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Team:Tsinghua/Design
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Revision as of 04:09, 16 August 2009
| Project Original | Background | Brainsotrming | Design | Experiment | Results | Notebook |
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Contents |
Introduction
Although several successive gene therapeutic approaches have been reported[1,2], an ideal gene delivery system with targeted specificity, high efficiency and safety is still not available[1,3,4]. Generally speaking, these factors to a large extent depend on the gene therapy vectors used[5]. In other words, the gene delivery system is still a bottleneck as well as a universal problem in the practical fields of gene therapy.
Our project is aimed at applying the ideas of synthetic biology at the genomic level and building a targeted gene therapy vector that can be applied with respect to the certain need of specificity. Also, we intend to propose a procedure for selecting certain synthetic gene vectors with specificity of one’s interest based our design gene therapy vectors. Mathematic modeling focus on both the synthesis of the targeted gene therapy vectors as well as the evaluation of the specificity selection procedure.
Generally, we have two sub-projects. One is the synthetic biology approach in simulation with the established viral vectors. However, the synthetic gene therapy vector will not be as cytotoxic as the commonly applied viral vectors. The other is the synthetic biology approach on the basis of naked plasmid vectors, while the efficiency and specificity of the gene therapy vector can be ensured by synthetic biology modification. The two sub-projects are interconnected in that the flexible synthetic domains of the key proteins are truly interchangeable.
Project1
Basic Idea
Project1 is aimed at synthesizing a gene therapy vector which is structurally and functionally similar to a commonly used viral vector termed adenovirus vector[6]. However, in order to achieve the synthetic biology standard for human practice in the realm of gene therapy, this gene therapy vector should be industrially easy to manipulate in its production and genetically easy to modify in its specificity. Also, based on the social implication of synthetic biology[7,8], the synthetic gene therapy vector must be safe for possible clinical use.
We compared and contrasted the structure of viron between adenovirus and bacteriophage lambda, and found the following facts: 1) the shapes of the viron of both adenovirus and bacteriophage lambda is a regular icosohedron[9], while the adenovirus protein (Fiber) that determines its specificity is positioned on the vertices of the icosohedral viron, which can specifically bind with a receptor called CAR[10]; 2) CAR is widely distributed on the plasma membrane of various types of cells[11-13], which partially contributes to its poor specificity to the target cells as well as its potential cytotoxicity[9,10]; 3) the proliferation of bacteriophage lambda is solely on the basis of its host E.coli, while the production of adenovirus gene therapy vector normally depends on eukaryotic cell lines which are more cost-inefficient and time-consuming; 4) the vertices of adenovirus viron are composed of pentamer of protein III attached to trimer of protein fiber, while the vertices of bacteriophage lambda viron are composed of protein C encoded by lambda phage genome.
Thus, if we can manage to synthesize a bacteriophage-lambda-based gene therapy vector in simulation to the adenovirus vector but modified at the vertices position of its viron, then the production of the synthetic gene therapy vector can be simplified and much easier to manipulate. In addition, considering the low immunogenicity of lambda phage proteins[6,14], the safety of the gene therapy can be improved compared with conventional adenovirus vector.
Our Project1 is equivalent to apply synthetic biology concepts and standards at the genomic level, constructing a genome which is neither adenovirus nor bacteriophage lambda genome. This synthetic genome, however, is capable of producing standardized and targeted gene therapy vectors for human clinical practice. Also, we use the abstraction principle in our design of this genome in order to make the synthetic gene therapy vector easy to be further modified, improved and industrialized. Project1 implicates the evolvement of synthetic biology to a higher level of living organism, the genome, which meets the trends of synthetic biology innovation.
Synthesis of the Gene Therapy Production System
Based on the standard of synthetic biology, we apply two approaches to synthesize the genome of our gene therapy vector
Bottom-Up Approach
As for the bottom-up approach, we amplify the target genes from the target genome(mostly structural genes from lambda phage genome, and also L5 (fiber) gene from adenovirus genome) and recombine them into one or two molecular cloning vectors according to the biobrick standard. In the synthetic genome of the gene therapy vector, gene C (which encodes the protein at the vertices of the lambda viroin) and L5 should be fused according to certain standardiztion consideration (termed targeted biobrick).In front of the encoding region bacteriophage lambda strucural proteins and the targeted biobrick, a T7 promoter (from iGEM parts) will be inserted for IPTG-inducible control.
Top-Down Approach
As for the top-down approach, we transplant the whole gene expression element under the upsteam regulation of promoter R' into a molecular cloning vector. Fortunately, the lambda promoter R' can be strongly enhanced by protein Q in the late state of wildtype phage infection, which makes a feasible scheme to regulate the structural proteins on the synthetic genome.In front of the encoding region of protein Q, a T7 promoter (from iGEM parts) will be inserted for IPTG-inducible control.
Synthesis of the Targeted Biobrick
The synthesized genome will be capable of producing gene therapy "viroins" with protein C specially modified. This targeted biobrick enables the synthetic gene therapy vector to be targeted specifically for certain types of cells. We generally decouple this biobrick into four "modules" (will be expressed to different domains in the C-Fiber fusion protein).
Synthesis of the Therapeutic DNA
Here we introduce another molecular cloning vector to mimic the bacteriophage lambda genome which will be packaged into the viroin. However, the synthetic “bacteriophage lambda genome” contains the therapeutic gene(s) needed by the target cells, which will be transported via stimulated viral introduction.
We term this molecular cloning vector “Therapeutic DNA”, which consists of a cos site for the package into the viroin of targeted gene therapy vector, the therapeutic gene(s) for in vivo gene therapy and the replication origin of bacteriophage lambda (including O gene and P gene).
Production of the targeted gene therapy vector
After the construction of the synthetic genome and the Therapeutic DNA which are carried by two molecular cloning vectors(they will carry different origins of DNA replication), we will cotransform them into the E.coli for the production of the targeted gene therapy vector.
After the addition of IPTG at proper peroid of the transformed bacteria, the structural proteins of the gene therapy vector will be expressed, which are sufficient for the package of the Therapeutic DNA (with O and P) into the gene therapy vector viroin.
Given appropriate time for enough package yields(evaluated by modeling), the E.coli for production will be lysated manually or inducibly. Then the gene therapy vectors can be isolated and enriched by established protocol of viroin purification.
Functioning of the targeted gene therapy vector
An idealized model for the functioning of the targeted gene therapy vector is proposed. After the injection of the purified gene therapy vectors, the vectors will conveyed to target cells by circulation (specific situations should be discussed with respect to different types of cells and different diseases). The synthetic vectors will invade into the target cells in a manner similiar to the wildtype adenovirus-attachment and internalization. This function is empowered by the targeted biobrick in the synthetic genome.
Firstly, the tissue-specific peptide (selected by phage display) on the surface of the viroin will attach to the receptors on the surface of the target cells. Secondly, the RGD domian at the bottom of the engineered fiber will interact with integrin of the targeted cells, thus internalize the whole viroin by endocytosis.
Project2
Basic Idea
Gene therapy is a new type of treatment developed rapidly in recent years, which is both efficient and safe. It is based on the fact that the target genes are transferred into the tissues with illness via vectors, and owing to the proteins translated by target genes, the illness is cured with efficiency. Therefore, gene therapy is highly regarded because of the targeting.
However, the therapies already developed are still meeting with several difficulties, i.e. tissue-specificity, the capacity of the target gene and the cell toxicity. Take the adenovirus vector for example. Recognition and infection by the adenovirus have no tissue-specification, and it can infect any kind of tissues. So serious immune responses are given, and those lead to poor result of therapies. On the other hand, plasmid vectors that cause less immune response have the disadvantage of low transformation efficiency and small capacity. Therefore, we are now seeking for a better designed vector, lambda phage, which is more specific to tissues. At the same time, the high effective lambda phage is also productive and has a large capacity.
Function of the System
Innovation of our Project
By comparing the structural similarity of bacteriophage lambda and adenovirus, this project applies the synthetic biology strategy at the genomic level, transplanting the complex process of viral gene therapy vector production into prokaryotic cells. This approach makes the industrial production of gene therapy vector easier to standardize and manipulate, which coordinates the standardization, abstraction and decoupling principles of synthetic biology. As for the synthetic engineering of the targeted biobrick, we decouple the process of viral infection into two sub-steps, attachment and internalization, which are ensured by the synthetic targeted peptides and RGD domain respectively. Viewed from the overall project, this approach is equivalent to synthesizing a viable form (coinciding with Craig Venter’s approach on synthetic biology) that actually contributes to clinical uses. This reflects the synthetic biology’s extension in human practice by learning from nature.
References(Project1)
[1] David A.Williams, and Christopher Baum. Gene Therapy—New Challenges Ahead. Science. 2003, 302, 400-401.
[2] Marina Cavazzana-Calvo et al.. Immunodeficiency (SCID)-X1 Disease Gene Therapy of Human Severe Combined. Science. 2000, 288, 669-672.
[3] Esmail D. Zanjani, and W. French Anderson. Prospects for in utero human gene therapy. Science. 1999, 285, 2084-2088.
[4] Leland H. Hartwell, Leroy Hood, Michael L. Goldberg, Ann E. Reynolds, Lee M. Silver, Ryth C. Veres. Genetics: From Genes to Genome. McGrawHall, 3rd edition, 2008.
[5] http://en.wikipedia.org/wiki/Gene_therapy
[6] Jerry Guo, and Hao Xin. Splicing out the West?. Science. 2007, 314, 1232-1235.
[7] Chopra Paras, and Akhil Kamma. Engineering life through Synthetic Biology. In Silico Biology 6. http://www.bioinfo.de/isb/2006/06/0038. Retrieved on 2008-06-09.
[8] http://www.syntheticbiology.org
[9] Michael T. M., John M. M., and Jack P. Brock Biology of Microorganisms. Prentice Hall, 12th edition, 2008.
[10] Glen RN, and Phoebe LS. Role of αv integrins in adenovirus cell entry and gene delivery. Microbiology and Molecular Biology reviews. 1999, 63, 725-734.
[11] Yuanming Zhang, and Jeffrey M. Bergelson. Adenovirus Receptors. J. Virol. 2005, 79, 12125–12131.
[12] Miyazawa N, Crystal RG, and Leopold PL. Adenovirus serotype 7 retention in a late endosomal compartment prior to cytosol escape is modulated by fiber protein. J. Virol. 2001, 75, 1387–1400.
[13] Shayakhmetov DM, Eberly AM, Li ZY, and Lieber A. Deletion of penton RGD motifs affects the efficiency of both the internalization and the endosome escape of viral particles containing adenovirus serotype 5 or 35 fiber knobs. J. Virol. 2005, 79, 1053–1061.
