Team:Tsinghua/Project Original

Overall project
A relatively significant procedure in gene therapy is to construct a vector to infect target cells and deliver cure gene into them. As a result, the vectors act as big role. And till now researchers use Adeno-associated viruses to do a good job, but the problems of high cost and low production of the virus has not been solved. That is why we attempted to build a highly productive carrier into bacteria. We transformed the structure genes of the phage into bacteria with specific chimera genes attached to the structural genes. We attempted to simulate the Adeno-associated viruses by the phage, for they share the similar structure. Fiber has been attached to the phage to enhance transformation efficiency. Not only we use the cosmid in the phage to carry the cure gene, which has great capability to carry large and multiple genes, but also the cure genes are tissue specific.

Gene Therapy
Gene therapy is defined as the introduction of genes into tissues or cells via gene transfer, with the purpose of deriving a therapeutic or preventative benefit from the function of these genes. It is the insertion of genes into an individual's cells and tissues to treat a disease, such as a hereditary disease in which a deleterious mutant allele is replaced with a functional one[1]. Although the technology is still in its infancy, it is one of the most promising and active research fields in medicine[1,2]. Antisense therapy is not strictly a form of gene therapy, but is a genetically-mediated therapy and is often considered together with other methods[1].

Vectors in Gene Therapy
Despite substantial progress, a number of key technical issues need to be resolved before gene therapy can be safely and effectively applied in the clinic, and an ideal gene delivery vector is one of the bottlenecks in gene therapy application[3]. Generally, vectors applied in gene therapy can be classified into viral or non-viral.

Viral Methods
All viruses bind to their hosts and introduce their genetic material into the host cell as part of their replication cycle. This genetic material contains basic 'instructions' of how to produce more copies of these viruses, hijacking the body's normal production machinery to serve the needs of the virus[1].

Adenovirus.

Adeno-associated virus.

Retroviruses.

Non-Viral Methods
Non-viral methods present certain advantages over viral methods, with simple large scale production and low host immunogenicity being just two. Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage; however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses[1].

Naked DNA.

Oligonucleotides.

Lipoplexes and polyplexes.

Therapeutic microRNA Delivery
Therapeutic strategies based on modulation of microRNA (miRNA) activity hold great promise due to the ability of these small RNAs to potently inﬂuence cellular behavior[4].

Cancer Gene Therapy
As a primary threat of human health, cancer causes about 13% of all human deaths[5]. According to the American Cancer Society, 7.6 million people died from cancer in the world during 2007[6]. Current treatments often have far reaching negative side effects[7]. The systemic toxicity of chemotherapy regimens still often result in acute and delayed nausea, mouth ulcerations and mild cognitive impairments[8].

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.

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.

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.