Sustained Gene Expression Research Articles

Recombinant adeno-associated virus with anti-tumor necrosis factor-alpha in an experimental autoimmune uveitis model.

Uveitis treatment is associated with side effects and inconsistent outcomes. Existing treatments often fail to provide targeted and sustained relief; thus, novel therapeutic approaches are needed. Among these, gene therapy using adeno-associated virus (AAV) vectors target specific retinal cells, show low immunogenicity, and demonstrate sustained gene expression, making it a potential advancement in uveitis treatment. Therefore, we utilized a AAV2 system encapsulating encoded anti-tumor necrosis factor-alpha (TNF-α) antibody to assess its efficacy in the treatment of experimental autoimmune uveitis (EAU) in mice. Compared with the AAV2-GFP group, AAV2-ADA-injected mice showed significantly reduced clinical, OCT, and histopathological scores in EAU with lower percentages of Th1 and Th17 cells in the eyes and higher percentages of Treg cells in the draining lymph nodes (LN). This study demonstrated the safety and effects of AAV2-ADA in EAU treatment, providing a promising therapeutic strategy for uveitis.

Polymer-Based Nanoparticles as Efficient Non-Viral Vectors for Gene Delivery in CAR-T Cell Therapy

Chimeric Antigen Receptor (CAR)-T cell therapy has emerged as a revolutionary method in cancer immunotherapy, achieving notable success in treating hematological malignancies. However, its effectiveness in solid tumors is limited, and the therapy faces challenges such as severe toxicities and high production costs. Polymer-based nanoparticles (PNPs) are gaining attention as a non-viral gene delivery system that can help address these issues, providing greater safety, scalability, and cost-effectiveness compared to viral vectors. This mini-review delves into various types of PNPs, including cationic, biodegradable, and stimuli-responsive polymers, showcasing their mechanisms for cellular uptake and sustained gene expression in CAR-T cells. Innovations like lipid-polymer hybrid nanoparticles and combination therapies improve gene delivery efficiency and therapeutic outcomes. Despite progress, challenges, including toxicity and immunogenicity, remain, prompting strategies such as surface modifications and targeting ligands. Recent advancements illustrate the potential of hybrid nanoparticles in enhancing CAR-T therapies, particularly for solid tumors, highlighting their critical role in cancer immunotherapy advancements.

Chitin-Functionalized Cell-Free System Enables Sustainable Biocatalysis and Gene Expression

Chitin-Functionalized Cell-Free System Enables Sustainable Biocatalysis and Gene Expression

Thermo-amplifier circuit in probiotic E. coli for stringently temperature-controlled release of a novel antibiotic

Peptide drugs have seen rapid advancement in biopharmaceutical development, with over 80 candidates approved globally. Despite their therapeutic potential, the clinical translation of peptide drugs is hampered by challenges in production yields and stability. Engineered bacterial therapeutics is a unique approach being explored to overcome these issues by using bacteria to produce and deliver therapeutic compounds at the body site of use. A key advantage of this technology is the possibility to control drug delivery within the body in real time using genetic switches. However, the performance of such genetic switches suffers when used to control drugs that require post-translational modifications or are toxic to the host. In this study, these challenges were experienced when attempting to establish a thermal switch for the production of a ribosomally synthesized and post-translationally modified peptide antibiotic, darobactin, in probiotic E. coli. These challenges were overcome by developing a thermo-amplifier circuit that combined the thermal switch with a T7 RNA Polymerase. Due to the orthogonality of the Polymerase, this strategy overcame limitations imposed by the host transcriptional machinery. This circuit enabled production of pathogen-inhibitory levels of darobactin at 40 °C while maintaining leakiness below the detection limit at 37 °C. Furthermore, the thermo-amplifier circuit sustained gene expression beyond the thermal induction duration such that with only 2 h of induction, the bacteria were able to produce pathogen-inhibitory levels of darobactin. This performance was maintained even in physiologically relevant simulated conditions of the intestines that include bile salts and low nutrient levels.Graphical

Development of an RNA virus-based episomal vector with artificial aptazyme for gene silencing

RNA virus-based episomal vector (REVec), engineered from Borna disease virus, is an innovative gene delivery tool that enables sustained gene expression in transduced cells. However, the difficulty in controlling gene expression and eliminating vectors has limited the practical use of REVec. In this study, we overcome these shortcomings by inserting artificial aptazymes into the untranslated regions of foreign genes carried in vectors or downstream of the viral phosphoprotein gene, which is essential for vector replication. Non-transmissive REVec carrying GuaM8HDV or the P1-F5 aptazyme showed immediate suppression of gene expression in a guanine or theophylline concentration-dependent manner. Continuous compound administration also markedly reduced the percentage of vector-transduced cells and eventually led to the complete elimination of the vectors from the transduced cells. This new REVec is a safe gene delivery technology that allows fine-tuning of gene expression and could be a useful platform for gene therapy and gene-cell therapy, potentially contributing to the cure of many genetic disorders.Key points• We developed a bornavirus vector capable of silencing transgene expression by insertion of aptazyme• Transgene expression was markedly suppressed in a compound concentration-dependent manner• Artificial aptazyme systems allowed complete elimination of the vector from transduced cells

Adeno‐associated virus vectors for gene therapy—focusing on melanoma

AbstractAdeno‐Associated Virus (AAV) vectors have been found to have great potential in the field of gene therapy due to their unique properties. These nonpathogenic vectors exhibit high tissue specificity, low immunogenicity, and sustained gene expression, enhancing their efficacy for targeted delivery. This review explores the application of AAV vectors in gene therapy. It introduces the application and development of AAV in hemophilia and Duchenne Muscular Dystrophy. The review also highlights the use of AAV vectors in melanoma gene therapy, focusing on four key areas: cancer growth intervention, targeting cancer cells, mediating immune responses, and modulating key signaling pathways. However, despite its many advantages, rAAV faces significant challenges, including host immune responses and biological barriers that limit its effectiveness. Such complexities need to be addressed; therefore, this review also discusses the application of several polymers, such as hydrogels, nanoparticles, and solid scaffolds, in the controlled delivery of AAV.

Dynamics of BMP signaling and stable gene expression in the early Drosophila embryo.

In developing tissues, morphogen gradients are thought to initialize gene expression patterns. However, the relationship between the dynamics of morphogen-encoded signals and gene expression decisions is largely unknown. Here we examine the dynamics of the Bone Morphogenetic Protein (BMP) pathway in Drosophila blastoderm-stage embryos. In this tissue, the BMP pathway is highly dynamic: it begins as a broad and weak signal on the dorsal half of the embryo, then 20-30 min later refines into a narrow, intense peak centered on the dorsal midline. This dynamical progression of the BMP signal raises questions of how it stably activates target genes. Therefore, we performed live imaging of the BMP signal and found that dorsal-lateral cells experience only a short transient in BMP signaling, after which the signal is lost completely. Moreover, we measured the transcriptional response of the BMP target gene pannier in live embryos and found it to remain activated in dorsal-lateral cells, even after the BMP signal is lost. Our findings may suggest that the BMP pathway activates a memory, or 'ratchet' mechanism that may sustain gene expression.

Targeting YES1 Disrupts Mitotic Fidelity and Potentiates the Response to Taxanes in Triple-Negative Breast Cancer.

Clinical trials examining broad-spectrum Src family kinase (SFK) inhibitors revealed significant dose-limiting toxicities, preventing advancement for solid tumors. SFKs are functionally heterogeneous, thus targeting individual members is a potential strategy to elicit antitumor efficacy while avoiding toxicity. Here, we identified that YES1 is the most highly overexpressed SFK in triple-negative breast cancer (TNBC) and is associated with poor patient outcomes. Disrupting YES1, genetically or pharmacologically, induced aberrant mitosis, centrosome amplification, multipolar spindles, and chromosomal instability. Mechanistically, YES1 sustained FOXM1 protein levels and elevated expression of FOXM1 target genes that control centrosome function and are essential for effective and accurate mitotic progression. In both in vitro and in vivo TNBC models, YES1 suppression potentiated the efficacy of taxanes, cornerstone drugs for TNBC that require elevated chromosomal instability for efficacy. Clinically, elevated expression of YES1 was associated with worse overall survival of patients with TNBC treated with taxane and anthracycline combination regimens. Together, this study demonstrates that YES1 is an essential regulator of genome stability in TNBC that can be leveraged to improve taxane efficacy. Significance: YES1 is a sentinel regulator of genomic maintenance that controls centrosome homeostasis and chromosome stability through FOXM1, revealing this pathway as a therapeutic vulnerability for enhancing taxane efficacy in triple-negative breast cancer.

Altered X-chromosome inactivation predisposes to autoimmunity.

In mammals, males and females show marked differences in immune responses. Males are globally more sensitive to infectious diseases, while females are more susceptible to systemic autoimmunity. X-chromosome inactivation (XCI), the epigenetic mechanism ensuring the silencing of one X in females, may participate in these sex biases. We perturbed the expression of the trigger of XCI, the noncoding RNA Xist, in female mice. This resulted in reactivation of genes on the inactive X, including members of the Toll-like receptor 7 (TLR7) signaling pathway, in monocyte/macrophages and dendritic and B cells. Consequently, female mice spontaneously developed inflammatory signs typical of lupus, including anti-nucleic acid autoantibodies, increased frequencies of age-associated and germinal center B cells, and expansion of monocyte/macrophages and dendritic cells. Mechanistically, TLR7 signaling is dysregulated in macrophages, leading to sustained expression of target genes upon stimulation. These findings provide a direct link between maintenance of XCI and female-biased autoimmune manifestations and highlight altered XCI as a cause of autoimmunity.

Transcription Factor Binding Site in Promoter Determines the Pattern of Plasmid-Based Transgene Expression In Vivo.

Understanding the regulation of transgene expression is critical for the success of plasmid-based gene therapy and vaccine development. In this study, we used two sets of plasmid vectors containing secreted embryonic alkaline phosphatase or the mouse IL-10 gene as a reporter and investigated the role of promoter elements in regulating transgene expression in vivo. We demonstrated in mice that hydrodynamic transfer of plasmids with the CMV promoter resulted in a high level of reporter gene expression that declined rapidly over time. In contrast, when plasmids with albumin promoters were used, a lower but sustained gene expression pattern was observed. We also found that plasmids containing a shorter CMV promoter sequence with fewer transcription factor binding sites showed a decrease in the peak level of gene expression without changing the overall pattern of reporter gene expression. The replacement of regulatory elements in the CMV promoter with a single regulatory element of the albumin promoter changed the pattern of transient gene expression seen in the CMV promoter to a pattern of sustained gene expression identical to that of a full albumin promoter. ChIP analyses demonstrated an elevated binding of acetylated histones and TATA box-binding protein to the promoter carrying regulatory elements of the albumin promoter. These results suggest that the strength of a promoter is determined by the number of appropriate transcription factor binding sites, while gene expression persistence is determined by the presence of regulatory elements capable of recruiting epigenetic modifying complexes that make the promoter accessible for transcription. This study provides important insights into the mechanisms underlying gene expression regulation in vivo, which can be used to improve plasmid-based gene therapy and vaccine development.

Self-amplifying RNAs generated with the modified nucleotides 5-methylcytidine and 5-methyluridine mediate strong expression and immunogenicity in vivo

Abstract When utilized in therapeutic applications, synthetic self-amplifying RNA (saRNA) can lead to higher and more sustained expression than standard messenger RNA (mRNA). This feature is particularly important for gene replacement therapy applications where prolonged expression could reduce the dose and frequency of treatments. The inclusion of modified nucleotides in synthetic non-amplifying mRNA has been shown to increase RNA stability, reduce immune activation and enhance gene expression. Preclinical and clinical studies with saRNA have so far exclusively relied on RNA containing the canonical nucleotides adenosine, cytidine, guanosine and uridine. For the first time, we show that noncanonical nucleotides, such as 5-methylcytidine (m5C) and 5-methyluridine (m5U), are sufficiently compatible with a replicon derived from Venezuelan equine encephalitis alphavirus mediating protein translation in vitro, while those containing N1-methylpseudouridine in place of uridine show no detectable expression. When administered in vivo, saRNA generated with m5C or m5U mediates sustained gene expression of the luciferase reporter gene, with those incorporating m5U appearing to lead to more prolonged expression. Finally, distinct antigen-specific humoral and cellular immune responses were induced by modified saRNA encoding the model antigen ovalbumin. The use of modified nucleotides with saRNA-based platforms could enhance their potential to be used effectively in a variety of applications.

Evaluation of Lentiviral Based Gene Delivery System in Adherent and Suspension in vitro Cell Models

Lentiviruses are a highly robust gene delivery system capable of in vitro and in vivo gene transfer into multiple cell types. Recent fourth-generation lentiviral systems have been designed for enhanced safety, however, the increased recombination events required to produce infective lentiviral particles may reduce production efficiency. A set protocol for all types of target cells is not recommended and optimization of conditions for gene transfer into different target cells is required. In this study, we aim to evaluate the efficiency and reproducibility of lentiviral production using a fourth-generation lentiviral packaging system and identify optimal parameters for successful transduction in two different cell models, adherent and suspension cells. Lentiviral production, effect of viral volume, sustained gene expression and transduction adjuvants on adherent and suspension gene- cell models were evaluated. Transfection and transduction efficiency of lentiviruses was evaluated by fluorescence microscopy and flow cytometry. This study demonstrates that production of green fluorescent protein (GFP)- lentiviruses using the fourth-generation lentiviral packaging is consistent and reproducible. Optimal transduction of adherent cell types is achieved at lower multiplicity of infection (MOI) compared to suspension cells and produces GFP-expressing cells with higher intensity. Expression of GFP is sustained in all cell types over multiple passages. Polycation DEAE-dextran was determined to improve transduction in suspension cells, however, provides similar transduction efficiency as polybrene in adherent cells. In conclusion, fourth generation lentiviral system reproducibly generates high titre lentiviruses capable of infecting multiple cell types, however transduction protocols for different cell types require further optimization.

Differential gene expression of Wharton's jelly-derived mesenchymal cells mediated by graphene oxide in basal and osteo-induced media.

Graphene oxide (GO) is widespread in scaffold engineering owing to its extraordinary properties such as multiple oxygen functional groups, high hydrophilicity ability and biocompatibility. It is known to promote differentiation in mesenchymal stem cells, but concomitant comparison of its modulation on the expression profiles of Wharton's jelly (WJ)-MSC surface markers, lineage differentiation, and epigenetic regulatory genes in basal and induced condition are still lacking. Unraveling the fundamental mechanisms is essential for the effective utilization of WJ-MSCs incorporated with GO in therapy. This study aims to explore the unique gene expression profiles and epigenetic characteristics of WJ-MSCs influenced by GO. The characterized GO-coated coverslip served as a substrate for culturing WJ-MSCs. In addition to investigating the impact of GO on cell proliferation and differentiation, we conducted a gene expression study using PCR array, while epigenetic control was assessed through bisulfite sequencing and Western blot analysis. Our findings indicate that the presence of GO maintained the proliferation and survival of WJ-MSCs. In the absence of induction, GO led to minor lipid and glycosaminoglycan deposition in WJ-MSCs. This was evidenced by the sustained expression of pluripotency and lineage-specific genes, demethylation at the OCT4 promoter, and a decrease in H3K9 methylation. In osteo-induced condition, the occurrence of osteogenesis appeared to be guided by BMP/TGF and ERK pathway activation, accompanied by the upregulation of osteogenic-related genes and downregulation of DNMT3b. GO in osteo-induced condition create a favorable microenvironment that promotes the osteogenesis of WJ-MSCs by influencing genetic and epigenetic controls. This helps in advancing our knowledge on the use of GO as priming platform and WJ-MSCs an alternate source for bone repair and regeneration.

Eomes-dependent mitochondrial regulation promotes survival of pathogenic CD4+ T cells during inflammation.

The mechanisms whereby Eomes controls tissue accumulation of T cells and strengthens inflammation remain ill-defined. Here, we show that Eomes deletion in antigen-specific CD4+ T cells is sufficient to protect against central nervous system (CNS) inflammation. While Eomes is dispensable for the initial priming of CD4+ T cells, it is required for long-term maintenance of CNS-infiltrating CD4+ T cells. We reveal that the impact of Eomes on effector CD4+ T cell longevity is associated with sustained expression of multiple genes involved in mitochondrial organization and functions. Accordingly, epigenetic studies demonstrate that Eomes supports mitochondrial function by direct binding to either metabolism-associated genes or mitochondrial transcriptional modulators. Besides, the significance of these findings was confirmed in CD4+ T cells from healthy donors and multiple sclerosis patients. Together, our data reveal a new mechanism by which Eomes promotes severity and chronicity of inflammation via the enhancement of CD4+ T cell mitochondrial functions and resistance to stress-induced cell death.

Generation of Cell Lines Stably Expressing a dCas9-Fusion or sgRNA to Address Dynamics of Long-Term Effects of Epigenetic Editing.

Epigenetic modifications play a crucial role in regulating gene expression patterns. Through epigenetic editing approaches, the chromatin structure is modified and the activity of the targeted gene can be reprogrammed without altering the DNA sequence. By using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic repeats) platform with nuclease-deactivated dCas9 proteins to direct epigenetic effector domains (EDs) to genomic regulatory regions, the expression of the targeted gene can be modulated. However, the long-term stability of these effects, although demonstrated, remains unpredictable. The versatility and flexibility of (co-)targeting different genes with multiple epigenetic effectors has made the CRISPR/dCas9 platform the most widely used gene modulating technology currently available. Efficient delivery of large dCas9-ED fusion constructs into target cells, however, is challenging. An approach to overcome this limitation is to generate cells that stably express sgRNA(s) or dCas9-ED constructs. The sgRNA(s) or dCas9-ED stable cell lines can be used to study the mechanisms underlying sustained gene expression reprogramming by transiently expressing the other of the two constructs. Here, we describe a detailed protocol for the engineering of cells that stably express CRISPR/dCas9 or sgRNA. Creating a system where one component of the CRISPR/dCas9 is stably expressed while the other is transiently expressed offers a versatile platform for investigating the dynamics of epigenetic reprogramming.

Exploring the Potential of Cytomegalovirus-Based Vectors: A Review.

Viral vectors have emerged as powerful tools for delivering and expressing foreign genes, playing a pivotal role in gene therapy. Among these vectors, cytomegalovirus (CMV) stands out as a promising viral vector due to its distinctive attributes including large packaging capacity, ability to achieve superinfection, broad host range, capacity to induce CD8+ T cell responses, lack of integration into the host genome, and other qualities that make it an appealing vector candidate. Engineered attenuated CMV strains such as Towne and AD169 that have a ~15 kb genomic DNA deletion caused by virus passage guarantee human safety. CMV's large genome enables the efficient incorporation of substantial foreign genes as demonstrated by CMV vector-based therapies for SIV, tuberculosis, cancer, malaria, aging, COVID-19, and more. CMV is capable of reinfecting hosts regardless of prior infection or immunity, making it highly suitable for multiple vector administrations. In addition to its broad cellular tropism and sustained high-level gene expression, CMV triggers robust, virus-specific CD8+ T cell responses, offering a significant advantage as a vaccine vector. To date, successful development and testing of murine CMV (MCMV) and rhesus CMV (RhCMV) vectors in animal models have demonstrated the efficacy of CMV-based vectors. These investigations have explored the potential of CMV vectors for vaccines against HIV, cancer, tuberculosis, malaria, and other infectious pathogens, as well as for other gene therapy applications. Moreover, the generation of single-cycle replication CMV vectors, produced by deleting essential genes, ensures robust safety in an immunocompromised population. The results of these studies emphasize CMV's effectiveness as a gene delivery vehicle and shed light on the future applications of a CMV vector. While challenges such as production complexities and storage limitations need to be addressed, ongoing efforts to bridge the gap between animal models and human translation continue to fuel the optimism surrounding CMV-based vectors. This review will outline the properties of CMV vectors and discuss their future applications as well as possible limitations.

Immunomodulatory contribution of mast cells to the regenerative biomaterial microenvironment

Bioactive immunomodulatory biomaterials have shown promise for influencing the immune response to promote tissue repair and regeneration. Macrophages and T cells have been associated with this response; however, other immune cell types have been traditionally overlooked. In this study, we investigated the role of mast cells in the regulation of the immune response to decellularized biomaterial scaffolds using a subcutaneous implant model. In mast cell-deficient mice, there was dysregulation of the expected M1 to M2 macrophage transition typically induced by the biomaterial scaffold. Polarization progression deviated in a sex-specific manner with an early transition to an M2 profile in female mice, while the male response was unable to properly transition past a pro-inflammatory M1 state. Both were reversed with adoptive mast cell transfer. Further investigation of the later-stage immune response in male mice determined a greater sustained pro-inflammatory gene expression profile, including the IL-1 cytokine family, IL-6, alarmins, and chemokines. These results highlight mast cells as another important cell type that influences the immune response to pro-regenerative biomaterials.

Comparative transcriptional and co-expression network analysis of two upland cotton accessions with extreme phenotypic differences reveals molecular mechanisms of fiber development.

Upland cotton (Gossypium hirsutum) is the main source of natural fiber in the global textile industry, and thus its fiber quality and yield are important parameters. In this study, comparative transcriptomics was used to analyze differentially expressed genes (DEGs) due to its ability to effectively screen candidate genes during the developmental stages of cotton fiber. However, research using this method is limited, particularly on fiber development. The aim of this study was to uncover the molecular mechanisms underlying the whole period of fiber development and the differences in transcriptional levels. Comparative transcriptomes are used to analyze transcriptome data and to screen for differentially expressed genes. STEM and WGCNA were used to screen for key genes involved in fiber development. qRT-PCR was performed to verify gene expression of selected DEGs and hub genes. Two accessions of upland cotton with extreme phenotypic differences, namely EZ60 and ZR014121, were used to carry out RNA sequencing (RNA-seq) on fiber samples from different fiber development stages. The results identified 704, 376, 141, 269, 761, and 586 genes that were upregulated, and 1,052, 476, 355, 259, 702, and 847 genes that were downregulated at 0, 5, 10, 15, 20, and 25 days post anthesis, respectively. Similar expression patterns of DEGs were monitored using short time-series expression miner (STEM) analysis, and associated pathways of DEGs within profiles were investigated. In addition, weighted gene co-expression network analysis (WGCNA) identified five key modules in fiber development and screened 20 hub genes involved in the development of fibers. Through the annotation of the genes, it was found that the excessive expression of resistance-related genes in the early fiber development stages affects the fiber yield, whereas the sustained expression of cell elongation-related genes is critical for long fibers. This study provides new information that can be used to improve fibers in newly developed upland cotton genotypes.

An Enhancer Within Abcb11 Regulates G6pc2 in C57BL/6 Mouse Pancreatic Islets.

G6PC2 is predominantly expressed in pancreatic islet beta cells where it encodes a glucose-6-phosphatase catalytic subunit that modulates the sensitivity of insulin secretion to glucose by opposing the action of glucokinase, thereby regulating fasting blood glucose (FBG). Prior studies have shown that the G6pc2 promoter alone is unable to confer sustained islet-specific gene expression in mice, suggesting the existence of distal enhancers that regulate G6pc2 expression. Using information from both mice and humans, and knowledge that single nucleotide polymorphisms (SNPs) both within and near G6PC2 are associated with variations in FBG in humans, we identified several putative enhancers 3' of G6pc2. One region, herein referred to as enhancer I, resides in the 25th intron of Abcb11 and binds multiple islet-enriched transcription factors. CRISPR-mediated deletion of enhancer I in C57BL/6 mice had selective effects on the expression of genes near the G6pc2 locus: in isolated islets G6pc2 and Spc25 expression were reduced ~50%, and Gm13613 expression was abolished, whereas Cers6 and Nostrin expression were unaffected. This partial reduction in G6pc2 expression enhanced islet insulin secretion at basal glucose concentrations but did not affect FBG or glucose tolerance in vivo, consistent with the absence of a phenotype in G6pc2 heterozygous C57BL/6 mice.

Inflammatory Licensed hMSCs Exhibit Enhanced Immunomodulatory Capacity in a Biomaterial Mediated Manner.

Craniomaxillofacial (CMF) bone injuries represent particularly challenging environments for regenerative healing due to their large sizes, irregular and unique defect shapes, angiogenic requirements, and mechanical stabilization needs. These defects also exhibit a heightened inflammatory environment that can complicate the healing process. This study investigates the influence of the initial inflammatory stance of human mesenchymal stem cells (hMSCs) on key osteogenic, angiogenic, and immunomodulatory criteria when cultured in a class of mineralized collagen scaffolds under development for CMF bone repair. We previously showed that changes in scaffold pore anisotropy and glycosaminoglycan content can significantly alter the regenerative activity of both MSCs and macrophages. While MSCs are known to adopt an immunomodulatory phenotype in response to inflammatory stimuli, here, we define the nature and persistence of MSC osteogenic, angiogenic, and immunomodulatory phenotypes in a 3D mineralized collagen environment, and further, whether changes to scaffold architecture and organic composition can blunt or accentuate this response as a function of inflammatory licensing. Notably, we found that a one-time licensing treatment of MSCs induced higher immunomodulatory potential compared to basal MSCs as observed by sustained immunomodulatory gene expression throughout the first 7 days as well as an increase in immunomodulatory cytokine (PGE2 and IL-6) expression throughout a 21-day culture period. Further, heparin scaffolds facilitated higher osteogenic cytokine secretion but lower immunomodulatory cytokine secretion compared to chondroitin-6-sulfate scaffolds. Anisotropic scaffolds facilitated higher secretion of both osteogenic protein OPG and immunomodulatory cytokines (PGE2 and IL-6) compared to isotropic scaffolds. These results highlight the importance of scaffold properties on the sustained kinetics of cell response to an inflammatory stimulus. The development of a biomaterial scaffold capable of interfacing with hMSCs to facilitate both immunomodulatory and osteogenic responses is an essential next step to determining the quality and kinetics of craniofacial bone repair.