The COVID-19 pandemic has brought to our attention the lack of fast, affordable, and sensitive diagnostic tests available on the market. The majority of commercial diagnostic tools are expensive, despite being quick and sensitive. This conundrum has brought attention to the necessity for developing high-quality tests at an affordable cost, emphasizing the importance of accessible diagnostics that are both rapid and sensitive. The Yager lab has been developing detection tools for infectious diseases, mainly based on isothermal nucleic acid amplification tests, using loop-mediated isothermal amplification (LAMP). The LAMP assay includes a DNA polymerase known as Bst. It was found that assay sensitivity improved when the samples were pretreated with HUDSON, which consists of TCEP (a reducing agent) and EDTA. I am investigating the improvement of Bst DNA polymerase activity with TCEP, using a commercial enzyme kinetics kit (EvaEZ) to quantify the Bst polymerase activity. The EvaEZ assay allows quantitative comparison between conditions by measuring fluorescence intensity indicative of DNA amplification. This was achieved by assessing primer binding, enzymatic extension, and EvaGreen dye intercalation, enabling comparison of the rates of fluorescence generation to evaluate amplification efficiency across positive, negative, and experimental control conditions. This project is still ongoing, and preliminary findings suggest promising results. This study demonstrates the potential for using reducing agents to optimize enzyme efficiency and improve detection sensitivity. This technique can readily improve detection speed and sensitivity both simply and affordably. We are able to be better prepared for the next pandemic.

Myeloproliferative neoplasms (MPNs) are blood cancers that can arise from the constitutive activation of the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathway. One mutation in this pathway, JAK2^V617F, is especially prevalent among MPN patients. JAK2^V617F alters the JAK2 protein structure so that the kinase is persistently phosphorylated and JAK-STAT signaling is perpetually active. However, the role of JAK2^V617F in MPN development is not well defined and selective therapeutic strategies to target JAK2^V617F remain elusive. To address these challenges, my project’s goal is to selectively degrade the JAK2^V617F protein and evaluate its downstream effects using the Ba/F3 cell system. Ba/F3 cells are interleukin-3 (IL-3) dependent for survival but can become IL-3 independent in the presence of an oncogene. I hypothesize that JAK2^V617F expression in Ba/F3 cells will confer IL-3 independence, while degradation of the mutant protein will revert the cells to their native, IL-3-dependent phenotype. To model pharmacological degradation of JAK2^V617F, I applied the degradation tag (dTAG) system, which harnesses the cellular ubiquitin-proteasome machinery to degrade proteins fused with an FKBP12^F36V tag using dTAG molecules. I first cloned plasmids to lentivirally express FKBP12^F36V-JAK2^V617F in Ba/F3 cells. Following viral transductions, I treated the transduced cells expressing FKBP12^F36V-JAK2^V617F with vehicle control or dTAG molecules to induce degradation. Western blots were used to confirm FKBP12^F36V-JAK2^V617F expression and dose-dependent degradation. To evaluate whether JAK2^V617F confers IL-3 independence, I cultured the Ba/F3 cells expressing FKBP12^F36V-JAK2^V617F without IL-3, treated them with vehicle control or dTAG molecules, and assessed cell viability using CellTiter-Glo luminescence assays. I anticipate IL-3 independent cell survival of the Ba/F3 cells expressing FKBP12^F36V-JAK2^V617F to decrease with increasing dTAG molecule treatment. The results from this project will build a new pipeline for studying oncogenes and contribute to the evaluation of JAK2^V617F degradation as a novel therapeutic approach against MPNs.

Bacteriophage MS2 phage-like particles (PLPs) are artificially constructed viral-like particles. The similarity of the particle to a virus allows the particles to be used as a control system for molecular detection and drug delivery systems. The capsid is made up of single chain coat protein dimers (SCCPD) and a singular maturase, and is able to package mRNA within the protein coat due to the dimerization of coat proteins spontaneously forming the capsid structure in the presence of a packaging signal. In addition, the PLPs also have the ability to display what is being packaged on the surface. Currently, it has been shown that PLPs are able to be purified via His-tag affinity, due to fusion of the His-tag onto the SCCPDs. However, if a peptide is displayed at the SCCPD site, the His-tag must be attached elsewhere on the PLP to ensure the PLP can be purified. To address this, I designed a new PLP by plasmid engineering via site-directed mutagenesis to display a peptide on AB-loops within the SCCPD, whilst packaging the corresponding mRNA within the capsid. I purified the PLP via a His-tag attached to the maturase protein. To verify correct particle formation, I ran SDS-PAGE to observe the density of bands corresponding to SCCPD and maturase. I designed a reverse transcriptase polymerase chain reaction and carried out a nuclease protection assay to verify mRNA packaging. Preliminary data of SDS-PAGE has shown the particle has successfully purified, and is correctly forming due to the observed maturase to SCCPD band density ratio on the gel meeting the expected ratio.

Protein nanoparticles are useful for the design of novel vaccines. We can use these nanomaterials for display of antigens; however, antigens tested thus far have been homooligomers—consisting of a single unique component, and existing protein nanoparticle assemblies are not well-suited for the display of heterooligomeric antigens (such as HCV E1E2). We attempt to solve this problem through designing uniformly symmetric icosahedral nanoparticles that contain two distinct protein chains within their fundamental geometric component—referred to as the asymmetric unit; this allows us to retain the particle symmetry and double the number of accessible linkage points, or chain termini. This doubling of termini could allow us to fuse heterodimeric antigens to our cages. To accomplish this design goal, I utilized RFdiffusion—a generative machine learning model—to generate two-component icosahedral protein backbones, which were then filtered by evaluating subunit packing through a set of contact distance calculations. ProteinMPNN, a DL-based sequence design method, was used to assign candidate sequences to each of the filtered backbones. Finally, complete designs were filtered by using AlphaFold2 to evaluate fidelity to the original design model. I expressed the top 96 designs in E. coli, but saw minimal protein with no indication of assembly. In an attempt to maximize my chances of forming successful cages, I have elected to conditionally generate backbones that favor alpha-helical secondary structure. In this new design round, I hope to see favorable improvements in inter-chain packing; this will lead to an increase in passing design candidates and hopefully allow my computationally generated structures to have a higher chance of assembly in lab. This work serves to streamline the development of a therapeutic platform that can display multi-component antigens, which could enable the creation of new vaccines.

Subtherapeutic drug levels can lead to the failure of antiretroviral therapy (ART) regimens used in Human Immunodeficiency Virus (HIV) treatment and prevention. However, gold-standard HIV drug level monitoring techniques—such as mass spectrometry—require bulky and expensive instruments that are not widely accessible at the point-of-need. Our group developed the REverSe TRanscrIptase Chain Termination (RESTRICT) enzymatic assay to rapidly (30 min) and inexpensively measure tenofovir diphosphate (TFV-DP), a nucleotide analog used in >90% of oral ART regimens and in all approved prevention regimens. However, RESTRICT currently requires trained operators to perform multiple time-sensitive liquid-handling steps. To reduce user intervention and minimize the need for laboratory equipment, we harnessed 3D-printed capillaric microfluidics to self-propel liquids using only surface tension effects encoded in microchannel geometry and surface chemistry. Specifically, we translated the manual tube-based RESTRICT to an automated microfluidic protocol by using autonomous trigger valves to pre-load multiple RESTRICT assay reagents and serpentine channels to control assay timing. Currently, RESTRICT reactions are incubated for 30 minutes at 37˚C, but we decreased the reaction time to 15 minutes and removed the need for an external heating source by incubating at room temperature (25˚C). There was only a 15% decrease in overall signal intensity in the faster, room temperature assays, and measured readout was distinguishable between clinically relevant concentrations of TFV-DP. Our results represent a first step towards integrating RESTRICT reactions and fluorescence readout onto a rapidly fabricated microfluidic chip. We hope to achieve a device that increases the accessibility of HIV drug level monitoring at the point of need without specialized equipment or highly trained operators.

Chimeric antigen receptor (CAR) T-cell therapy is a revolutionary cancer treatment with promising clinical efficacy in treating hematological cancers. Yet, current CAR T cells are designed to target a single antigen and have had little success treating solid-tumors due to tumor-antigen heterogeneity. To address these limitations, we have engineered a universal SpyCatcher003 CAR T-cell system (DB5 CARs) that utilizes synthetic targeting-intermediates conjugated onto CARs to bind multiple cancer-antigens and mediate tumor cell killing. My project aims to utilize a design based approach to engineer and optimize the serum stability of the SpyTag003-peptide to improve T-cell activation for in vivo cancer treatment applications. In preliminary studies, we demonstrate the loading of a synthetic, high-affinity biomaterial folate-SpyTag003 peptide chimera onto DB5 CAR T cells. In addition, folate-SpyTag003 binds with high-affinity and specificity to folate receptor alpha positive (FOLR1⁺) KB tumor cells. CD8⁺ and CD4⁺ DB5 CAR T cells labeled with folate-SpyTag003 chimera showed a robust increase in cytotoxic activity and cytokine expression when incubated with FOLR1⁺ KB cells. Thus, we aim to optimize the SpyTag003-peptide chimera to improve binding to FOLR1 for targeted killing of FOLR1⁺ tumor cells (e.g., ovarian and breast tumors) as an in vivo cancer treatment application. By iteratively improving the design of the folate-SpyTag003 intermediate and DB5 CARs, our approach for universal CAR T-cell therapy could provide safe, cost-effective, and broad-targeting treatments for patients with heterogeneous solid-tumors.