Targeting the ERK signalling pathway in cancer

SPECIFIC AIMS: This project centers around developing a novel technique for targeting the extracellular signal-regulated kinase (ERK) signaling pathway in cancer cells to inhibit their growth and proliferation. In order to achieve this goal, first an aptamer will be conjugated to a chitosan nanoparticle which will contain a pH responsive DNA origami box. This origami box encapsulates CRISPR/CAS9 to target the ERK signaling cascade of the cell encoded in the DNA. Aptamers can target certain receptors indicative of cancer cells in order to be endocytosed, thus allowing for the delivery of CRISPR/CAS9 technology into the cytosol of the targeted cells. CRISPR/CAS9 can ultimately shut down the ERK pathway by editing the genome of cancer cells to prevent the production of this protein. Lack of the ERK protein should result in decreased cell viability. 

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Research into this complex field will allow for significant advances into today’s fight with cancer. This project develops a novel approach for selectively targeting cancer cells while shutting down an integral pathway to their survival. This approach allows for further exploration into the capabilities of gene editing and eventually more advances into personalized medicine. It can allow for additional expansion into broader treatments and targeting approaches in drug delivery. Our long-term vision is to develop a more generalized drug delivery model derived from our current approach. We hope to expand our approach to target all cells with overexpressed ERK signaling pathways instead of limiting our design to focusing on HER1 and HER3 receptors. Currently, our approach is limited to delivering a genome that will prevent the cell from further proliferation. It is anticipated that the cell will no longer be viable after depletion of this genomic sequence editing drugs such as CRISPR/CAS9 and selectively targeting cancer cells that overexpress HER1 and HER3; but in the future, this approach can be expanded to other gene related diseases or can even be expanded to targeting other types of cells. The objective is to produce HER1/HER3 targeting aptamers conjugated to chitosan nanoparticles that contain pH responsive DNA origami boxes which encapsulate CRISPR/CAS9 that will selectively shut down the ERK signaling cascade. Thecentralhypothesis is that the aptamer will target HER1/HER3 for cellular uptake which will enter the cytosol via endosomal escape as the chitosan nanoparticles will swell in response to the acidic environment, thus releasing the DNA box which will in turn release the CRISPR/CAS9 into the cytosol to shut down ERK and starve cancer cells. The rationale for this work is that by using the CRISPR/CAS9 to terminate the ERK signaling cascade the cell will be deprived of the various signals necessary for cell proliferation and differentiation, resulting in cell death. Specific Aims are:

1.       Identify a nanoparticle to encapsulate a DNA origami box which contains CRISPR/CAS9, and its potential to be released according to their respective pH sensitivities. The working hypothesis is that the chitosan nanoparticle will hold the DNA origami box to shield it from the body and surrounding environment. Since chitosan is a cationic nanoparticle, it will be constricted at the slightly basic pH of the bloodstream, but it will expand and swell in response to acidic pHs such as those of the cellular endosome. Once the chitosan nanoparticle expands and the endosome bursts, the DNA origami box will be released and exposed to the acidic contents of the endosome. The box will then open and expand in response to the acidic pH and release its contents to the cytosol. Each chitosan nanoparticle will be formulated at sizes between 250 to 350 nm which can hold around three 70 to 90 nm DNA boxes.
2.       Evaluate the ability of the aptamer to target HER1/HER3 for cellular uptake and the CRISPR/CAS9 to shutdown the ERK signaling cascade. The working hypothesis is that the aptamer targets HER1 and HER3 for cellular uptake to expose the contents of the cell to the CRISPR/CAS9. The CRISPR/CAS9 can then diffuse into the nucleus and knockdown expression of ERK.

This work is innovative becausethere are no current gene editing drug delivery systems that involve the use of DNA origami to encapsulate CRISPR/CAS9 combined with an aptamer and nanoparticle encapsulation.This project will yield the following expected outcomes. First, we will reproduce a double aptamer that targets HER1/HER3 and is carboxylated to chitosan nanoparticles. Second, the chitosan nanoparticles will burst the cellular endosome due to swelling. Third, the contents of the nanoparticle will be released into the bursted endosomal environment, thus exposing the DNA origami boxes to a slightly acidic pH. Fourth, these boxes will open and release the CRISPR/CAS9 to the cytosol. Fifth, the CRISPR/CAS9 will travel to the nucleus and target the appropriate ERK sequence for knockdown of this pathway. Sixth, cell viability should be reduced. Although the current focus is on the clinical application of cancer treatment, this platform has the potential to be a fundamental tool in the drug delivery space.

SIGNIFICANCE: This project poses great significance in terms of its potential clinical applications. According to the NIH, in 2018 an estimated 1.7 million cases of cancer will be diagnosed.1 HER1 is overexpressed on many different types of cancer cells including: anal cancers, epithelial tumors of the head and neck, and breast cancer.2-4 Moreover, increased expression of HER1/HER3 is related to poor prognosis in most cancers studied.5 It is estimated that in 2018, anal cancer, skin cancer, and breast cancer will affect at least 368,520 people.6 Finding an effective way to insert CRISPR/CAS9 into cancer cells through the targeting of HER1 and HER3 would serve as a platform for gene therapy in a variety of cancers. 

 Furthermore, the project has broader significance within the field of drug delivery. In terms of drug delivery, this approach can expand into a more generalized therapy by switching the conjugated aptamer to target any receptor of interest that is indicative of certain cell types or disease states. Through this aptamer targeting, it allows for increased bioavailability while reducing delivery of drug to any undesired areas. Additionally, a multilayered approach such as our own allows for protection of our drug against the body. If a drug such as CRISPR/CAS9 was just allowed to freely float in the bloodstream, it would elicit an immune response or be rapidly cleared/eliminated. Protective nanoparticles or coatings hide and protect therapies to increase circulation time and thus increase the efficiency of the drug.7 Cationic nanoparticles such as chitosan will enable increased cellular uptake as well due to their strong cellular interactions.8

 The importance of suppressing the ERK signaling pathway has significant influence in drug therapy for cancer treatment. There are currently multiple inhibitors that have reached clinical trial stage including BAY 43-9006 (Raf Inhibitor), PD184352, PD032591, and ARRY-142886 (MEK 1/2 inhibitors).9 In tumors, the ERK pathway is upregulated and controls the cell’s ability to burgeon, differentiate, and survive, making inhibition of this pathway a prominent target for drug therapies.9 Our innovation does not seek to inhibit the ERK signaling pathway; rather, we will seek to delete the further production of the protein from its genome.

INNOVATION: The combination of a nucleic acid aptamer conjugated with a chitosan nanoparticle that encapsulates a DNA origami box provides an innovative way to protect and transport CRISPR/CAS9 to the interior of the cell. Currently, a major restriction associated with the CRISPR/CAS9 system is the limited ability to introduce its components to the nucleus of the cell. Many methods that have been attempted for delivery have triggered defense responses of the cell, which prevent the components from reaching the nucleus and thus inhibiting treatment efficacy. Thus, the nanoparticle and DNA origami box encapsulation system is a novel approach for transporting the CRISPR/CAS9 technology across the cell membrane for successful delivery to the nucleus, provided the nanoparticle itself is designed to be able to cross without triggering a negative cellular response. Methods of nanoparticle encapsulation have been reported as successful previously, though the exact nanoparticle formulation and rational differs from the one explored in this project.10

APPROACH:  Aim 1: Identify a nanoparticle to encapsulate a DNA origami box which contains CRISPR/CAS9, and its potential to be released according to their respective pH sensitivities.The working hypothesis is that the chitosan nanoparticle will hold the DNA origami box to shield it from the body and surrounding environment. We formulated this hypothesis based on cationic nanoparticles constricting in neutral/basic pHs such as that of the bloodstream and the ability for nanoparticles to be efficiently loaded with various materials. Once taken into the cell, the chitosan nanoparticle will swell in response to an acidic pH, creating pores for the origami boxes to escape and bursting the endosome. The DNA origami box should encapsulate CRISPR/CAS9 and open in response to the acidic pH11 of the endosome which will allow for diffusion of CRISPR/CAS9 out of the box and into the cytosol. Within this aim, we expect to develop a chitosan based nanoparticle that swells in response to acidic pHs and releases DNA origami boxes which in turn release CRISPR/CAS9 in response to an acidic pH as well.

Experimental Design: Producing Chitosan Nanoparticle: The chitosan nanoparticles will be prepared according to the protocol and loaded with the DNA origami boxes (these boxes will be loaded with CRISPR/CAS9) as opposed to protein outlined by Rampino, et al.12 These nanoparticles need to be in the size range of 250-350 nm (unswelled) in order to encapsulate multiple DNA origami boxes (~70-90 nm). In order to analyze the correct size range, particles will be selectively filtered and sized via dynamic light scattering (DLS). Additionally, these nanoparticles will be analyzed in terms of release rate and swelling potential (up to 1 µm) to burst the endosome and to ensure the origami boxes can diffuse out at acidic pHs (4.5 to 6.5). SEM will be used to image the nanoparticles with ImageJ to quantify the diameter and pore sizes, and DLS will be utilized again. Loading efficiency of the particles can be assessed by quantifying the concentration of DNA in the supernatant through UV-vis spectroscopy. Release rate of the boxes will need to be quantified and should occur within seconds. Gel electrophoresis of the particles will be utilized to ensure the box is loaded. Obtaining CRISPR/CAS9: The CRISPR/CAS9 utilized in this study will be purchased from Santa Cruz Biotechnology.13 This product should knockout the ERK signaling cascade; we will test the success of this product by transfecting it into MCF-7 human breast cancer cells and then quantifying the gene knockout and cell viability.Constructing DNA Origami Box:  The DNA origami boxes will be produced by following the protocol outlined by Burns, et al.11 We will modify this protocol by loading the box with CRISPR/CAS9 as opposed to green fluorescence protein, and the size and loading efficiency will be assessed according to their protocol as well.11 Since their boxes were 25 nm in length,11 we will increase the box size to our desired 70-90 nm by adding base pairs. Release rate of CRISPR/CAS9 will also need to be assessed and needs to occur rapidly (within seconds) by exposing the loaded boxes to an acidic environment and measuring CRISPR/CAS9 concentration in the supernatant over time. Gel electrophoresis of the boxes will be utilized to ensure the CRISPR is loaded.

Expected Outcomes: Using the above methodology, the CRISPR/CAS9 will be successfully encapsulated within a DNA origami box. These boxes will be protected by the chitosan nanoparticle. Upon swelling of the nanoparticle, we expect the release of CRISPR/CAS9 to occur within seconds. Delivery of the nanoparticle to the cell will be further explained in Aim 2.

Alternative Strategies: Potential problems arising from utilizing chitosan, or any cationic polymer, as the nanoparticle is that it may disrupt the cell membrane, leading to cell death.8 If chitosan fails to safely deliver the DNA box, alternative cationic polymers will be explored. Additionally, if the mesh size of the swollen nanoparticle is not large enough to accommodate the size of the DNA origami box, the nanoparticle will need to be made to swell more or the boxes will need to be made smaller.  If the purchased CRISPR/CAS9 does not significantly decrease MCF-7 cell viability, research into other CRISPR products will be researched.

Aim 2: Evaluate the ability of the aptamer to target HER1/HER3 for cellular uptake and the CRISPR/CAS9 to shutdown the ERK signaling cascade. The working hypothesis is that the aptamer targets HER1 and HER3 for cellular uptake to expose the contents of the cell to the CRISPR/CAS9. We formulated this hypothesis based on the success of previous studies involving a triple aptamer to target HER1, HER2, and HER3 receptors for endocytosis and cellular uptake.14,16 Within this aim, we expect to modify previously established methods14 to synthesize an aptamer to target HER1 and HER3 in order to achieve endocytosis of our nanoparticle.

Experimental Design: Our double aptamer complex modified from Yu, et al, will be carboxylated to our previously developed chitosan nanoparticles (loaded with DNA boxes that contain CRISPR/CAS9).14,17 To ensure carboxylation occurred, FTIR will be used to determine if the appropriate peak is expressed. We will be using an alamar blue assay to measure the viability of the cell line after treatment. We will also use a fluorescent tag to CAS9 to observe if it reached the cell nucleus. Additionally, we will sequence and quantify the DNA of the cells before and after treatment through rt-PCR in order to assess the ability of the CRISPR/CAS9 technology to delete the desired genome sequence.

Expected Outcomes: We expect that the aptamer will successfully target the HER1 and HER3 receptors and increase cellular uptake of our nanoparticle. Once inside the endsome, the nanoparticle will swell, bursting the endosome and releasing the DNA origami box into the cytosol. The acidic contents of the endosome will open the origami box which in turn should allow the CRISPR/CAS9 to escape and delete the cascade of the ERK signaling pathway from the genome. Resulting in eventual termination of the cell.

Alternative Strategies: If the aptamer does not target the correct receptor or promote cellular uptake of our complex, then additional aptamer strategies and development will need to be researched. Our design may need to target a new receptor if endocytosis of our nanoparticle does not occur. If the aptamer is not successfully carboxylated to our nanoparticle, then other conjugation methods such as amide linkages will be evaluated or other cationic nanoparticles will be evaluated for carboxylation.

REFERENCE LIST

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(2) Walker, Francine, et al. “Growth Factor Receptor Expression in Anal Squamous Lesions: Modifications Associated with Oncogenic Human Papillomavirus and Human Immunodeficiency Virus.” Human Pathology, W.B. Saunders, 27 Aug. 2009, www.sciencedirect.com/science/article/pii/
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(3) Kumar, Vinay, et al. Robbins Basic Pathology. Elsevier/Saunders, 2013. p. 179

(4) Tovey, Sian M, et al. “Outcome and Human Epithelial Growth Factor Receptor (HER) 1-4 Status in Invasive Breast Carcinomas with Proliferation Indices Evaluated Using Bromodeoxyuridine (BrdU) Labelling.” Breast Cancer Research, BioMed Central, 4 Mar. 2004, breast-cancer-research.biomedcentral.com
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(7) Zhan, Jiayin, et al. “The Research Progress of Targeted Drug Delivery Systems.” IOP Conference Series: Materials Science and Engineering, vol. 207, 2017, doi:10.1088/1757-899x/207/1/012017.

(8) Bilensoy, Erem. “Cationic Nanoparticles for Cancer Therapy.” Expert Opinion on Drug Delivery, vol. 7, no. 7, June 2010, pp. 795–809., doi:10.1517/17425247.2010.485983.

(9) Kohno, Michiaki, and Jacques Pouyssegur. “Targeting the ERK Signaling Pathway in Cancer Therapy.” Annals of Medicine, vol. 38, no. 3, 2006, pp. 200–211., doi:10.1080/07853890600551037.

(10) Addison, Frances. “How to Get CRISPR into Cells.” How to Get CRISPR into Cells | Front Line Genomics, Front Line Genomics, 9 Feb. 2017, www.frontlinegenomics.com/news/9942/delivering-crispr-cells/.

(11) Burns, Jonathan, et al. “DNA Origami Inside-Out Viruses.” ACS Synthetic Biology, 7 Feb. 2018, cdn-pubs.acs.org/doi/10.1021/acssynbio.7b00278.

(12) Rampino, Antonio, et al. “Chitosan Nanoparticles: Preparation, Size Evolution and Stability.” International Journal of Pharmaceutics, Elsevier, 22 July 2013, www.sciencedirect.com/science/article/pii/
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(13) “ERK 1 CRISPR Knockout and Activation Products (h).” SCBT – Santa Cruz Biotechnology, www.scbt.com/scbt/product/erk-1-crispr-knockout-and-activation-products-h/;jsessionid=KNPz_nTRJLf3xFYFNa88XHUijDzwZk3QfOKAmje-X7HLPWKk6kaH!1538204823.

(14) Yu, Xiaolin, et al. “Targeting EGFR/HER2/HER3 with a Three-in-One Aptamer-SiRNA Chimera Confers Superior Activity against HER2 Breast Cancer.” Molecular Therapy – Nucleic Acids, vol. 10, 2018, pp. 317–330., doi:10.1016/j.omtn.2017.12.015.

(15) Ceresa, Brian P. “Spatial Regulation of Epidermal Growth Factor Receptor Signaling by Endocytosis.” MDPI, Multidisciplinary Digital Publishing Institute, 20 Dec. 2012, www.mdpi.com/1422-0067/14/1/72.

(16) Shilova, O. N., et al. “Internalization and Recycling of the HER2 Receptor on Human Breast Adenocarcinoma Cells Treated with Targeted Phototoxic Protein DARPinminiSOG.” Acta Naturae, A.I. Gordeyev, July 2015, www.ncbi.nlm.nih.gov/pmc/articles/PMC4610174/.

(17) Sayari, E., et al. “MUC1 Aptamer Conjugated to Chitosan Nanoparticles, an Efficient Targeted Carrier Designed for Anticancer SN38 Delivery.” International Journal of Pharmaceutics, Elsevier, 3 June 2014, www.sciencedirect.com/science/article/pii/S0378517314003810.