Matthew Steven MacDougall

All Publications

• Immunodeficiency: Gene therapy for primary immune deficiency. Allergy and asthma proceedings Afzal, S. Y., MacDougall, M. S., McGhee, S. A. 2024; 45 (5): 384-388

  Abstract

  Current gene therapy for inborn errors of immunity have involved the use of gene addition approaches with viral delivery. This main strategy has had demonstrated success mainly in severe combined immune deficiency, Wiskott-Aldrich syndrome, and chronic granulomatous disease. Despite the increasing success of gene therapy, there are limitations of gene therapy, and, therefore, hematopoietic stem cell transplantation continues to be the preferred option. With improvements in viral delivery through next-generation lentiviral vectors and the advent of gene editing with CRISPR-Cas9, the efficacy and safety of gene therapy may soon surpass hematopoietic stem cell transplantation. Furthermore, these advances improve the viability of gene therapy for inborn errors of immunity primarily through decreased risk of transplantation-related complications. Therefore, despite current limitations, gene therapy for inborn errors of immunity is poised to continue to expand to more patients and indications.

  View details for DOI 10.2500/aap.2024.45.240054

  View details for PubMedID 39294901

• CD39 delineates chimeric antigen receptor regulatory T cell subsets with distinct cytotoxic & regulatory functions against human islets FRONTIERS IN IMMUNOLOGY Wu, X., Chen, P., Whitener, R. L., MacDougall, M. S., Coykendall, V. M. N., Yan, H., Kim, Y., Harper, W., Pathak, S., Iliopoulou, B. P., Hestor, A., Saunders, D. C., Spears, E., Sevigny, J., Maahs, D. M., Basina, M., Sharp, S. A., Gloyn, A. L., Powers, A. C., Kim, S. K., Jensen, K. P., Meyer, E. H. 2024; 15: 1415102

  Abstract

  Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases, including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs, most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets, may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes, a human β cell line and human islet β cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet β cells. In exploring subsets and mechanisms that may explain this pattern, we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39- CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly, β cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased β cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet β cells in the setting of CAR immunotherapy. In summary, introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction.

  View details for DOI 10.3389/fimmu.2024.1415102

  View details for Web of Science ID 001266095000001

  View details for PubMedID 39007132

  View details for PubMedCentralID PMC11239501

• Sequential Activation of Guide RNAs to Enable Successive CRISPR-Cas9 Activities MOLECULAR CELL Clarke, R., Terry, A. R., Pennington, H., Hasty, C., MacDougall, M. S., Regan, M., Merrill, B. J. 2021; 81 (2): 226-+

  Abstract

  Currently, either highly multiplexed genetic manipulations can be delivered to mammalian cells all at once or extensive engineering of gene regulatory sequences can be used to conditionally activate a few manipulations. Here, we provide proof of principle for a new system enabling multiple genetic manipulations to be executed as a preprogrammed cascade of events. The system leverages the programmability of the S. pyogenes Cas9 and is based on flexible arrangements of individual modules of activity. The basic module consists of an inactive single-guide RNA (sgRNA)-like component that is converted to an active state through the effects of another sgRNA. Modules can be arranged to bring about an algorithmic program of sequential genetic manipulations without the need for engineering cell-type-specific promoters or gene regulatory sequences. With the expanding diversity of available tools that use spCas9, this sgRNA-based system provides multiple levels of interfacing with mammalian cell biology.

  View details for DOI 10.1016/j.molcel.2020.12.003

  View details for Web of Science ID 000672600500005

  View details for PubMedID 33378644

• Intracellular Ca2+ Homeostasis and Nuclear Export Mediate Exit from Naive Pluripotency CELL STEM CELL MacDougall, M. S., Clarke, R., Merrill, B. J. 2019; 25 (2): 210-+

  Abstract

  Progression through states of pluripotency is required for cells in early mammalian embryos to transition away from heightened self-renewal and toward competency for lineage specification. Here, we use a CRISPR mutagenesis screen in mouse embryonic stem cells (ESCs) to identify unexpected roles for nuclear export and intracellular Ca2+ homeostasis during the exit out of the naive state of pluripotency. Mutation of a plasma membrane Ca2+ pump encoded by Atp2b1 increased intracellular Ca2+ such that it overcame effects of intracellular Ca2+ reduction, which is required for naive exit. Persistent self-renewal of ESCs was supported both in Atp2b1-/-Tcf7l1-/- double-knockout ESCs passaged in defined media alone (no LIF or inhibitors) and in wild-type cells passaged in media containing only calcitonin and a GSK3 inhibitor. These new findings suggest a central role for intracellular Ca2+ in safeguarding naive pluripotency.

  View details for DOI 10.1016/j.stem.2019.04.015

  View details for Web of Science ID 000478084700008

  View details for PubMedID 31104942

  View details for PubMedCentralID PMC6685429

• Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks MOLECULAR CELL Clarke, R., Heler, R., MacDougall, M. S., Yeo, N., Chavez, A., Regan, M., Hanakahi, L., Church, G. M., Marraffini, L. A., Merrill, B. J. 2018; 71 (1): 42-+

  Abstract

  The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double-stranded break (DSB), Cas9 remains stably bound to DNA. Here, we show persistent Cas9 binding blocks access to the DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches from one direction toward the Cas9-DSB complex. By exploiting these RNA-polymerase/Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of protospacer adjacent motif (PAM) sequences and a simple method of improving selection of highly active sgRNAs for genome editing.

  View details for DOI 10.1016/j.molcel.2018.06.005

  View details for Web of Science ID 000438142200007

  View details for PubMedID 29979968

  View details for PubMedCentralID PMC6063522

• Co-incident insertion enables high efficiency genome engineering in mouse embryonic stem cells NUCLEIC ACIDS RESEARCH Shy, B. R., MacDougall, M. S., Clarke, R., Merrill, B. J. 2016; 44 (16): 7997-8010

  Abstract

  CRISPR/Cas9 nucleases have enabled powerful, new genome editing capabilities; however, the preponderance of non-homologous end joining (NHEJ) mediated repair events over homology directed repair (HDR) in most cell types limits the ability to engineer precise changes in mammalian genomes. Here, we increase the efficiency of isolating precise HDR-mediated events in mouse embryonic stem (ES) cells by more than 20-fold through the use of co-incidental insertion (COIN) of independent donor DNA sequences. Analysis of on:off-target frequencies at the Lef1 gene revealed that bi-allelic insertion of a PGK-Neo cassette occurred more frequently than expected. Using various selection cassettes targeting multiple loci, we show that the insertion of a selectable marker at one control site frequently coincided with an insertion at an unlinked, independently targeted site, suggesting enrichment of a sub-population of HDR-proficient cells. When individual cell events were tracked using flow cytometry and fluorescent protein markers, individual cells frequently performed either a homology-dependent insertion event or a homology-independent event, but rarely both types of insertions in a single cell. Thus, when HDR-dependent selection donors are used, COIN enriches for HDR-proficient cells among heterogeneous cell populations. When combined with a self-excising selection cassette, COIN provides highly efficient and scarless genome editing.

  View details for DOI 10.1093/nar/gkw685

  View details for Web of Science ID 000384687000043

  View details for PubMedID 27484482

  View details for PubMedCentralID PMC5027516

• Evaluating the effect of age and comorbidity on dupilumab-dependent immunoglobulin E reduction in children with food allergy. Allergy and asthma proceedings Afzal, S. Y., MacDougall, M. S., Lu, D., McGhee, S. A. 2026; 47 (5): 328-335

  Abstract

  Background: Current therapies for food allergy prevention, oral immunotherapy and omalizumab, increase reaction threshold, but their curative potential remains unknown. Dupilumab has emerged as a promising treatment for several atopic conditions. We observed that many children with food allergy who are on dupilumab had profound reductions in food-specific immunoglobulin E (sIgE) levels over time. Objective: We sought to determine whether sIgE levels decrease in children with food allergy who are on dupilumab and whether changes over time were influenced by allergen type, race, sex, indication for therapy, or age of initiation of therapy. Methods: A retrospective analysis of 44 children with food allergy who were on dupilumab was conducted for longitudinal assessment of sIgE. Results: A statistically significant decrease in sIgE levels was observed across multiple allergens in the 44 children treated with dupilumab for a mean of 1.4 years. A stratified analysis identified a statistically significant decrease (p < 0.05) in sIgE levels in children receiving treatment for both atopic dermatitis (AD) and eosinophilic esophagitis (EoE). This reduction remained significant across all subgroups regardless of race, sex, or age of initiation of dupilumab. The rate of change in sIgE levels over time was greater in children with AD than with EoE, but this difference did not reach statistical significance. Notably, age < 6 years at dupilumab initiation was associated with steeper declines and greater absolute reductions in sIgE levels over time (-1.281 versus -0.784 log₂ IgE/year), with this difference approaching statistical significance (p = 0.0873), despite the small cohort size. Conclusion: We hypothesize that dupilumab may modify clinical tolerance in younger children during a window of immune plasticity and may increase reaction thresholds in older children. Even if this hypothesis is not confirmed, there is clinical utility in reductions of IgE levels, especially in children who are pansensitized, in whom these reductions likely improve signal-to-noise for clinically relevant sensitization. We advocate for prospective early-intervention trials that evaluate dupilumab as adjunctive therapy in children with comorbid atopy to determine its potential role in preventing food allergy development.

  View details for DOI 10.2500/aap.2026.47.260057

  View details for PubMedID 42698168

• Case Report: Effective IL-4/IL-13 axis suppression and resolution of severe atopic dermatitis by dupilumab monotherapy in a patient with IPEX syndrome. Frontiers in immunology Alshanti, M., MacDougall, M. S., Gokbak, M. N., Ramachandran, A., Alzaabi, A. A., Alexander, J. L., Bacchetta, R., Shendi, H. M. 2026; 17: 1923415

  Abstract

  The treatment of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome has largely focused on immunosuppression with little impact on the atopic manifestations of the disease. Dupilumab therapy has been used successfully in primary atopic diseases and immune regulatory disorders to control type 2 inflammation. Indeed, it has been reported in two prior IPEX cases, but in combination with immunosuppressive agents. Here, we are the first to report the clinical and immunologic impact of dupilumab monotherapy in IPEX.A 4-year-old boy with a confirmed hemizygous Forkhead box protein 3 (FOXP3) missense variant (c.1150G>A; p.Ala384Thr) presented with severe, treatment-refractory atopic dermatitis beginning in the newborn period, failure to thrive, lymphadenopathy, food and environmental allergies, asthma, recurrent febrile neutropenia, and type 1 diabetes mellitus, with a peak IgE of 74,625 IU/mL. Following initiation of dupilumab monotherapy at age 2, the patient had a marked improvement in atopic dermatitis by Eczema Area and Severity Index (EASI) score, a rapid decline in serum IgE, improved growth, reduced respiratory hospitalizations, and a profoundly improved quality of life.Immunologically, dupilumab effectively suppressed IgE and plasma interleukin-4 (IL-4) and IL-13 to healthy control levels. In addition, C-C motif chemokine ligand 27 (CCL27), important for cutaneous T-cell trafficking, was detected at healthy control levels. Upstream alarmins, eosinophil mediators, T helper 1 (Th1) cytokines, and Treg-specific demethylated region (TSDR) demethylation remained elevated-indicating ongoing systemic immune dysregulation not fully addressed by dupilumab alone.This case demonstrates that dupilumab monotherapy can effectively suppress the IL-4/IL-13 axis and substantially improve atopic manifestations in IPEX, potentially by restoring skin-specific immune tolerance through rebalancing cutaneous T-cell trafficking. These findings support dupilumab as a targeted adjunctive therapy for the atopic features of IPEX and provide a rationale for the systematic study of dupilumab alone or in combination with other immunosuppression regimens.

  View details for DOI 10.3389/fimmu.2026.1923415

  View details for PubMedID 42676327

  View details for PubMedCentralID PMC13526569

• Integration of the naive pluripotency gene network with the response to GSK3 inhibition by Tcf7l1-driven enhancer decommissioning. Stem cell reports Kwon, J., Zhang, J. Y., Shy, B. R., MacDougall, M. S., Leung, E. H., Chronis, C., Gounari, F., Merrill, B. J. 2026: 102980

  Abstract

  The transition out of naive pluripotency requires the simultaneous dissolution of the naive gene regulatory network as well as a redirection of transcriptional response to WNT/β-CATENIN signaling. Here, we show that Tcf7l1 functions as a linchpin factor that connects the two outcomes via effects at TCF7L1-bound cis-regulatory elements in mouse pluripotent stem cells. A combination of gene knockout and inducible expression lines of ESCs was generated to experimentally isolate effects of Tcf/Lef factors on pluripotency. Naive cis-regulatory elements were specifically bound by and rapidly decommissioned by TCF7L1 upon releasing cells from GSK3 inhibition. These findings suggest that Tcf7l1-mediated enhancer decommissioning serves as a critical mechanism by which pluripotent cells rapidly alter the response to signaling pathways in preparation for gastrulation, and it may be a mechanism used in diverse cell types for modulating the effects of WNT/β-CATENIN signaling pathway in a cell-specific manner to stimulate a variety of cell fate decisions.

  View details for DOI 10.1016/j.stemcr.2026.102980

  View details for PubMedID 42392086

• Infant in extremis: respiratory failure secondary to lower airway infantile hemangioma. BMC pediatrics MacDougall, M. S., Afzal, S. Y., Freedman, M. S., Han, P. 2022; 22 (1): 744

  Abstract

  Infantile hemangiomas (IHs) are vascular tumors that commonly affect infants and usually regress spontaneously or can be easily treated as an outpatient with topical beta-blockers. However, IHs that present in the airway may cause life-threatening symptoms due to airway obstruction or risk of bleeding. Here we present the first documented case of an infant with rapid deterioration and acute respiratory failure secondary to a lower airway hemangioma.This 3-month-old male initially presented in respiratory distress with symptoms consistent with a viral respiratory infection, however showed no clinical improvement with standard therapies. An urgent CT scan revealed a mass occluding the right mainstem bronchus. Upon transfer to a tertiary care facility, he developed acute respiratory failure requiring emergent intubation and single lung ventilation. The availability of multiple subspecialists allowed for stabilization of a critically ill child, expedited diagnosis, and ultimately initiation of life-saving treatment with beta blockers. After 17 total hospital days, he was extubated successfully and discharged home in good condition.While IH is a rare cause of infantile respiratory distress, we present multiple pearls for the general pediatrician for management of IHs of the airway.

  View details for DOI 10.1186/s12887-022-03821-1

  View details for PubMedID 36581920

• Topical Vapocoolant-Associated Vaso-occlusive Event in a 10-year-old with Sickle Cell Disease. Pain management nursing : official journal of the American Society of Pain Management Nurses Kothamasu, A., MacDougall, M. S., Mallard, J., Hsu, L. L. 2021; 22 (5): 631-633

  Abstract

  Vapocoolant sprays are convenient forms of cold temperature analgesia. These sprays may not be suitable for all patients with particular concern for patients with sickle cell disease. To prevent any further cases from occurring, we propose adding a more specific cautionary statement to the manufacturer guidelines. We also hope that medical personnel can help patients with sickle cell avoid topical and environmental cold temperature triggers for sickle vaso-occlusive pain and reduce the suffering in this rare disease.

  View details for DOI 10.1016/j.pmn.2021.04.008

  View details for PubMedID 34090800

• The NAMPT Promoter Is Regulated by Mechanical Stress, Signal Transducer and Activator of Transcription 5, and Acute Respiratory Distress Syndrome-Associated Genetic Variants AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Sun, X., Elangovan, V., Mapes, B., Camp, S. M., Sammani, S., Saadat, L., Ceco, E., Ma, S., Flores, C., MacDougall, M. S., Quijada, H., Liu, B., Kempf, C. L., Wang, T., Chiang, E. T., Garcia, J. G. N. 2014; 51 (5): 660-667

  Abstract

  Increased nicotinamide phosphoribosyltransferase (NAMPT) transcription is mechanistically linked to ventilator-induced inflammatory lung injury (VILI), with VILI severity attenuated by reduced NAMPT bioavailability. The molecular mechanisms of NAMPT promoter regulation in response to excessive mechanical stress remain poorly understood. The objective of this study was to define the contribution of specific transcription factors, acute respiratory distress syndrome (ARDS)-associated single nucleotide polymorphisms (SNPs), and promoter demethylation to NAMPT transcriptional regulation in response to mechanical stress. In vivo NAMPT protein expression levels were examined in mice exposed to high tidal volume mechanical ventilation. In vitro NAMPT expression levels were examined in human pulmonary artery endothelial cells exposed to 5 or 18% cyclic stretch (CS), with NAMPT promoter activity assessed using NAMPT promoter luciferase reporter constructs with a series of nested deletions. In vitro NAMPT transcriptional regulation was further characterized by measuring luciferase activity, DNA demethylation, and chromatin immunoprecipitation. VILI-challenged mice exhibited significantly increased NAMPT expression in bronchoalveolar lavage leukocytes and in lung endothelium. A mechanical stress-inducible region (MSIR) was identified in the NAMPT promoter from -2,428 to -2,128 bp. This MSIR regulates NAMPT promoter activity, mRNA expression, and signal transducer and activator of transcription 5 (STAT5) binding, which is significantly increased by 18% CS. In addition, NAMPT promoter activity was increased by pharmacologic promoter demethylation and inhibited by STAT5 silencing. ARDS-associated NAMPT promoter SNPs rs59744560 (-948G/T) and rs7789066 (-2,422A/G) each significantly elevated NAMPT promoter activity in response to 18% CS in a STAT5-dependent manner. Our results show that NAMPT is a key novel ARDS therapeutic target and candidate gene with genetic/epigenetic transcriptional regulation in response to excessive mechanical stress.

  View details for DOI 10.1165/rcmb.2014-0117OC

  View details for Web of Science ID 000347419400007

  View details for PubMedID 24821571