Team:Heidelberg/Project/Introduction

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Introduction


Motivation

Introduction

Motivation Referring to the team abstracts, most of the iGEM Teams are still dealing with what we would call "classical synthetic biology", meaning bacterial engineering. Since the last year's project established to some degree "mammalian synthetic biology", they paved the way for entering the real medical track focussing on gene therapy. The most important issue in terms of gene therapy is regulation of transgene expression. After last year's team characterized synthetic promoters for tuning of gene expression in vitro, we have taken one step further, introducing synthetic microRNAs and their binding sites as a new level of expression control.


Background

Micro RNAs (miRNAs), a class of abundant small noncoding RNAs, are key regulators in all kinds of organisms ranging from viruses to mammals. By binding to target sequences most commonly found in the 3' untranslated region (UTR) of the mRNA, miRNAs inhibit the translation of their target mRNAs and thereby adjust the expression of many proteins related to the miRNA expression in a cell (Brenecke et al. 2005). The importance of miRNA-mediated gene regulation is impressively reflected by the fact that roughly 1% of the human genome codes for miRNAs which target 20-25% of all protein coding genes (Lewis et al. 2005). Therefore, a large proportion of the transcriptome may be subjected to miRNA-mediated control (Lau et al. 2001). The broad regulatory scope of miRNAs underlines their key roles in a wide range of biological processes including proliferation, apoptosis, hematopoeisis and oncogenesis (Bushati and Cohen, 2007). The expression patterns of miRNAs in different cell types, tissues and developmental stages of a cell vary highly, but remain relatively constant within a certain single cell type in a certain stage (Brown et al. 2006 , Gangaragu and Lin 2009). - The special properties of miRNA binding sites and the inhibiting character of miRNAs are excellent devices to be exploited for gene therapy. Not only the fine-tuning capabilities of varying miRNA binding sites, but also the possibility to distinguish different cell types by their miRNA expression profile make synthetic miRNA binding sites an excellent tool to regulate translation in vitro and in vivo.


miRNAs are a class of small regulatory RNAs derived from endogenously expressed primary micro-RNA (pri-miRNA) [8-10]. After being processed in the nucleus and transported in pre-miRNA form to the cytoplasm where it forms a hairpin structure that is recognized by Dicer and cleaved into 22nt long mature miRNA. The miRNA is then loaded into the RNA-induced silencing complex (RISC) which recognizes a specific binding site sequence in the 3' untranslated region (3'-UTR) of a target mRNA and inhibit its translation, thereby downregulating the overall protein expression of the target gene. Endogenous miRNAs can have thousands of gene targets and are therefore considered to be key players in expression regulation.


, 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 its 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 infected 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. 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.


For the first time, we could show miRNA mediated fine tuning and cell targeting in vitro and in vivo. Furthermore we wanted to create a library of synthetic viruses that could be evolved to enhance target specificity of our approach. In the combination of the two, we see doors open to the future of RNA based gene regulatory therapy.
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 its 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 infected 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.

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)