project

description

What is our project?

We aim to design a microRNA-based detection system for non-Hodgkin's B-cell lymphoma that would be more cost-effective, efficient, and accessible than current methods.

b-cell lymphoma

Non-Hodgkin's B-cell Lymphoma is part of the Leukemia and Myeloma family of aggressive blood cancers with increasing rates of diagnosis and notable importance in modern oncological research. Blood cancers account for 8.6% of all new cancer diagnoses in the United States. Every 9 minutes, a person in the United States dies of Lymphoma, Leukemia, or Myeloma with an estimated 60,300 deaths in 2001 alone (Public Law 107-172, 2002). B-cell Lymphoma specifically kills more than 1 in every 100,000 people, and while diagnosis and death rates are decreasing, this disease still kills a significant population every year (Winkelmann, N.). B-cell Lymphoma has also garnered attention in legislation, with the Hematological Cancer Research Investment and Education Act of 2002 inspiring a swell of research surrounding blood cancers. Despite this, a disparity exists between class and race in both diagnosis of the disease and survival rate. In a cohort of 223,709 patients diagnosed with Large Diffuse B-cell Lymphoma from the National Cancer Database between 2004 and 2018, 87% of patients were White, 8% were Black, and 5% were other ethnicities. In patients 60 years or younger, overall survival outcomes were 76 months in White patients, but only 46 months in Black patients (Ermann et al., 2022).

current diagnoses

Currently, diagnosis of B-cell Lymphoma is difficult and costly. Often, procedures involve invasive techniques such as lymph node and bone marrow biopsies, immunophenotyping, flow cytometry, cytogenetic analysis, and gene expression profiling (Leukemia and Lymphoma Society), as well as X-ray, PET imaging, and slow physical examination requiring the development of a tumor prior to diagnosis (Cincinnati Children's Hospital). Current blood tests for B-cell Lymphoma require immunophenotyping and flow cytometry which require cytometers with most costing $75,000-$100,000, a prohibitively expensive amount for many people (Cell analysis at the bench). Not only are these tests prohibitively expensive but they also cannot detect B-cell Lymphoma early enough to maximize treatment opportunities.

Lily's Legacy...

The project has its origins in an idea to try to diagnose canine B-cell lymphoma earlier. This idea started because of the story of a dog belonging to one of the team leaders. The dog's name was Lily and she had been diagnosed with B-cell lymphoma. Her survival for nearly a year despite a poor initial prognosis inspired our idea to create a test to diagnose the cancer earlier. Such a test is especially relevant for B-cell and non-Hodgkin's lymphomas, which usually spread undetected and do not present symptoms until they have become difficult to treat. By creating a test that would detect lymphoma earlier, it will be possible to increase patients'chances of survival. Ultimately, while we shifted our focus from dogs to humans, our goal remains: to increase the chances of survival for B-cell lymphoma patients by diagnosing them as early as possible.

...to Lifesaving Innovations

As the project developed, we made changes based on a desire to advance in the field. Our first idea was to use an E. coli chassis and RNA or protein secondary structures to create a switch that would activate GFP upon binding to biomarker microRNA (or miRNA). We chose miRNA-326, which research has correlated with B-cell lymphoma. As we realized the difficulties of creating such secondary structures, we attempted to integrate how miRNA operates naturally into our design. This focus led us to make a circuit including AGO2, the protein that silences genes in humans and eukaryotes. In our circuit, AGO2 will use miRNA-326 to downregulate repressor proteins for GFP, thus allowing GFP expression only when miR-326 is present. While the AGO2 protein has been cloned in bacteria before, this would be the first use of AGO2 to regulate gene translation in E. coli using a specific microRNA as an inducer, an advance in the field that we hope will encourage more use of miRNA to achieve practical, diagnostic, and experimental results in synthetic biology.

our solution

We hope to address this problem by creating an effective, portable, and cheaper alternative to traditional B-cell Lymphoma diagnosis utilizing a synthetic biology blood test which detects microRNA. MicroRNA, or miRNA, is a small non-coding RNA responsible for translational gene regulation. Changes in blood concentration have been shown to correlate with the presence or absence of different diseases. Specifically for B-cell lymphoma, miRNA has been shown to detect it up to 2 and half years before proper diagnosis (Jørgensen, S., et al.).

our solution

To take advantage of this, we aim to utilize E.coli-based cell-free systems with an engineered gene circuit that will equip them to detect microRNA associated with B-cell Lymphoma, specifically miRNA-326. This will be done with two plasmids: one of human Argonaute 2 (AGO2), and the other of transcriptional repressor LacI, translation repressor L7Ae, and GFP. Without miRNA, the LacI and L7Ae work as a dual-regulation system to prevent the expression of GFP. However, in the presence of miRNA, AGO2 binds with the miRNA, forming an RNA-induced silencing complex (RISC). miRNA acts as the guide strand, and these two core elements can perform the main activities of the RISC complex. The RISC then cleaves mRNA that is complementary to the miRNA guide strand. In our system, the complementary mRNA segment will be placed in the second plasmid to cut and prevent LacI and L7Ae from repressing GFP expression. Therefore, the E.coli cells are allowed to glow, indicating that miRNA is present. Since miRNA requires near-perfect base complementarity to bind to a target sequence in mRNA, the resulting high specificity can ensure our system accurately detects the presence of the miRNA of interest and minimize the risk of false positive and negative results and thus allow precise diagnostics.

our solution

We aim to test our system in actual E. Coli cells first, by transforming miRNA into our engineered E.coli. This represents a relatively novel and undocumented process. Thus, success would be a significant contribution to research surrounding miRNA and RNA activity in cells. Combined with a microfluidic assay whose development is beyond the scope of this project, this method can be used in a novel diagnostic option, only utilizing a small blood sample. Such a diagnostic, possibly taking the form of a simple finger prick blood test, would be minimally invasive and can help expand diagnosis to a greater population.

references

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Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G., & Tuschl, T. (2004). Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Molecular cell, 15(2), 185-197. https://doi.org/10.1016/j.molcel.2004.07.007

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Salvatore, V., Potenza, N., Papa, U., Nobile, V., & Russo, A. (2010). Bacterial expression of mouse argonaute 2 for functional and mutational studies. International journal of molecular sciences, 11(2), 745-753. https://doi.org/10.3390/ijms11020745

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