X-Linked Agammaglobulinemia (XLA) is a devastating genetic disorder in which the BTK gene is non-functional, producing B-cells that are unable to fight infection. We employ adenine base editing (ABE) to restore function in the BTK gene in autologous hematopoietic stem and progenitor cells (HSPCs). This method has strong potential for correcting BTK mutations, as ABEs cause direct conversion of A-T to G-C base pairs while minimizing insertions or deletions, resulting in a higher success rate at producing a base-perfect product compared to wildtype BTK genes, something homology-directed repair through CRISPR-Cas9 cannot do consistently. We hypothesize that ABE may act as a one-time treatment that can restore normal B-cell development in XLA patients. We are currently testing the base editor on mutations C1684T, C763T, and G1761A, evaluating various protospacer adjacent motifs (PAMs) and guide RNA (gRNA) combinations for editing efficiency. We found high levels of editing at the correct target site with minimal bystander editing at adjacent adenines, particularly with C1684T, especially when compared to predictions from BE-HIVE. Following this, functional analysis of protein reconstitution was performed via Western blot. We also found 40% editing in healthy donor CD34+ cells transduced with BTK harboring lentiviruses. Going forward, we aim to continue validating ABE usability in HSPCs, determine functional restoration following ABE introduction, and begin testing in mice engrafted with edited HSPCs.

The protozoan parasite Toxoplasma gondii has established chronic infection in nearly one-third of the world’s population, largely due to its broad array of host immune evasion mechanisms induced through the secretion of effector proteins. Mitochondrial association factor 1b (TgMAF1b) is an effector protein that tethers the parasite vacuole to host mitochondria via direct interaction with mitochondrial import receptor TOM70. Mitochondria are hubs of innate immunity, with TOM70 playing a role in immune signaling by enhancing mitochondrial antiviral signaling (MAVS) protein-mediated activation of IRF3 and NF-κB and downstream interferon (IFN) responses. To investigate whether TgMAF1b modulates immune responses by interacting with TOM70, TgMAF1b was exogenously expressed in murine embryonic fibroblasts and immune responses were quantified after immunostimulation using luciferase reporter assays. Preliminary data indicates that expression of TgMAF1b amplified levels of IFN-γ signaling but dampened levels of Type-I IFN and NF-κB activity, suggesting the effector protein can interfere with the MAVS signalosome. Further, expression of a TgMAF1b variant harboring a C-terminal deletion that disrupts its ability to bind TOM70 fails to inhibit Type-I IFN responses, indicating the binding interaction between TgMAF1b and TOM70 is required to dampen innate immune responses. The immune modulation strategy explored here reveals host-pathogen dynamics which may be conserved in other infectious agents that interact with host mitochondria.

Skeletal muscle stem cells (satellite cells) inhabit a specialized niche beneath the basal lamina of myofibers, where they remain quiescent until activated by muscle damage. While human pluripotent stem cell-derived skeletal muscle progenitor cells (hPSC-SMPCs) are potential candidates for treating muscular dystrophies, their capacity to colonize the adult muscle stem cell niche is less effective than that of primary adult MuSCs. This discrepancy may stem from developmental variations or a lack of compatibility between the transplanted progenitors and the recipient environment. We hypothesized that the age of the host environment influences niche occupancy, suggesting that neonatal muscle might be more accommodating to immature progenitors. To investigate this, we transplanted human SMPCs into the tibialis anterior muscles of neonatal and adult mice without prior injury. Following a 28-day period, we evaluated engraftment by measuring total human cell presence, niche occupancy, and the restoration of dystrophin. Our results indicated that the neonatal recipients exhibited significantly higher counts of dystrophin positive fibers, implying enhanced myogenic integration within the younger muscle context. Ongoing research is focused on optimizing delivery methods and cell dosages to further clarify how host age impacts niche colonization.

Tumor-infiltrating lymphocytes and immune checkpoint blockade therapies have shown marked clinical success; however, only a subset of patients demonstrate durable responses. T-cell receptor (TCR) engineered T cell therapy is a promising approach for non-responsive patients, leveraging T-cell cytotoxicity against tumor neoantigens. However, the TCR properties that confer sustained cytotoxic responses remain poorly understood. Our laboratory has developed panels of neoantigen-specific TCRs derived from melanoma patients treated with immune checkpoint inhibitors and corresponding autologous melanoma cell lines. This study leverages a panel of TCRs that recognize the same tumor neoantigen but exhibit distinct cytotoxic profiles in repetitive antigen challenge assays against the autologous melanoma cell line. For TCR characterization, we developed in-house peptide-MHC-beta-2-microglobulin single-chain trimers (pMHC-SCTs). These pMHC-SCTs were validated by Western blot, and their binding to specific TCRs was confirmed by flow cytometry. The constructs were then used to assess T-cell anti-tumor function across the panel, including measurements of functional affinity and avidity, cytokine production by intracellular cytokine staining, and signaling dynamics by phosphoflow. Ultimately, in-house generation of these reagents supports mechanistic studies aimed at defining TCR-dependent pathways, providing a foundation for engineering strategies to enhance the durability of TCR-based therapies in solid tumors.

The Aguilar lab is doing research on viruses with very high mortality rates (the identities of the tested virus was anonymized to protect unpublished data) in attempt to develop vaccines using self-amplifying RNA (saRNA) to protect humans from these viruses. saRNA is harvested via hamster sera, which contains antibodies generated in response to the vaccine. This research aims to investigate whether the vaccinated-hamster sera from the saRNA-based platform bind to cells expressing viral fusion proteins and to make pseudotyped particles with high titers to infect Vero cells.