Team:Heidelberg/Project/Introduction

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Our project delivers a whole new cassette of tools around miRNA including miMeasure, miTuner, and miBEAT:
Our project delivers a whole new cassette of tools around miRNA including miMeasure, miTuner, and miBEAT:
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1) We design a standard measurement construct (miMeasure), which enables an accurate and comparable measurement of silencing strengths of miRNAs or miRNAs derived from shRNAs. The miMeasure construct allows a convenient exchange of miRNA binding sites. To test the sensitivity of miMeasure we create randomly assembled (raPCR) patterns of binding sites for miR-122 - a species of miRNA, which is specifically upregulated in hepatocytes.
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1) We design a standard measurement construct (miMeasure), which enables an accurate and comparable measurement of silencing strengths of miRNAs or miRNAs derived from shRNAs. The miMeasure construct allows a convenient exchange of miRNA binding sites. To test the sensitivity of miMeasure we create randomly assembled (miRaPCR) patterns of binding sites for miR-122 - a species of miRNA, which is specifically upregulated in hepatocytes.
2) We design a shRNA expression construct (miTuner), which allows a convenient exchange of shRNA genes and to tune the expression level of ist target gene in a precise way.
2) We design a shRNA expression construct (miTuner), which allows a convenient exchange of shRNA genes and to tune the expression level of ist target gene in a precise way.

Revision as of 17:27, 27 October 2010

Introduction


Motivation

RNA interference (RNAi) is a powerful technology to specifically silence the expression of target genes in eukaryotic cells [1-4]. Many of the underlying mechanisms such as gene regulation by endogenous micro RNAs (miRNAs), gene silencing by short interfering RNAs (siRNAs), as well as the endogenous expression of small hairpin RNAs (shRNAs) to mimic miRNAs have been rapidly elucidated over the last decade. First approaches to use RNAi for targeted gene therapy have been very promising. In some cases they already reached clinical phase I [5-7]. It is therefore very timely to introduce the methodology of engineering RNAi into synthetic biology with the goal of establishing an entirely new approach for gene regulation in human cells.

Background

miRNA is derived from endogenously expressed primary micro-RNA (pri-miRNA) [8-10], which is cleaved in the nucleus by the endoribonuclease Drosha to pre-miRNA. In the cytoplasm this pre-miRNA self-hybridizes via a hairpin loop and is further processed by the enzyme Dicer, leaving a double stranded and approximately 22 nucleotide long miRNA with characteristic overhangs of 2 nucleotides at each side. The miRNA is recognized by and loaded onto the RNA-induced silencing complex (RISC) enabling RISC to recognize a specific sequence of the 3' untranslated region (3'-UTR) of a target mRNA. A miRNA can have hundreds of gene targets, because the sequence homology with the target is highly imperfect. This is possible because the nucleotides two to eight (5') are mainly responsible for target binding (seed region). It hampers in this way RISC to nick the bound mRNA strand and results in various but moderate strengths of post-transcriptional gene silencing [9-12].

siRNA is artificially synthesized and exogenously transfected into cells; it resembles miRNA in its structure [2,10]. It is usually designed to perfectly match a sequence in the 3'-UTR or the open reading frame (ORF) of a target gene and allows RISC to cleave the mRNA strand [13,14]. Intracellular RNases are then attracted for complete degradation resulting in high post-transcriptional gene silencing. siRNA is however not stable in serum and not taken up by target cells in an organism [7,15]. These burdens can be overcome by endogenous expression of shRNA, which resembles pre-miRNA and is analogously processed by Dicer [5].

Overall Project Aim

Our project delivers a whole new cassette of tools around miRNA including miMeasure, miTuner, and miBEAT:

1) We design a standard measurement construct (miMeasure), which enables an accurate and comparable measurement of silencing strengths of miRNAs or miRNAs derived from shRNAs. The miMeasure construct allows a convenient exchange of miRNA binding sites. To test the sensitivity of miMeasure we create randomly assembled (miRaPCR) patterns of binding sites for miR-122 - a species of miRNA, which is specifically upregulated in hepatocytes.

2) We design a shRNA expression construct (miTuner), which allows a convenient exchange of shRNA genes and to tune the expression level of ist target gene in a precise way.

3) We analyze the silencing strengths for a variety of imperfect miRNA/binding sites in order to understand the quantitative structure-activity relationship (QSAR). The data is used to train a model (miBEAT), which allows the design of shRNA/binding site sequences to deliberately adjust the expression rate of any other target gene.

Since it is not yet shown in the literature to what extent imperfect miRNA sequences can be utilized for controlled gene regulation, our project provides a fundamentally new insight into the nature of mi/siRNA regulation. To complement our "miTechnology" for a complete gene therapy approach, we use the adeno-associated virus (AAV) for transduction of the shRNA gene into target cells and encounter the challenge of tissue specific gene delivery [16-19]:

4) We follow two virus shuffling approaches to re-engineer the AAV capsid gene by directed evolution. This is used for production of a virus that is specifically integrated into hepatocytes.

5) We test the hepatocyte specificity of the fittest AAV clone via a luciferase reporter in living mice. We show that organ specific targeting is readily achieved in our mouse model.

In summary, we have constructed a miRNA tool kit as a solid basis for future teams to conduct engineering work based on the powerful technology of RNA interference. We have further derived a model, which improves the scientific understanding of the nature of miRNA regulation and helps to rationally design our tuning constructs. We have finally improved and used methods to engineer AAV capsids for specific infection of hepatocytes. The following project sections give more background on the individual topics and provide detailed overviews on the corresponding results. A detailed documentation of the laboratory work can further be found in the notebook.

References

[1] A. Fire et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature 391:806-811 (1998)
[2] S. M. Elbashir et al., Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature 411:494-498 (2001)
[3] G. Meister et al., Mechanisms of gene silencing by double-stranded RNA, Nature 431:343-349 (2004)
[4] G. J. Hannon et al., Unlocking the potential of the human genome with RNA interference, Nature 431:371-378 (2004)
[5] D. H. Kim et al., Strategies for silencing human disease using RNA interference, Nat. Rev. Genet. 8:173-184 (2007)
[6] D. Castanotto et al., The promises and pitfalls of RNA-interference-based therapeutics, Nature 457:426-433 (2009)
[7] M. A. Behlke et al., Chemical modification of siRNAs for in vivo use, Oligonucleotides 18:305-319 (2008)
[8] M. Lagos-Quintana et al., Identification of novel genes coding for small expressed RNAs, Science 294:853-858 (2001)
[9] R. W. Carthew et al., Origins and mechanisms of miRNAs and siRNAs, Cell 136:642-655 (2009)
[10] P. Brodersen et al., Revisiting the principles of microRNA target recognition and mode of action, Nat.Rev. Mol. Cell Biol. 10:141-148 (2009)
[11] D. P. Bartel et al., MicroRNAs: target recognition and regulatory functions, Cell 136:215-233 (2009)
[12] W. Filipowicz et al., Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Gen. 9:102-114 (2008)
[13] G. Hutvagner et al., Argonaute proteins: key players in RNA silencing, Nat. Rev., Mol. Cell Biol. 9:22-32 (2008)
[14] Y. Pei et al., On the art of identifying effective and specific siRNAs, Nat. Methods 3:670-676 (2006)
[15] D. R. Corey et al., Chemical modification: the key to clinical application of RNA interference?, J. Clin. Invest. 117:3613-3622 (2007)
[16] D. Grimm et al., RNAi and gene therapy: a mutual attraction, Hematology 473-481 (2007)
[17] D. Grimm et al., Adeno-associated virus vectors for short hairpin RNA expression, Methods Enzymol. 392:381-405 (2005)
[18] D. Grimm et al., From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy, Curr. Gene Ther. 3:281-304 (2003)
[19] D. Grimm et al, Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways, Nature 441:537-541 (2006)