Deciphering regulation of FOXP3 expression in human conventional T cells.

FOXP3 is a lineage-defining transcription factor that controls differentiation and maintenance of suppressive function of regulatory T cells (Tregs). Foxp3 is exclusively expressed in Tregs in mice. However, in humans, FOXP3 is not only constitutively expressed in Tregs; it is also transiently expressed in stimulated CD4+CD25- conventional T cells (Tconvs)1-3. Mechanisms governing the expression of FOXP3 in human Tconvs are not understood. Here, we performed CRISPR interference (CRISPRi) screens using a 15K-member gRNA library tiling 39 kb downstream of the FOXP3 transcriptional start site (TSS) to 85 kb upstream of the TSS in Treg and Tconvs. The FOXP3 promoter and conserved non-coding sequences (CNS0, CNS1, CNS2 and CNS3), characterized as enhancer elements in murine Tregs, were required for maintenance of FOXP3 in human Tregs. In contrast, FOXP3 in human Tconvs depended on regulation at CNS0 and a novel Tconv-specific noncoding sequence (TcNS+) located upstream of CNS0. Arrayed validations of these sites identified an additional repressive cis-element overlapping with the PPP1R3F promoter (TcNS-). Pooled CRISPR knockouts revealed multiple transcription factors required for proper expression of FOXP3 in Tconvs, including GATA3, STAT5, IRF4, ETS1 and DNA methylation-associated regulators DNMT1 and MBD2. Analysis of ChIP-seq and ATAC-seq paired with knock-out (KO) of GATA3, STAT5, IRF4, and ETS1 revealed regulation of CNS0 and TcNS+ accessibility. Collectively, this work identified Treg-shared and Tconv-specific cis-elements and the trans-factors that interact with them, building a network of regulators controlling FOXP3 expression in human Tconvs.

Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells.

Cis-regulatory elements (CREs) interact with trans regulators to orchestrate gene expression, but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of the adjacent costimulatory genes CD28, CTLA4 and ICOS, encoding regulators of T cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells, both conventional and regulatory subsets, uncovered gene-, cell subset- and stimulation-specific CREs. Integration with CRISPR knockout screens and assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. We then discovered a critical CCCTC-binding factor (CTCF) boundary that reinforces CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets, this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex, multi-gene locus critical to immune homeostasis.

Non-coding sequence variation reveals fragility within interleukin 2 feedback circuitry and shapes autoimmune disease risk.

Genetic variants associated with human autoimmune diseases commonly map to non-coding control regions, particularly enhancers that function selectively in immune cells and fine-tune gene expression within a relatively narrow range of values. How such modest, cell-type-selective changes can meaningfully shape organismal disease risk remains unclear. To explore this issue, we experimentally manipulated species-conserved enhancers within the disease-associated IL2RA locus and studied accompanying changes in the progression of autoimmunity. Perturbing distinct enhancers with restricted activity in conventional T cells (Tconvs) or regulatory T cells (Tregs)-two functionally antagonistic T cell subsets-caused only modest, cell-type-selective decreases in IL2ra expression parameters. However, these same perturbations had striking and opposing effects in vivo , completely preventing or severely accelerating disease in a murine model of type 1 diabetes. Quantitative tissue imaging and computational modelling revealed that each enhancer manipulation impinged on distinct IL-2-dependent feedback circuits. These imbalances altered the intracellular signaling and intercellular communication dynamics of activated Tregs and Tconvs, producing opposing spatial domains that amplified or constrained ongoing autoimmune responses. These findings demonstrate how subtle changes in gene regulation stemming from non-coding variation can propagate across biological scales due to non-linearities in intra- and intercellular feedback circuitry, dramatically shaping disease risk at the organismal level.

A Localized Materials-Based Strategy to Non-Virally Deliver Chondroitinase ABC mRNA Improves Hindlimb Function in a Rat Spinal Cord Injury Model.

Spinal cord injury often results in devastating consequences for those afflicted, with very few therapeutic options. A central element of spinal cord injuries is astrogliosis, which forms a glial scar that inhibits neuronal regeneration post-injury. Chondroitinase ABC (ChABC) is an enzyme capable of degrading chondroitin sulfate proteoglycan (CSPG), the predominant extracellular matrix component of the glial scar. However, poor protein stability remains a challenge in its therapeutic use. Messenger RNA (mRNA) delivery is an emerging gene therapy technology for in vivo production of difficult-to-produce therapeutic proteins. Here, mineral-coated microparticles as an efficient, non-viral mRNA delivery vehicles to produce exogenous ChABC in situ within a spinal cord lesion are used. ChABC production reduces the deposition of CSPGs in an in vitro model of astrogliosis, and direct injection of these microparticles within a glial scar forces local overexpression of ChABC and improves recovery of motor function seven weeks post-injury.

Single-dose mRNA therapy via biomaterial-mediated sequestration of overexpressed proteins.

Nonviral mRNA delivery is an attractive therapeutic gene delivery strategy, as it achieves efficient protein overexpression in vivo and has a desirable safety profile. However, mRNA's short cytoplasmic half-life limits its utility to therapeutic applications amenable to repeated dosing or short-term overexpression. Here, we describe a biomaterial that enables a durable in vivo response to a single mRNA dose via an "overexpress and sequester" mechanism, whereby mRNA-transfected cells locally overexpress a growth factor that is then sequestered within the biomaterial to sustain the biologic response over time. In a murine diabetic wound model, this strategy demonstrated improved wound healing compared to delivery of a single mRNA dose alone or recombinant protein. In addition, codelivery of anti-inflammatory proteins using this biomaterial eliminated the need for mRNA chemical modification for in vivo therapeutic efficacy. The results support an approach that may be broadly applicable for single-dose delivery of mRNA without chemical modification.

Functionalization of microparticles with mineral coatings enhances non-viral transfection of primary human cells.

Gene delivery to primary human cells is a technology of critical interest to both life science research and therapeutic applications. However, poor efficiencies in gene transfer and undesirable safety profiles remain key limitations in advancing this technology. Here, we describe a materials-based approach whereby application of a bioresorbable mineral coating improves microparticle-based transfection of plasmid DNA lipoplexes in several primary human cell types. In the presence of these mineral-coated microparticles (MCMs), we observed up to 4-fold increases in transfection efficiency with simultaneous reductions in cytotoxicity. We identified mechanisms by which MCMs improve transfection, as well as coating compositions that improve transfection in three-dimensional cell constructs. The approach afforded efficient transfection in primary human fibroblasts as well as mesenchymal and embryonic stem cells for both two- and three-dimensional transfection strategies. This MCM-based transfection is an advancement in gene delivery technology, as it represents a non-viral approach that enables highly efficient, localized transfection and allows for transfection of three-dimensional cell constructs.

Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery.

Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled-release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine.