T cell development occurs in the thymus, where thymic progenitors differentiate into various cell types, including regulatory T (Treg) cells. The transcription factor FOXP3 is essential for Treg development and immune regulation, directing lineage specification; mutations cause severe autoimmune diseases such as IPEX syndrome. To study the impact of FOXP3 in vitro, the artificial thymic organoid (ATO) system enables recapitulation of T cell development and is currently the only model that generates mature, positively selected T cells. This system incorporates induced pluripotent stem cells (iPSCs) that undergo hematopoietic specification. However, robust generation of FOXP3+ Tregs in the ATO remains elusive. To explore the effects of FOXP3 expression, specific isoforms can be overexpressed in iPSCs, including the canonical full-length isoform, FOXP3fl, and FOXP3Δ2, which lacks the exon 2 nuclear export signal. By engineering CRISPR-Cas-9 constructs for a knock-in approach to insert FOXP3 into the endogenous ZBTB7B (ThPOK) gene locus, we can restrict FOXP3fl expression to CD4+ T cells. The Cas9-guide ribonucleoprotein complex and the FOXP3 donor template were delivered via electroporation and heterozygous and homozygous FOXP3fl edited clones were isolated after. We will now study the impact of delayed FOXP3fl expression on development within the ATO. This research seeks to advance our understanding of T cell development, with potential implications for novel therapeutic strategies for autoimmune diseases.

X-linked agammaglobulinemia is a disease affecting 1 in 200,000 males caused by a mutation of the Bruton tyrosine kinase gene, responsible for the development of B cells. Since B cells produce antibodies, patients affected by XLA have an increased susceptibility to infections. Allogeneic transplant of bone marrow from a healthy donor to the patient would result in a cure. However, it poses the threat of graft vs host disease. Alternatively, gene editing the patient’s hematopoietic stem cells would allow for correction of the mutation and restore the HSCs ability to differentiate into functional B cells. Before clinical applications, this must be tested and optimized in vitro. We focused on the use of viral vectors when delivering a healthy BTK donor sequence. Adeno-associated virus (AAV) have previously led to high integration of the BTK donor, however it was toxic to cells in vitro. On the other hand, integration-defective lentiviral vectors (IDLV) had less integration potential, but preserved cell viability. In order to test optimal conditions for gene editing, we electroporated HSCs with a CRISPR/Cas9 complex and transduced them with a DNA donor using either AAV or IDLV. Then, we compared BTK integration by ddPCR and BTK expression using flow cytometry. Preliminary findings concluded that AAV integrated BTK more efficiently and had higher BTK expression, but more optimizations of both vectors must be done to confirm. Once done successfully, it will be possible to select the best vector for delivery in patient cells.

Duchenne muscular dystrophy (DMD) is a severe, X-linked neuromuscular disorder characterized by muscle degeneration. This project examines how Ataluren treatment, a nonsense mutation read-through drug, alters muscle cellular composition and transcriptional states in DMD using single-nucleus RNA sequencing (snRNA-seq) of patient muscle biopsies collected pre-treatment, after nine months, and after four years, alongside healthy controls. Nuclei were clustered with Seurat to define major cell types and mapped onto a 43-library DMD reference using Symphony to identify healthy-like and DMD-like subclusters. Ataluren treatment was associated with progressive shifts across cell types towards healthy-like states, particularly in samples that received long-term treatment. Key changes included a partial restoration of quiescent satellite-cell pools, reduced commitment to dysregulated satellite-cell states, a shift of type I myofibers towards healthy-like states, and increased differentiation into myocytes. Gene set enrichment analysis using Enrichr demonstrated activation of myogenesis, muscle contraction, and tRNA aminoacylation pathways in several cell types, suggesting enhanced protein synthesis and muscle tissue recovery. Overall, Ataluren treatment in nonsense-mutation DMD is associated with progressive, cell-specific remodeling of the muscle environment towards healthier cellular and transcriptional states over time, supporting Ataluren’s potential to modify disease progression at the cellular level.

Epigenetic regulators play critical roles in controlling lymphocyte function. UTX, a canonical histone demethylase, has been shown to be essential for natural killer (NK) cell responses, but its mechanism remains unclear. Notably, loss of the demethylase domain in UTX does not impair NK cell function, suggesting that UTX regulates NK cells through a mechanism other than its demethylase activity. Loss-of-function mutations in UTX and KMT2D, another histone modifier, cause Kabuki syndrome, a disorder characterized by growth abnormalities and increased susceptibility to viral infections. Using NK-cell-specific knockout mouse models, we found that loss of either UTX or KMT2D results in impaired effector function (i.e. reduced effector molecule production and degranulation measured by flow cytometry). In addition, deletion of one protein leads to reduced levels of the other, suggesting a stabilizing relationship. As such, we hypothesize that KMT2D forms a stabilizing complex with UTX to regulate NK cell function. To test this, we are transducing a UTX stabilization domain (identified in the Drosophila homolog of KMT2D) into KMT2D-deficient NK cells and assessing whether this rescues NK effector function. This work defines a demethylase-independent mechanism of epigenetic regulation in NK cells and can provide insight into immune dysfunction in Kabuki syndrome, which may inform future therapeutic strategies.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus linked to several diseases, including Kaposi sarcoma, primary effusion lymphoma, and multicentric Castleman disease. It establishes lifelong infection by alternating latent and lytic cycles, permitting immune evasion and host defense exploitation. Since KSHV is primarily transmitted via a salivary-mucosal route, preventing infections at mucosal surfaces is critical. Mucosal vaccines aim to elicit immune protection at entry sites, such as the oral and nasal cavity, with intranasal immunization conferring to oral protection through shared mucosal immune trafficking and secretory IgA in saliva. Despite this potential, few studies have examined mucosal vaccines against KSHV, highlighting a gap in prevention strategies. Here, we evaluated lipid nanoparticles (LNPs) carrying KSHV open reading frame 4 (ORF4) mRNA, which encodes a complement control protein, for intranasal immunization. In a murine model, intranasal immunization was well tolerated and elicited systemic and mucosal antibody responses. Indirect ELISA of serum and saliva detected ORF4-specific IgA and IgG, indicating activation of both mucosal and systemic immunity. These findings underscore the potential for mRNA-LNP vaccination delivered intranasally to induce mucosal responses against KSHV and reduce KSHV-associated diseases.