Lorem Sit Amet Dolor
Researcher: Lorem Sit Amet

123 abc lane, Townsville, ZZ 00000, USA
Funding Progress: $§ / $§§§§§

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A novel CRISPR/Cas9 approach to target the EWS-FLI1 fusion gene in Ewing’s sarcoma
Translational
Ewing Sarcoma (Bone Cancer)
Lay Summary

Ewing’s sarcoma (EWS) is a devastating pediatric cancer of bone and soft tissues where the treatments used are typically very toxic, resulting in profound morbidities. Furthermore, these intense and non-specific treatments often lead to secondary tumors later in life. Thus, a less toxic and more specific treatment is critically needed to impact this dreadful disease. In this proposal, we will explore a novel precision approach to specifically target and kill EWS cells without affecting normal cells. The principle of this approach is to assemble a toxic endonuclease at a chromosome rearrangement junction (CRJ) that only exists in the cancer cells. In EWS cells we will specifically target the fusion gene EWS-FLI1, formed by a chromosome rearrangement bringing one part of the EWS on chromosome 11 together with one part of the FLI1 gene on chromosome 22. This fusion gene is oncogenic and EWS cells depend on it for survival. We hypothesize that using our precision approach to target the CRJ in the fusion gene will be toxic to the EWS cells both by inducing a toxic DNA double strand break (DSB) as well as inhibiting the expression of the fusion protein, while normal cells should not be affected.

Executive Summary

Executive Summary

1. Scientific Merit

Ewing’s sarcoma (EWS) is an aggressive pediatric cancer that affects bone and soft tissues. Despite intense therapeutic regiments, the survival rates for patients with metastasis are dismal. Furthermore, the intense treatments used are very toxic resulting in profound morbidities that negatively impact on both the quality and quantity of life for survivors. Thus, new and less toxic precision therapies targeting Ewing’s sarcomas are critically needed. The novel idea to be tested in this proposal is to use the CRISPR/Cas9 technology to target the EWS-FLI1 fusion gene, which is the “Achilles heel” of EWS cells, and specifically kill these cells without affecting normal cells. We will utilize a dCas9 fused to the endonuclease Fok1 that will be brought to each side of the chromosome rearrangement junction (CRJ) in the EWS-FLI1 fusion gene, with the aid of a pair of CRJ-targeting gRNAs. This allows for the activation of Fok1 endonuclease by dimerization causing a DNA double strand break (DSB) leading to loss of cell fitness. Our hypothesis to be tested is that by targeting the CRJ of the fusion gene with the Fok1-dCas9 endonuclease complex, cancer-specific toxicity will occur both from the induced DSB as well as from loss of expression of the fusion protein on which the cancer cells are dependent on. Thus, our novel CRISPR/Cas9 approach may be especially well suited for the treatment of Ewing’s sarcoma because of its dependence on the oncogenic EWS-ETS fusion proteins that can be specifically targeted with this approach.

2. Feasability

Our preliminary data show “proof-of-principle” that we can successfully generate and localize a functional endonuclease to cancer specific CRJs, which generates targeted DSBs leading to reduced cancer cell fitness. In this Cure Starts Now grant application we propose to apply this CRISPR-based technique to specifically target the CRJ in the EWS-FLI1 fusion gene in pediatric Ewing’s sarcoma cells. We will also initiate efforts to assess CRISPR-Gold nanoparticles as a means to deliver Fok1-dCas9 and gRNA RNPs use nanoparticles to deliver the Fok1-dCas9 and gRNAs to target CRJs in EWS cells in vitro. Importantly, normal cells should be unaffected by this approach since dimerization of the Fok1 endonuclease would only occur at the CRJs in the cancer cells. Our long-term goal is to apply this novel approach in mouse models of Ewing’s sarcoma and eventually in the clinic for individualized precision cancer treatment. This novel personalized approach should be applicable to any cancer and could be transformative.

3. Expertise

The two research teams that together will perform the proposed experiments in this Cure Starts Now application, are very complementary to each other and possess all the expertise to successfully execute this proposal. Dr. Ljungman and his team developed the “Precision KLIPP Therapy” approach that will be used in this proposal to specifically target the oncogenic EWS-FLI1 fusion gene in EWS cells. Dr. Huibin Yang is a senior Research Associate in the Ljungman lab who has many years of experience in molecular biology. He successfully developed all the reagents to obtain the “proof-of-principle” data for this approach in HCT116 colon cancer cells and is now generating reagents to target the EWS-FLI1 fusion gene in EWS cells. Dr. Lawlor is a world leader in the field of Ewing’s sarcoma and has devoted her career to find a cure for this dreadful disease. She and her lab will bring valuable expertise on Ewing’s sarcoma biology to this project. Drs. Ljungman and Lawlor have published scientific articles together and are currently collaborating on an R01 grant on Ewing's sarcoma biology.

Description of Research Proposal

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Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Donec faucibus, turpis sit amet maximus dapibus, sapien nisl bibendum turpis, pharetra commodo tellus libero vel nulla. Sed nec velit viverra, congue sapien et, gravida libero. Proin eget ante eget turpis egestas accumsan. Aliquam arcu nibh, aliquam rhoncus vulputate in, pellentesque at sem. Maecenas cursus tempus nibh id tempus. Mauris dolor sapien, lacinia sit amet condimentum at, dapibus eget lectus. Phasellus vel pellentesque ex. Nunc aliquam in ligula at tristique. In mollis suscipit felis eu finibus. Nullam non dignissim nibh, nec suscipit ex. Suspendisse tincidunt et mauris id finibus. Aliquam vehicula a sem quis venenatis.

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Budget

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer gravida non felis non euismod. Fusce finibus aliquet consequat. Nam ac metus bibendum, iaculis purus sed, suscipit ligula. Proin et nisi libero. Mauris non urna urna. Nullam augue eros, fringilla sed mauris vitae, porta tincidunt risus. Aliquam sed tincidunt sem. Quisque lacinia quam tortor, imperdiet efficitur odio iaculis in. Sed ultricies condimentum volutpat. Vivamus dignissim faucibus porta.

Curabitur ut ipsum non odio malesuada vulputate. Morbi maximus, est eu lobortis molestie, tortor sapien hendrerit nisi, in cursus odio diam ut odio. Fusce pulvinar volutpat velit. Aliquam erat volutpat. Integer rhoncus mollis suscipit. Praesent non ipsum mollis, finibus nunc a, scelerisque nibh. In feugiat iaculis velit, eu semper lacus dignissim nec. Praesent vitae nisi leo. Cras venenatis dictum magna ut semper. Sed eget eros nibh. Sed vitae quam sed dolor faucibus elementum. Curabitur interdum porttitor finibus. Nullam tincidunt odio lectus, sit amet rhoncus libero dapibus sed. Sed mollis egestas enim, vel porta tortor volutpat eget.

Morbi orci urna, ornare non pretium eget, pulvinar eget magna. Ut consectetur efficitur varius. Fusce ac aliquet mauris, at mattis ligula. Quisque est libero, interdum id orci et, ornare luctus diam. Proin commodo lectus id accumsan blandit. Nulla eu turpis interdum, luctus ante ac, imperdiet tellus. In semper enim eu tristique aliquam.

Collaborations and Conflicts of Interest

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Curabitur ut ipsum non odio malesuada vulputate. Morbi maximus, est eu lobortis molestie, tortor sapien hendrerit nisi, in cursus odio diam ut odio. Fusce pulvinar volutpat velit. Aliquam erat volutpat. Integer rhoncus mollis suscipit. Praesent non ipsum mollis, finibus nunc a, scelerisque nibh. In feugiat iaculis velit, eu semper lacus dignissim nec. Praesent vitae nisi leo. Cras venenatis dictum magna ut semper. Sed eget eros nibh. Sed vitae quam sed dolor faucibus elementum. Curabitur interdum porttitor finibus. Nullam tincidunt odio lectus, sit amet rhoncus libero dapibus sed. Sed mollis egestas enim, vel porta tortor volutpat eget.