The right approach to human therapy

The RIGHT (RNA Interference Technology as Human Therapeutic Tool) consortium consists of 18 research institutions and four companies from nine European countries. The project has been funded as an integrated project by the European Commission’s Sixth Framework Programme for Research and Development (FP6) since January 2005. Thomas F. Meyer from the Max Planck Institute for Infection Biology in Berlin is coordinating this European research project that aims at exploiting the vast potential of RNA interference (RNAi) for human therapy.

RNA interference is a technology where short (21-23 bp) double-stranded RNA is used to induce the sequence-specific degradation of mRNA and thereby block the synthesis of the corresponding protein. This technology revolutionised basic research, by making the analysis of gene function relatively easy. As a technique to control gene expression, it also has the potential to specifically regulate the production of disease-associated genes.

For many severe, unvanquished diseases, such as cancer or HIV, research is mainly oriented towards either chemical drug development, usually based on an empirical ‘trial and error’ approach or vaccinology. Vaccinology has been key in the treatment and prevention of selected diseases, but did not reach the general applicability necessary to defeat the majority of human diseases.

RNAi allows now for an entirely novel therapeutic approach with an immense potential for effective human therapies. Based on the knowledge of roles that specific gene sequences play in disease, RNAi molecules can be generated for down-regulating the expression of distinct dysfunctional genes, enabling a rational targeted therapy. It is highly effective and sensitive since it targets an early gene functional level (mRNA). Additionally, RNAi is regarded to have a minimum of toxic or other side effects due to its sequence specificity. The availability of RNA-based tools would dramatically impact the efficiency and timely development of new therapies for severe diseases caused by gene malfunctions such as are prevalent in cancer or viral diseases, in particular where they are triggered by rapidly mutating viruses such as HIV.

Application of RNAi as a therapeutic tool in vivo requires that some technical problems first be overcome, such as insufficient uptake or low stability of the inhibitors, undesired interferon response or unspecific silencing of other genes. Recently, Grimm et al. (Nature 2006) could show that shRNAs can lead to fatality in mice due to a competition of these shRNAs with the cellular miRNA processing machinery. The RIGHT project is divided in five competence domains to address the multiple facets of therapy development: Molecular mechanisms and technologies, Chemical tools, Genetic tools, Pharmacokinetic assessment of RNAi applications in living animals and Cell biology and disease models. In RIGHT, experts from these synergistic competence areas are working together to exploit the vast potential of this interesting new technology and to make the application of RNAi as therapeutic tool possible in a multidisciplinary way.

Basic research is necessary to deepen the understanding of the molecular mechanisms of RNAi and microRNAs as a basis to use this new technology for therapeutic approaches. In addition, strategies and tools are needed to share this new knowledge base with the scientific community. The groups of Annick Harel-Bellan at CNRS in Villejuif, France and of Irene Bozzoni at the University of Rome, Italy, are working successfully on the identification and characterisation of miRNAs. It could be shown that miR-181 participates in muscle cell differentiation (Naguibneva et al. 2005) and that miR-223 is involved in human granulocyte differentiation (Fazi et al., 2005). Furthermore, a Web-based miRNA browser based on multiple genome sequence alignments has been developed in Jørgen Kjems’ group at Aarhus University, Denmark and will soon be publicly available. This group also developed a miRNA detection system based on padlock probes and rolling circle amplification (Jonstrup et al., 2006).

In the groups of Jørgen Kjems in Aarhus, Marino Zerial at the Max Planck Institute for Molecular Cell Biology and Genetics in Germany and Olli Kallioniemi at the VTT Technical Research Centre of Finland in Turku, techniques are being developed for screening and optimisation of RNAi constructs.

There are two main possibilities to introduce RNAi into the organism: either one can use small interfering RNAs (siRNAs) that are delivered directly into cells as naked siRNAs or with the help of a transfection reagent. The other possibility is to use shRNAs that are delivered on a plasmid into the cell and are thereby expressed using the machinery of the cell itself. Chemists from Sweden, Denmark, Germany and Belgium are working on ‘chemical tools’ to improve the properties of siRNAs. Here chemical modifications on already tested inhibitor sequences are synthesised to increase the stability of the siRNAs in vivo. Such modifications are the introduction of oxetane or azetedine nucleosides (Bogucka et al. 2005; Honcharenko et al 2006), modifications synthesised in the group of Jyoti Chattopadhyaya at the University of Uppsala, Sweden. One other possibility to increase the stability of the inhibitors is the introduction of LNAs, a technique used in the group of Jesper Wengel at Southern Denmark University in Odense (Kauppinen et al., 2006). Furthermore, other modifications or lipid-conjugations are applied to achieve a better delivery to and uptake by targeted organs or cells, thereby increasing the efficiency of the inhibitors in vivo. Novel delivery strategies are also being tested by the group of Jørgen Kjems at Aarhus University, who is developing chitosan/siRNA nanoparticles to use as a siRNA delivery methodology for the more efficient application of RNA-mediated therapy to systemic and mucosal diseases (Howard et al., 2006). All of these efforts should help to enhance the efficiency of siRNAs in vivo and thereby improve the potential of RNAi as therapeutic tool.

As an alternative, especially for the treatment of chronic diseases, gene therapeutic approaches are being taken into consideration. To improve the applicability of shRNAs, ‘genetic tools’ can be employed. Different viral and non-viral RNAi-vectors are being developed to improve the plasmid uptake and the targeting of inhibitors to specific organs. The group of Luigi Naldini at the Fondazione Centro San Raffaele del Monte Tabor in Milano, Italy, uses lentiviral vectors that include miRNA target sequences to regulate gene expression in different organs (Brown et al., 2006). With these vectors, the delivery and segregated expression of shRNAs among different tissues is possible. Their studies on endogenous miRNA regulation have provided them a novel framework to develop more efficient and safe vectors to express shRNAs.

It is necessary to evaluate all these novel chemically modified siRNAs and new vector systems for shRNA expression in cell culture systems and animal models to assess not only their efficiency in knocking-down the targeted gene, but to assess their pharmacokinetic properties and possible side effects. Here the group of Marino Zerial at the Max Planck Institute for Molecular Cell Biology and Genetics in Dresden, Germany, focuses on uptake and intracellular trafficking of the inhibitors. The group of Olli Kallioniemi at VTT in Turku studies possible off-target effects by applying microarray technology. Using proven and generally accepted protocols in the field, a detailed pharmacological analysis of the RIGHT RNAi applications in animals will be performed.

The assessment of the inhibitors is first performed with standard targets, such as eGFP or luciferase in cell culture and the corresponding transgenic animal models. A subsequent evaluation of the most promising RNAi strategies will then be performed in several disease models to approach the final goal of developing effective disease-specific RNAi tools. At the University of Torino, Italy, the groups of Carola Ponzetto and Giorgio Inghirami are working on the treatment of different forms of cancer with RNAi strategies. In the context of gastric cancer, it could be demonstrated that the inhibition of Tpr-Met expression by RNAi suppresses Tpr-Met-mediated transformation and tumorigenesis in a cell culture model (Taulli et al., 2005). And in the context of anaplastic large-cell lymphomas, the ablation of anaplastic lymphoma kinase (ALK) via RNAi leads to a reversion of the transformed phenotype in ALK+ mouse embryonic fibroblasts (Piva et al., 2006). In addition to the intrinsic disease models, different infectious disease models are being developed. Here two different strategies are followed for an effective cure. Either the pathogen is targeted directly by RNAi or cell components are identified that are essential for the survival of the pathogen which become the focus of the RNAi treatment. The second strategy is of particular interest for the treatment of infections with highly variable pathogens, including HIV or influenza. Here, effective therapies require the accurate prediction of the pathogenic variant. Drugs directed against viral components can rapidly lose their effectiveness because of mutations in the pathogen.

At the Biomedical Sciences Research Center ‘Alexander Fleming’ in Vari, Greece, the group of George Mosialos is working on an RNAi-based therapy directed against EBV. The groups of Jørgen Kjems at Aarhus University, Denmark and of Guido Kroemer at CNRS, Villejuif, France, are developing RNAi strategies to combat HIV. A first step was the successful identification of target sides for the design of possible inhibitors in the HIV genome (Jakobsen et al., 2004). The group of Guido Kroemer focuses on the cellular processes involved in HIV infection and subsequent cell death (Perfettini et al. 2005). Novel approaches of RNAi to treat influenza infections are applied in the group of Thomas F. Meyer at the Max Planck Institute for infection biology. These diverse cell culture and animal models should deepen the insights into the mechanisms and potential of RNAi, and should help to develop new strategies for the application of RNAi in human therapies.

Clinical trials are not part of the RIGHT project, but pharmaceutical companies outside of RIGHT have initial results illustrating that the application of RNAi as therapeutic tool is promising. Proof-of-principle for the therapeutic use of siRNA has been documented for diseases of the eye by Acuity pharmaceuticals. They plan to enter into Phase II clinical trials next year. Sirna Therapeutics finished a Phase I trial with siRNA to treat age–related macular degeneration, and plan to enter into Phase II studies in 2006. These studies encourage the RIGHT consortium to further develop RNA for human therapies.

References

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Brown BD, Venneri MA, Zingale A, Sergi LS and Naldini L: Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer; Nat Med. 2006;12(5):585-91

Fazi F, Rosa A, Fatica A, Gelmetti V, De Marchis ML, Nervi C, Bozzoni I: A minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis. Cell 2005; 123(5):819-31.

Grimm D, Streetz KL, Jopling, CL, Storm TL, Pandey K, Davis CR, Marion P, Salazar F and Kay MA: Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 2006; 441, 537-541

Honcharenko D, Varghese OP, Plashkevych O, Barman J, Chattopadhyaya J: Synthesis and structure of novel conformationally constrained 1′,2′-azetidine-fused bicyclic pyrimidine nucleosides: their incorporation into oligo-DNAs and thermal stability of the heteroduplexes. J Org Chem. 2006; 71(1):299-314.

Howard KA, Rahbek UL, Liu X, Damgaard CK, Glud SZ, Andersen MO, Hovgaard MB, Schmitz A, Nyengaard JR, Besenbacher F, Kjems J: RNA Interference in Vitro and in Vivo Using a Novel Chitosan/siRNA Nanoparticle System. Mol Ther. 2006 Jul 7; [Epub ahead of print]

Jakobsen MR, Damgaard CK, Andersen ES, Podhajska A, Kjems J: A genomic selection strategy to identify accessible and dimerization blocking targets in the 5′-UTR of HIV-1 RNA. Nucleic Acids Res. 2004; 32(7)

Jonstrup SP, Koch J, Kjems J: A microRNA detection system based on padlock probes and rolling circle amplification. RNA. 2006; 12(9):1747-52.

Kauppinen S, Vester B, Wengel J: Locked nucleic acid: high-affinity targeting of complementary RNA for RNomics. Handb Exp Pharmacol. 2006;(173):405-22

Naguibneva I, Ameyar-Zazoua M, Polesskaya A, Ait-Si-Ali S, Groisman R, Souidi M, Cuvellier S, Harel-Bellan A: The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation. Nat Cell Biol. 2006; 8(3):278-84.

Perfettini JL, Castedo M, Nardacci R, Ciccosanti F, Boya P, Roumier T, Larochette N, Piacentini M, Kroemer G.: Essential role of p53 phosphorylation by p38 MAPK in apoptosis induction by the HIV-1 envelope. J Exp Med. 2005; 201(2):279-89

Piva R, Chiarle R, Manazza AD, Taulli R, Simmons W, Ambrogio C, D’Escamard V, Pellegrino E, Ponzetto C, Palestro G, Inghirami G: Ablation of oncogenic ALK is a viable therapeutic approach for anaplastic large-cell lymphomas. Blood. 2006; 107(2):689-97

Taulli R, Accornero P, Follenzi A, Mangano T, Morotti A, Scuoppo C, Forni PE, Bersani F, Crepaldi T, Chiarle R, Naldini L, Ponzetto C.: RNAi technology and lentiviral delivery as a powerful tool to suppress Tpr-Met-mediated tumorigenesis. Cancer Gene Ther. 2005; 12(5):456-63.