Biology subjects

Spiller, K. L.

Publications and source records attributed to Spiller, K. L..

6 recordsLinked to original sources

bioRxiv2026

Integrative Multiomic Analysis Reveals How Non-Viral Delivery System Selection Shapes CRISPR Gene Editing Outcomes in Stem Cells.

The clinical translation of CRISPR gene editing is challenged by the lack of delivery systems that are both safe and efficient in therapeutically relevant cell types such as mesenchymal stem cells (MSCs). Non-viral delivery avoids the immunogenicity and genomic integration risks of viral vectors but faces fundamental trade-offs between editing efficiency and cytotoxicity. Here, we present a comprehensive multiomic analysis of four non-viral CRISPR delivery modalities including cell-penetrating peptide- (CPP), lipid-, and polymer-based nanoparticles and electroporation; across mRNA and ribonucleoprotein (RNP) molecular formats. We systematically evaluate each modality, demonstrating that lipid-based delivery achieved the highest editing rates at the cost of genomic instability risks, interferon pathway activation, and a pro-inflammatory shift in MSC paracrine activity. Alternatively, CPPs yield moderate editing rates while reducing these unintended side-effects, whereas polymers and electroporation consistently yielded the lowest efficiencies. CRISPR molecular format and delivery method interacted in a stress-dependent manner, with RNP delivery reducing editing rates under high-stress systems while improving them in lower-stress modalities such as CPP and electroporation. These findings establish that editing efficiency alone is an insufficient metric for delivery system selection, and that genomic stability, transcriptomic dysregulation, and inflammatory response must be treated as primary design criteria for CRISPR therapies.

bioengineering↗

bioRxiv2025

Bioinspired Oxidized mRNA Lipid Nanoparticles for Ex Vivo Engineering of Chimeric Antigen Receptor Macrophages Targeting Solid Tumors

Solid tumors remain difficult to treat via conventional and novel therapeutic strategies. Immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy have been remarkably effective in treating hematological cancers, but their efficacy is limited in solid tumors. Recently, CAR macrophages (CAR-Ms) have emerged as a promising solid tumor immunotherapy, primarily for their intrinsic tumor infiltration and effector functions. However, CAR-Ms are engineered using viral transduction, which is associated with aberrant immunogenicity and toxicity. To overcome these challenges, we developed a bioinspired oxidized lipid nanoparticle (LNP) platform for mRNA-based engineering of human CAR-Ms. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for delivery to human macrophages. The composition of the top LNP was subsequently optimized using an orthogonal design of experiments (DoE) and physicochemical properties, such as size and mRNA encapsulation, were tuned via optimization of microfluidic mixing parameters, yielding a particle that significantly outperformed a gold standard C12-200 LNP. Utilizing small molecule and antibody inhibitors, we demonstrate that uptake of optimized LNPs into macrophages is driven by apolipoprotein E (ApoE) independent macropinocytosis, which is further supported by potent extrahepatic spleen tropism upon intravenous administration to mice. Lastly, we demonstrate the translatability of this LNP platform and utilize it to engineer functional primary human HER2-CAR-Ms ex vivo with potent antigen-specific tumor killing, validated in an ex vivo co-culture with ovarian cancer cells. This bioinspired oxidized LNP platform can potentially be utilized to engineer a range of human CAR-M immunotherapies to treat various types of solid tumors.

bioengineering↗

bioRxiv2022

Gene Expression Changes in Therapeutic Ultrasound-Treated Human Chronic Wound Tissue

Low-frequency, low-intensity ultrasound has been previously shown to promote healing of chronic wounds in humans, but mechanisms behind these effects are poorly understood. The purpose of this study was to evaluate gene expression differences in debrided human venous ulcer tissue from patients treated with low-frequency (20 kHz), low-intensity (100 mW/cm2) ultrasound compared to a sham treatment in an effort to better understand the potential biological mechanisms. Debrided venous ulcer tissue was collected from 32 subjects one week after sham treatment or low-frequency, low-intensity ultrasound treatment. Of these samples, 7 samples (3 ultrasound treated and 4 sham treated) yielded sufficient quality total RNA for analysis by ultra-high multiplexed PCR (Ampliseq) and expression of more than 24,000 genes was analyzed. 477 genes were found to be significantly different between the ultrasound and sham groups using cut-off values of p<0.05 and fold change of 2. Gene set enrichment analysis identified 20 significantly enriched gene sets from upregulated genes and 4 significantly enriched gene sets from downregulated genes. Most of the enriched gene sets from upregulated genes were related to cellcell signaling pathways. The most significantly enriched gene set from downregulated genes was the inflammatory response gene set. These findings show that therapeutic ultrasound influences cellular behavior in chronic wounds as early as one week after application. Considering the well-known role of chronic inflammation in impairing wound healing in chronic wounds, these results suggest that a downregulation of inflammatory genes is a possible biological mechanism of ultrasound-mediated venous chronic wound healing. Such increased understanding may ultimately lead to the enhancement of ultrasound devices to accelerate chronic wound healing and increase patient quality of life.

bioengineering↗

bioRxiv2021

Prior Pro-inflammatory Polarization Changes the Macrophage Response to IL-4

Tissue repair is largely regulated by diverse macrophage populations whose functions are timing- and context-dependent. The early phase of healing is dominated by pro-inflammatory macrophages, also known as M1, followed by the emergence of a distinct and diverse population that is collectively referred to as M2. The extent of the diversity of the M2 population is unknown. M2 macrophages may originate directly from circulating monocytes or from phenotypic switching of pre-existing M1 macrophages within the site of injury. The differences between these groups have not been investigated, but have major implications for understanding and treating pathologies characterized by deficient M2 activation, such as chronic wounds, which also exhibit diminished M1 macrophage behavior. This study investigated the influence of prior M1 activation on human macrophage polarization to an M2 phenotype in response to IL-4 treatment in vitro. Compared to unactivated (M0) macrophages, M1 macrophages upregulated several receptors that promote the M2 phenotype, including the primary receptor for IL-4. M1 activation also changed the macrophage response to treatment with IL-4, generating an M2-like phenotype with a distinct gene and protein expression signature compared to M2 macrophages prepared directly from M0 macrophages. Functionally, compared to M0-derived M2 macrophages, M1-derived M2 macrophages demonstrated increased migratory response to SDF-1, and conditioned media from these macrophages promoted increased recruitment of endothelial cells in transwell assays. Together, these findings indicate the importance of prior M1 activation in regulating subsequent M2 behavior, and suggest that augmentation of M1 behavior may be a therapeutic target in dysfunctional tissue repair. Summary sentenceM1 macrophages that are switched to an M2 phenotype exhibit a distinct functional phenotype compared to M2 macrophages derived directly from unactivated (M0) macrophages.

immunology↗

bioRxiv2020

Regulation of extracellular matrix assembly and structure by hybrid M1/M2 macrophages

Aberrant extracellular matrix (ECM) assembly surrounding implanted biomaterials is the hallmark of the foreign body response, in which implants become encapsulated in thick fibrous tissue that prevents their proper function. While macrophages are known regulators of fibroblast behavior, how their phenotype influences ECM assembly and the progression of the foreign body response is poorly understood. In this study, we used in vitro models with physiologically relevant macrophage phenotypes, as well as controlled release of macrophage-modulating cytokines from gelatin hydrogels implanted subcutaneously in vivo to investigate the role of macrophages in ECM assembly. Primary human macrophages were polarized to four distinct phenotypes, which have each been associated with fibrosis, including pro-inflammatory M1, pro-healing M2, and a hybrid M1/M2, generated by exposing macrophages to M1- and M2-promoting stimuli simultaneously. Additionally, macrophages were first polarized to M1 and then to M2 (M1[->]M2) to generate a phenotype typically observed during normal wound healing. Human dermal fibroblasts that were cultured in macrophage-conditioned media upregulated numerous genes involved in regulation of ECM assembly, especially in M2-conditioned media. Hybrid M1/M2 macrophage-conditioned media caused fibroblasts to produce a matrix with thicker and less aligned fibers, while M2 macrophage-conditioned media caused the formation of a more aligned matrix with thinner fibers. Gelatin methacrylate hydrogels containing interleukin-4 (IL4) and IL13-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles were designed to promote the M2 phenotype in a murine subcutaneous in vivo model. NanoString multiplex gene expression analysis of hydrogel explants showed that hydrogels with and without drug caused markers of both M1 and M2 phenotypes to be highly expressed, but the release of IL4+IL13 promoted upregulation of M2 markers and genes associated with regulation of ECM assembly, such as Col5a1 and Col6a1. Biochemical analysis and second harmonic generation microscopy showed that the release of IL4+IL13 increased total sulfated glycosaminoglycan content and decreased fibril alignment, which is typically associated with less fibrotic tissue. Together, these results show that hybrid M1/M2 macrophages regulate ECM assembly, and that shifting the balance towards M2 may promote architectural and compositional changes in ECM with enhanced potential for downstream remodeling.

bioengineering↗

bioRxiv2019

Non-Genetic Reprogramming of Monocytes via Microparticle Phagocytosis for Sustained Modulation of Macrophage Phenotype

Monocyte-derived macrophages orchestrate tissue regeneration by homing to sites of injury, phagocytosing pathological debris, and stimulating other cell types to repair the tissue. Accordingly, monocytes have been investigated as a translational and potent source for cell therapy, but their utility has been hampered by their rapid acquisition of a pro-inflammatory phenotype in response to the inflammatory injury microenvironment. To overcome this problem, we designed a cell therapy strategy where we collect and exogenously reprogram monocytes by intracellularly loading the cells with biodegradable microparticles containing an anti-inflammatory drug in order to modulate and maintain an anti-inflammatory phenotype over time. To test this concept, poly(lactic-co-glycolic) acid microparticles were loaded with the anti-inflammatory drug dexamethasone (Dex) and administered to primary human monocytes for four hours to facilitate phagocytic uptake. After removal of non-phagocytosed microparticles, microparticle-loaded monocytes differentiated into macrophages and stored the microparticles intracellularly for several weeks in vitro, releasing drug into the extracellular environment over time. Cells loaded with intracellular Dex microparticles showed decreased expression and secretion of inflammatory factors even in the presence of pro-inflammatory stimuli up to 7 days after microparticle uptake compared to untreated cells or cells loaded with blank microparticles. This study represents a new strategy for long-term maintenance of anti-inflammatory macrophage phenotype using a translational monocyte-based cell therapy strategy without the use of genetic modification. Because of the ubiquitous nature of monocyte-derived macrophage involvement in pathology and regeneration, this strategy holds potential as a treatment for a vast number of diseases and disorders.

bioengineering↗