Difference between revisions of "Team:ColumbiaNYC/Description"

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     <h2>Background</h2>
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     <h3>Some Background</h3>
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     <p>
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    <br>
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       The idea of using bacteria as a therapeutic for cancer has been around for about 150 years, beginning with William Coley,
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     <p>Cancer is a global scourge; it strikes without regard to age, race, gender, ethnicity, and socioeconomic class. Fortunately,
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       who gave cancer patients a mixture of heat-inactivated Streptococcus pyogenes and Serratia marcescens, known as “Coley’s
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       such treatment options as radiation therapy and chemotherapy have dramatically improved patient outcomes, prolonging
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       toxin,” to destroy tumors. This idea gradually evolved into reprogramming bacteria to fight cancer using recombinant
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       life in most patients and offering a cure to others. While radiotherapy has achieved sub-millimeter1 precision in targeting
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       DNA. The two most popular vectors for bacterial cancer therapy are Salmonella and E. coli. E. coli that produced shRNA
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      solid tumor cancers, chemotherapy is administered systemically, resulting in off-target effects in healthy dividing
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       can knockdown an oncogene in colorectal cancer. The mechanism in which the bacteria silences the oncogene relies on
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       cells, often with considerable disruption to quality of life. Loss of white blood cell progenitors leads to a broken
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       transkingdom RNA interference, encoded in the bacteria's transkingdom RNAi plasmid (TRIP) (Xiang et al. 2006). The
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       immune system. Death of cells lining the digestive tract causes vomiting and diarrhea. Damage to cells of the hair
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       TRIP plasmid contains three major sections, the shRNA-producing gene, the invasin gene, and the hlyA gene. The shRNA-producing
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       follicle brings baldness and the immense toll on one’s body image.</p>
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       gene produces shRNA that corresponds to the mRNA sequence of the targeted oncogene. The invasin gene allows the bacteria
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    <br>
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       to invade the cancer cell and get encapsulated in an endosome. The bacteria lyse inside the endosome, and the hlyA
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    <h3>A Bit about our Project</h3>
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      gene encodes for listeriolysin O, which produces holes (pores) in the endosome so that the shRNA produced by the bacteria
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    <br>
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       can get out of the endosome to reach the mammalian cell’s cytoplasm. The released shRNA is then cleaved by the enzyme
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    <p> SilenshR is a synthetic biology solution that leverages the innate capacity of bacteria to colonize the hypoxic and immune-privileged
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       Dicer in the mammalian cell’s cytoplasm to become siRNA. The siRNA then associates with other proteins in the cytoplasm
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       cores of tumors, conferring specificity to a systemic therapeutic approach. Once the SilenshR E. coli reach a cell
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       to form the RISC complex. The RISC complex then binds the corresponding target mRNA in the cytoplasm and cleaves it,
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       density of 2*10
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       silencing the gene that the mRNA encodes. This mechanism has been proven to be an effective inhibitor of a cancerous
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      <sup>11</sup> colony forming units (cfu) per milliliter within the tumor, genetic circuits are activated allowing SilenshR
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       protein that has been very difficult to target with drugs. In this project, we optimize and expand the applications
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      recombinants to invade cancer cells and release a short hairpin RNA (shRNA) targeting an expressed oncogene thereby
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       of this mechanism. We will use quorum sensing as an additional safeguard to make sure that this mechanism only attacks
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       halting the unchecked cellular proliferation of cancer. By circumventing the need for systemic delivery of chemotherapy
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      cancer cells. With quorum sensing, this mechanism will only be activated when a certain bacterial cell density is reached.
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       and the inevitable off-target cytotoxicity, SilenshR keeps the immune system intact, spares the digestive epithelial
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      This density is only possible in very anaerobic environments, which is characteristic of tumors. Challenges to this
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       lining from damage and relieves anxiety surrounding hair loss.
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       include the natural anaerobic environment in the gut and whether the bacteria will proliferate in a healthy gut as
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    </p>
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       well. To resolve this, we are working to put the quorum sensing circuit under the control of a nitric oxide promoter.
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      Since nitric-oxide rich environments are only characteristic of areas of inflammation and are highly characteristic
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    <br>
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      of cancer, having the quorum sensing circuit under the control of a nitric-oxide promoter that is only activated in
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    <p>Our invasiveness circuit is mediated by diffusion of the small molecule AHL, or acylhomoserine lactone, produced by AHL
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       nitric-oxide rich environments would prevent the bacteria from invading healthy gut cells even when quorum is reached.
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       synthase. The presence of the AHL autoregulator in our bacteria promotes transcription at the lux promoter, regulating
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       A synthetic alternative to this mechanism that would be simpler to test in the laboratory is to control the quorum
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       AHL synthase in a positive feedback loop as well as the genes invasin and hlyA from Yersinia and Listeria, respectively.
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       circuit with a tet (tetracycline) on system where a tet repressor represses the expression of the invasion circuit
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       Invasin interacts with surface B1-integrin receptors on the cancer cell membrane, resulting in phagocytosis of the
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       when tetracycline and/or doxycycline is not present. When doxycycline/tetracycline is synthetically introduced to the
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       SilenshR bacteria. Within the phagosome, the HlyA protein, diffusible across membranes, inserts pores, compromising
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      environment, the tet repressor is repressed by the rtTA (reverse tetracycline-controlled transactivator), and transcription
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       the SilenshR’s containment within the vesicle.
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       of the invasion circuit occurs. The doxycycline/tetracycline can only be introduced to cancer sites. We are targeting
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    </p>
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      cervical cancer and prostate cancer. We will determine the effectiveness of this mechanism in each of these cancers
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    <br>
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       using proof-of-concept experiments by inhibiting eGFP. We will then target oncogenes in these cancers. </p>
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    <p>SilenshR is not just one recombinant bacterial strain or one sequence of DNA: SilenshR is a revised approach to cancer
 +
       treatment with greater specificity and less discomfort. Just as different chemotherapeutics might be prescribed depending
 +
       on the affected organ, the therapeutically active component of SilenshR, the shRNA sequence, can be altered depending
 +
       on the particular gene promoting tumor growth. For example, while EGFR (Epidermal Growth Factor Receptor) overexpression
 +
       contributes to tumor malignancy in lung cancer and c-MYC drives cell proliferation in breast cancer, SilenshR recombinants
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       can be used to treat both kinds of cancer with a simple restriction digest and swap of a 63 base pair stretch of DNA
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       encoding shRNA. In the cytosol, the shRNA is cleaved at the hairpin by the Dicer complex and the resulting siRNA is
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       complementary to a sequence in the mRNA of targeted protein product. Degradation of the mRNA by the RNA-Induced Silencing
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       Complex, or RISC, results in an inhibition of translation of the targeted gene.
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    </p>
 
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Revision as of 02:07, 2 November 2017

Project Description

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Some Background


Cancer is a global scourge; it strikes without regard to age, race, gender, ethnicity, and socioeconomic class. Fortunately, such treatment options as radiation therapy and chemotherapy have dramatically improved patient outcomes, prolonging life in most patients and offering a cure to others. While radiotherapy has achieved sub-millimeter1 precision in targeting solid tumor cancers, chemotherapy is administered systemically, resulting in off-target effects in healthy dividing cells, often with considerable disruption to quality of life. Loss of white blood cell progenitors leads to a broken immune system. Death of cells lining the digestive tract causes vomiting and diarrhea. Damage to cells of the hair follicle brings baldness and the immense toll on one’s body image.


A Bit about our Project


SilenshR is a synthetic biology solution that leverages the innate capacity of bacteria to colonize the hypoxic and immune-privileged cores of tumors, conferring specificity to a systemic therapeutic approach. Once the SilenshR E. coli reach a cell density of 2*10 11 colony forming units (cfu) per milliliter within the tumor, genetic circuits are activated allowing SilenshR recombinants to invade cancer cells and release a short hairpin RNA (shRNA) targeting an expressed oncogene thereby halting the unchecked cellular proliferation of cancer. By circumventing the need for systemic delivery of chemotherapy and the inevitable off-target cytotoxicity, SilenshR keeps the immune system intact, spares the digestive epithelial lining from damage and relieves anxiety surrounding hair loss.


Our invasiveness circuit is mediated by diffusion of the small molecule AHL, or acylhomoserine lactone, produced by AHL synthase. The presence of the AHL autoregulator in our bacteria promotes transcription at the lux promoter, regulating AHL synthase in a positive feedback loop as well as the genes invasin and hlyA from Yersinia and Listeria, respectively. Invasin interacts with surface B1-integrin receptors on the cancer cell membrane, resulting in phagocytosis of the SilenshR bacteria. Within the phagosome, the HlyA protein, diffusible across membranes, inserts pores, compromising the SilenshR’s containment within the vesicle.


SilenshR is not just one recombinant bacterial strain or one sequence of DNA: SilenshR is a revised approach to cancer treatment with greater specificity and less discomfort. Just as different chemotherapeutics might be prescribed depending on the affected organ, the therapeutically active component of SilenshR, the shRNA sequence, can be altered depending on the particular gene promoting tumor growth. For example, while EGFR (Epidermal Growth Factor Receptor) overexpression contributes to tumor malignancy in lung cancer and c-MYC drives cell proliferation in breast cancer, SilenshR recombinants can be used to treat both kinds of cancer with a simple restriction digest and swap of a 63 base pair stretch of DNA encoding shRNA. In the cytosol, the shRNA is cleaved at the hairpin by the Dicer complex and the resulting siRNA is complementary to a sequence in the mRNA of targeted protein product. Degradation of the mRNA by the RNA-Induced Silencing Complex, or RISC, results in an inhibition of translation of the targeted gene.