corresponding synthetic siRNAs that attack the same target sequence. One obvious difference between these two effector molecules is the hairpin loop of the shRNA. Most studies use the original shRNA design of the pSuper system, but no extensive study regarding optimization of the shRNA loop sequence has been performed. We tested the impact of different hairpin loop sequences, varying in size and structure, on the activity of a set of shRNAs targeting HIV-1. we were able to transform weak inhibitors into intermediate or even strong shRNA inhibitors by replacing the loop sequence. We demonstrate that the efficacy of these optimized shRNA inhibitors is improved significantly in different cell types due to increased siRNA production. These results indicate that the loop sequence is an essential part of the shRNA design. The optimized shRNA loop sequence is generally applicable for RNAi knockdown studies, and will allow us to develop a more potent gene therapy against HIV-1. (C) 2010 Elsevier B.V. All rights reserved

RNAi-based gene therapy is a powerful approach to treat viral infections because of its high efficiency and sequence specificity. The HIV-1-based lentiviral vector system is suitable for the delivery of RNAi inducers to HIV-1 susceptible cells due to its ability to transduce nondividing cells, including hematopoietic stem cells, and its ability for stable transgene delivery into the host cell genome. However, the presence of anti-HIV short hairpin RNA (shRNA) and microRNA (miRNA) cassettes can negatively affect the lentiviral vector titers. We show that shRNAs, which target the vector genomic RNA, strongly reduced lentiviral vector titers but inhibition of the RNAi pathway via saturation could rescue vector production. The presence of miRNAs in the vector RNA genome (sense orientation) results in a minor titer reduction due to Drosha processing. A major cause for titer reduction of miRNA vectors is due to incompatibility of the cytomegalovirus promoter with the lentiviral vector system. Replacement of this promoter with an inducible promoter resulted in an almost complete restoration of the vector titer. We also showed that antisense poly(A) signal sequences can have a dramatic effect on the vector titer. These results show that not all sequences are compatible with the lentiviral vector system and that care should be taken in the design of lentiviral vectors encoding RNAi inducers.

RNA interference (RNAi) is a widely used gene suppression tool that holds great promise as a novel antiviral approach. However, for error-prone viruses including human immunodeficiency virus type 1(HIV-1), a combinatorial approach against multiple conserved sequences is required to prevent the emergence of RNAi-resistant escape viruses. Previously, we constructed extended short hairpin RNAs (e-shRNAs) that encode two potent small interfering RNAs (siRNAs) (e2-shRNAs). We showed that a minimal hairpin stem length of 43 base pairs (bp) is needed to obtain two functional siRNAs. In this study, we elaborated on the e2-shRNA design to make e-shRNAs encoding three or four antiviral siRNAs. We demonstrate that siRNA production and the antiviral effect is optimal for e3-shRNA of 66 bp. Further extension of the hairpin stem results in a loss of RNAi activity. The same was observed for long hairpin RNAs (lhRNAs) that target consecutive HIV-1 sequences. Importantly, we show that HIV-1 replication is durably inhibited in T cells stably transduced with a lentiviral vector containing the e3-shRNA expression cassette. These results show that e-shRNAs can be used as a combinatorial RNAi approach to target error-prone viruses.

Human immunodeficiency virus type 1 (HIV-1) replication in T cells can be inhibited by RNA interference (RNAi) through short hairpin RNA (shRNA) expression from a lentiviral vector. However, for the development of a durable RNAi-based gene therapy against HIV-1, multiple shRNAs need to be expressed simultaneously in order to avoid viral escape. In this study, we tested a multiple shRNA expression strategy for different shRNAs using repeated promoters in a lentiviral vector. Although highly effective in co-transfection experiments, a markedly reduced activity of each expressed shRNA was observed in transduced cells. We found that this reduced activity was due to recombination of the expression cassette repeat sequences during the transduction of the lentiviral vector, which resulted in deletions of one or multiple cassettes. To avoid recombination, we tested different promoters for multiple shRNA expression. We compared the activity of the human polymerase III promoters U6, H1, and 7SK and the polymerase II U1 promoter. Activities of these promoters were similar, irrespective of which shRNA was expressed. We showed that these four expression cassettes can be combined in a single lentiviral vector without causing recombination. Moreover, whereas HIV-1 could escape from a single shRNA, we now show that HIV-1 escape can be prevented when four shRNAs are simultaneously expressed in a cell.

RNA interference (RNAi) can be induced by intracellular expression of a short hairpin RNA (shRNA). Processing of the shRNA requires the RNaseIII-like Dicer enzyme to remove the loop and to release the biologically active small interfering RNA (siRNA). Dicer is also involved in microRNA (miRNA) processing to liberate the mature miRNA duplex, but recent studies indicate that miR-451 is not processed by Dicer. Instead, this miRNA is processed by the Argonaute 2 (Ago2) protein, which also executes the subsequent cleavage of a complementary mRNA target. Interestingly, shRNAs that structurally resemble miR-451 can also be processed by Ago2 instead of Dicer. The key determinant of these “AgoshRNA” molecules is a relatively short basepaired stem, which avoids Dicer recognition and consequently allows alternative processing by Ago2. AgoshRNA processing yields a single active RNA strand, whereas standard shRNAs produce a duplex with guide and passenger strands and the latter may cause adverse off-target effects. In this study, we converted previously tested active anti-HIV-1 shRNA molecules into AgoshRNA. We tested several designs that could potentially improve AgoshRNA activity, including extension of the complementarity between the guide strand and the mRNA target and reduction of the thermodynamic stability of the hairpins. We demonstrate that active AgoshRNAs can be generated. However, the RNAi activity is reduced compared to the matching shRNAs. Despite reduced RNAi activity, comparison of an active AgoshRNA and the matching shRNA in a sensitive cell toxicity assay revealed that the AgoshRNA is much less toxic.

Recent evidence indicates the presence of alternative pathways for microRNA (miRNA) and short hairpin (shRNA) processing. Specifically, some of these molecules are refractory to Dicer-mediated processing, which allows alternative processing routes via the Ago2 endonuclease. The resulting RNA molecules differ in size and sequence and will thus trigger the silencing of different target RNAs. It is, therefore, important to understand these processing routes in mechanistic detail such that one can design exclusive RNA reagents for a specific processing route. The exact sh/miRNA properties that determine this routing toward Dicer or Ago2 are incompletely understood. The size of the base-paired stem seems an important determinant, but other RNA elements may contribute as well. In this study, we document the importance of a weak G-U or U-G base pair at the top of the hairpin stem.

RNA interference (RNAi) holds great promise as gene therapy approach against viral pathogens, including HIV-1. A specific anti-HIV-1 response can be induced via transfection of synthetic small interfering RNAs (siRNAs) or via intracellular transgene expression of short hairpin RNAs (shRNAs) or microRNAs (miRNAs). Both targeting of the viral mRNAs or the mRNAs for cellular co-factors that are required for viral replication have been shown successful in suppressing HIV-1 replication. However, like conventional mono-therapies, the use of a single anti-HIV-1 RNAi inducer results in the emergence of RNAi-escape mutants. To prevent viral escape, a combinatorial RNAi approach should be used in which multiple RNAi effectors against HIV-1 are simultaneously expressed. Although induced RNAi is able to trigger a robust and specific knockdown of virus replication, it is becoming apparent that RNAi therapeutics encounter difficulties concerning off-target effects, cellular toxicity and specific delivery to the right cells. This review covers the recent progress in combinatorial RNAi-based approaches against HIV-1 using lentiviral vectors as a delivery system. The potential for a clinical gene therapy application will be discussed.