Alun Vaughan Jackson

All Publications

• Programmable synthetic cytokine receptors polarize macrophages to user-defined functional states. bioRxiv : the preprint server for biology Lunger, J. C., Sant'Anna, L. E., Salcido-Alcántar, A., Hornero, R. A., Cho, W., Vaughan-Jackson, A., Gu, M., Liu, J. Y., Beckett, A. N., Parrilla-Garcia, J., Ramakrishna, S., Bassik, M. C., Daniels, K. G. 2026

  Abstract

  Technology that precisely controls macrophage polarization to distinct functional states would deepen our understanding of macrophage biology and enable the development of new macrophage cell therapies. Here, we use a synthetic cytokine receptor (SCR) platform with a programmable signaling domain to control the polarization of primary human macrophages. SCRs containing signaling motifs from the interferon-gamma (IFN-γ) or Interleukin-10 (IL-10) receptors mimic key features of pro-inflammatory or anti-inflammatory polarization, respectively. Random recombination of nine distinct signaling motifs to create new SCR signaling domains generates a diverse landscape of synthetic macrophage states with varied expression of inflammatory markers (CD80, CD40) and anti-inflammatory markers (CD163, CD206), and varied phagocytic capacity. SCRs programmed with multiple YLxQ motifs increase macrophage phagocytosis of E. coli and chimeric antigen receptor (CAR)-macrophage phagocytosis of cancer cells in mice, reducing tumor burden by 30-fold. The motif-dependent polarization is well-described by a two-state model, enabling quantitative prediction of macrophage polarization state from SCR signaling domain composition. Leveraging this model, we design an SCR that simultaneously enhances phagocytosis and maintains a macrophage pro-inflammatory state. Together, these findings establish a framework for synthetic programming of macrophage polarization states, with potential applications in cancer immunotherapy and other disease contexts.

  View details for DOI 10.64898/2026.05.12.724672

  View details for PubMedID 42182394

  View details for PubMedCentralID PMC13192837

• Programmable synthetic cytokine receptors polarize macrophages to user-defined functional states bioRxiv Lunger, J. C., Sant'Anna, L. E., Salcido-Alcantar, A., Arroyo Hornero, R., Cho, W., Vaughan-Jackson, A., Gu, M., Liu, J. Y., Beckett, A. N., Parrilla-Garcia, J., Ramakrishna, S., Bassik, M. C., Daniels, K. G. 2026

  View details for DOI 10.64898/2026.05.12.724672

• Reduced Cas9 transgene silencing by incorporation of intron sequences. Nature communications Arana, S., Du, P. P., Vaughan-Jackson, A., Enright, N., Spees, K., Valbuena, R., Garcia, C. A., Nguyen, T., Venida, A., Seczynska, M., Bintu, L., Lehner, P. J., Prolo, L. M., Bassik, M. C. 2025; 16 (1): 10656

  Abstract

  Silencing remains a significant challenge for exogenous gene expression, limiting both the penetrance and expressivity of transgenes. In particular, silencing of Cas9 expression is a major technical limitation for many gene editing and CRISPR screening applications. Here, we demonstrate that including introns in Cas9 expression cassettes significantly reduces silencing across multiple cell lines. Notably, the incorporation of an intron into a CRISPRa construct results in reduced silencing, increased expression levels, and markedly enhanced activation of target genes. We investigate diverse intron sequences and discover that T-rich introns over 2 kb confer the greatest protection against silencing. In addition, we find that introns can work synergistically with chromatin opening elements to further mitigate silencing, suggesting regulatory mechanisms are acting at both the DNA and RNA level to silence exogenous genes. Our work highlights the potential of introns to optimize genetic constructs for enhanced expression and improved cellular engineering requiring constitutive expression of large transgenes.

  View details for DOI 10.1038/s41467-025-65669-0

  View details for PubMedID 41309578

  View details for PubMedCentralID PMC12660324

• Macropinocytosis as a novel target for pediatric high grade glioma therapy Nguyen, T., Spees, K., Garcia, C., Arana, S., Du, P., Levin-Konigsberg, R., Vaughan-Jackson, A., Nettnin, E., Bassik, M., Prolo, L. OXFORD UNIV PRESS INC. 2025: v179-v180

  View details for DOI 10.1093/neuonc/noaf201.0709

  View details for Web of Science ID 001613298600026

• Development of compact transcriptional effectors using high-throughput measurements in diverse contexts. Nature biotechnology Tycko, J., Van, M. V., , DelRosso, N., Ye, H., Yao, D., Valbuena, R., Vaughan-Jackson, A., Xu, X., Ludwig, C., Spees, K., Liu, K., Gu, M., Khare, V., Mukund, A. X., Suzuki, P. H., Arana, S., Zhang, C., Du, P. P., Ornstein, T. S., Hess, G. T., Kamber, R. A., Qi, L. S., Khalil, A. S., Bintu, L., Bassik, M. C. 2024

  Abstract

  Transcriptional effectors are protein domains known to activate or repress gene expression; however, a systematic understanding of which effector domains regulate transcription across genomic, cell type and DNA-binding domain (DBD) contexts is lacking. Here we develop dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous target genes and test effector function for a library containing 5,092 nuclear protein Pfam domains across varied contexts. We also map context dependencies of effectors drawn from unannotated protein regions using a larger library tiling chromatin regulators and transcription factors. We find that many effectors depend on target and DBD contexts, such as HLH domains that can act as either activators or repressors. To enable efficient perturbations, we select context-robust domains, including ZNF705 KRAB, that improve CRISPRi tools to silence promoters and enhancers. We engineer a compact human activator called NFZ, by combining NCOA3, FOXO3 and ZNF473 domains, which enables efficient CRISPRa with better viral delivery and inducible control of chimeric antigen receptor T cells.

  View details for DOI 10.1038/s41587-024-02442-6

  View details for PubMedID 39487265

  View details for PubMedCentralID 4494013

• Deciphering the impact of genomic variation on function. Nature 2024; 633 (8028): 47-57

  Abstract

  Our genomes influence nearly every aspect of human biology-from molecular and cellular functions to phenotypes in health and disease. Studying the differences in DNA sequence between individuals (genomic variation) could reveal previously unknown mechanisms of human biology, uncover the basis of genetic predispositions to diseases, and guide the development of new diagnostic tools and therapeutic agents. Yet, understanding how genomic variation alters genome function to influence phenotype has proved challenging. To unlock these insights, we need a systematic and comprehensive catalogue of genome function and the molecular and cellular effects of genomic variants. Towards this goal, the Impact of Genomic Variation on Function (IGVF) Consortium will combine approaches in single-cell mapping, genomic perturbations and predictive modelling to investigate the relationships among genomic variation, genome function and phenotypes. IGVF will create maps across hundreds of cell types and states describing how coding variants alter protein activity, how noncoding variants change the regulation of gene expression, and how such effects connect through gene-regulatory and protein-interaction networks. These experimental data, computational predictions and accompanying standards and pipelines will be integrated into an open resource that will catalyse community efforts to explore how our genomes influence biology and disease across populations.

  View details for DOI 10.1038/s41586-024-07510-0

  View details for PubMedID 39232149

  View details for PubMedCentralID 7405896

• Hypoxia dampens innate immune signalling at early time points and increases Zika virus RNA levels in iPSC-derived macrophages JOURNAL OF GENERAL VIROLOGY Schilling, M., Vaughan-Jackson, A., James, W., McKeating, J. A. 2023; 104 (8)

  Abstract

  Type I interferons (IFNs) are the major host defence against viral infection and are induced following activation of cell surface or intracellular pattern recognition receptors, including retinoic-acid-inducible gene I (RIG-I)-like receptors (RLRs). All cellular processes are shaped by the microenvironment and one important factor is the local oxygen tension. The majority of published studies on IFN signalling are conducted under laboratory conditions of 18% oxygen (O2), that do not reflect the oxygen levels in most organs (1-5 % O2). We studied the effect of low oxygen on IFN induction and signalling in induced Pluripotent Stem Cell (iPSC)-derived macrophages as a model for tissue-resident macrophages and assessed the consequence for Zika virus (ZIKV) infection. Hypoxic conditions dampened the expression of interferon-stimulated genes (ISGs) following RLR stimulation or IFN treatment at early time points. RNA-sequencing and bio-informatic analysis uncovered several pathways including changes in transcription factor availability, the presence of HIF binding sites in promoter regions, and CpG content that may contribute to the reduced ISG expression. Hypoxic conditions increased the abundance of ZIKV RNA highlighting the importance of understanding how low oxygen conditions in the local microenvironment affect pathogen sensing and host defences.

  View details for DOI 10.1099/jgv.0.001885

  View details for Web of Science ID 001163665100006

  View details for PubMedID 37584553

  View details for PubMedCentralID PMC10877081

• Density dependent regulation of inflammatory responses in macrophages FRONTIERS IN IMMUNOLOGY Vaughan-Jackson, A., Stodolak, S., Ebrahimi, K. H., Johnson, E., Reardon, P. K., Dupont, M., Zhang, S., McCullagh, J. S. O., James, W. S. 2022; 13: 895488

  Abstract

  Macrophage distribution density is tightly regulated within the body, yet the importance of macrophage crowding during in vitro culture is largely unstudied. Using a human induced pluripotent stem cell (iPSC)-derived macrophage model of tissue resident macrophages, we characterize how increasing macrophage culture density changes their morphology and phenotype before and after inflammatory stimulation. In particular, density drives changes in macrophage inflammatory cytokine and chemokine secretion in both resting and activated states. This density regulated inflammatory state is also evident in blood monocyte derived-macrophages, the human monocytic THP-1 immortalized cell line, and iPSC-derived microglia. Density-dependent changes appear to be driven by a transferable soluble factor, yet the precise mechanism remains unknown. Our findings highlight cell plating density as an important but frequently overlooked consideration of in vitro macrophage research relevant to a variety of fields ranging from basic macrophage cell biology to disease studies.

  View details for DOI 10.3389/fimmu.2022.895488

  View details for Web of Science ID 000904954600001

  View details for PubMedID 36591218

  View details for PubMedCentralID PMC9800520

• A rapid antibody screening haemagglutination test for predicting immunity to SARS-CoV-2 variants of concern COMMUNICATIONS MEDICINE Ertesvag, N., Xiao, J., Zhou, F., Ljostveit, S., Sandnes, H., Lartey, S., Saevik, M., Hansen, L., Madsen, A., Mohn, K. G. I., Fjelltveit, E., Olofsson, J., Tan, T., Rijal, P., Schimanski, L., Oyen, S., Brokstad, K., Dunachie, S., Jamsen, A., James, W. S., Harding, A. C., Harvala, H., Dung Nguyen, Roberts, D., Patel, M., Gopal, R., Zambon, M., Wei, L., Gilbert-Jaramillo, J., Knight, M. L., Vaughan-Jackson, A., Dupont, M., Lamikanra, A. A., Klennerman, P., Barnes, E., Deeks, A., Johnson, S., Skelly, D., Stafford, L., Townsend, A., Tondel, C., Kuwelker, K., Blomberg, B., Bredholt, G., Onyango, T., Vahokoski, J., Bansal, A., Trieu, M., Amdam, H., Akselsen, P., Skorge, T., Okkenhaug, L., Linchausen, D., Langeland, N., Cox, R., PHE Virology Grp, Oxford Collaborative Grp, Bergen COVID-19 Res Grp 2022; 2 (1): 36

  Abstract

  Evaluation of susceptibility to emerging SARS-CoV-2 variants of concern (VOC) requires rapid screening tests for neutralising antibodies which provide protection.Firstly, we developed a receptor-binding domain-specific haemagglutination test (HAT) to Wuhan and VOC (alpha, beta, gamma and delta) and compared to pseudotype, microneutralisation and virus neutralisation assays in 835 convalescent sera. Secondly, we investigated the antibody response using the HAT after two doses of mRNA (BNT162b2) vaccination. Sera were collected at baseline, three weeks after the first and second vaccinations from older (80-99 years, n = 89) and younger adults (23-77 years, n = 310) and compared to convalescent sera from naturally infected individuals (1-89 years, n = 307).Here we show that HAT antibodies highly correlated with neutralising antibodies (R = 0.72-0.88) in convalescent sera. Home-dwelling older individuals have significantly lower antibodies to the Wuhan strain after one and two doses of BNT162b2 vaccine than younger adult vaccinees and naturally infected individuals. Moverover, a second vaccine dose boosts and broadens the antibody repertoire to VOC in naïve, not previously infected older and younger adults. Most (72-76%) older adults respond after two vaccinations to alpha and delta, but only 58-62% to beta and gamma, compared to 96-97% of younger vaccinees and 68-76% of infected individuals. Previously infected older individuals have, similarly to younger adults, high antibody titres after one vaccination.Overall, HAT provides a surrogate marker for neutralising antibodies, which can be used as a simple inexpensive, rapid test. HAT can be rapidly adaptable to emerging VOC for large-scale evaluation of potentially decreasing vaccine effectiveness.

  View details for DOI 10.1038/s43856-022-00091-x

  View details for Web of Science ID 001088892200002

  View details for PubMedID 35603265

  View details for PubMedCentralID PMC9053181

• Differentiation of human induced pluripotent stem cells to authentic macrophages using a defined, serum-free, open-source medium (vol 16, pg 1735, 2021) STEM CELL REPORTS Vaughan-Jackson, A., Stodolak, S., Ebrahimi, K. H., Browne, C., Reardon, P. K., Pires, E., Gilbert-Jaramillo, J., Cowley, S. A., James, W. S. 2021; 16 (12): 3093

  View details for DOI 10.1016/j.stemcr.2021.11.010

  View details for Web of Science ID 000731607400008

  View details for PubMedID 34910900

  View details for PubMedCentralID PMC8693763

• Differentiation of human induced pluripotent stem cells to authentic macrophages using a defined, serum-free, open-source medium STEM CELL REPORTS Vaughan-Jackson, A., Stodolak, S., Ebrahimi, K. H., Browne, C., Reardon, P. K., Pires, E., Gilbert-Jaramillo, J., Cowley, S. A., James, W. S. 2021; 16 (7): 1735-1748

  Abstract

  Human induced pluripotent stem cells (iPSCs) and macrophages derived from them are increasingly popular tools for research into both infectious and degenerative diseases. However, as the field strives for greater modeling accuracy, it is becoming ever more challenging to justify the use of undefined and proprietary media for the culture of these cells. Here, we describe a defined, serum-free, open-source medium for the differentiation of iPSC-derived macrophages. This medium is equally capable of maintaining these cells compared with commercial alternatives. The macrophages differentiated in this medium display improved terminally differentiated cell characteristics, reduced basal expression of induced antiviral response genes, and improved polarization capacity. We conclude that cells cultured in this medium are an appropriate and malleable model for tissue-resident macrophages, on which future differentiation techniques can be built.

  View details for DOI 10.1016/j.stemcr.2021.05.018

  View details for Web of Science ID 000674432500012

  View details for PubMedID 34171284

  View details for PubMedCentralID PMC8282471

• Efficacy of NS5A inhibitors against unusual and potentially difficult-to-treat HCV subtypes commonly found in sub-Saharan Africa and South East Asia. Journal of hepatology Nguyen, D., Smith, D., Vaughan-Jackson, A., Magri, A., Barnes, E., Simmonds, P. 2020; 73 (4): 794-799

  Abstract

  The efficacy of NS5A inhibitors against several less common subtypes of hepatitis C virus (HCV) is poorly characterised. Some subtypes including 3b, 3g, 6u and 6v commonly harbour amino acid residues in NS5A that may confer resistance to direct-acting antivirals (DAAs) in other common subtypes. Data from patients also suggest that 1l and 4r with amino acid substitutions at positions 28-31 and 93 in NS5A are relatively resistant to DAA therapy.In this study, we tested the efficacy of daclatasvir, elbasvir, ledipasvir, pibrentasvir and velpatasvir against these subtypes using the SGR-JFH1 replicon backbone.NS5A inhibitors showed different levels of efficacy with only pibrentasvir effective against all tested subtypes. Daclatasvir and ledipasvir were ineffective against 6u and 6v (half maximal effective concentration [EC50] values of 239-321 nM) while 3b and 3g were only susceptible to pibrentasvir. Analysis of effects of individual mutations indicated that Q30R in 1l increased the EC50 of ledipasvir by 18-fold, conferring intermediate resistance, while those of L31M and Y93H in 4r induced increases in EC50 values of 2,100- and 3,575-fold (high-level resistance).The high ledipasvir EC50 values of 1l with the Q30R substitution, 4r L31M and 4r Y93H may explain the treatment failure in patients who were infected with these viruses and treated with ledipasvir + sofosbuvir. This study also shows the ineffectiveness of the first generation NS5A inhibitors against 6u and 6v, and confirms the inherent resistance of 3b and 3g to most NS5A inhibitors. Clinical studies to confirm in vivo sensitivity to NS5A inhibitors are urgently needed so that rational, effective treatment strategies may be developed for unusual subtypes.Little is known about the efficacy of NS5A inhibitors against some "unusual" hepatitis C virus (HCV) subtypes including 1l, 3b, 3g, 4r, 6u and 6v. In this study, we manufactured HCV replicons which express the NS5A protein from the unusual HCV subtypes 1l, 3b, 3g, 4r, 6u, 6v. We then tested the effect of the NS5A inhibitors daclatasvir, elbasvir, ledipasvir, pibrentasvir and velpatasvir on blocking replication, using these replicons. We show that these replicons are resistant at some level to all NS5A inhibitors other than pibrentasvir.

  View details for DOI 10.1016/j.jhep.2020.05.029

  View details for PubMedID 32470499

• Pharmacological activation of the circadian component REV-ERB inhibits HIV-1 replication. Scientific reports Borrmann, H., Davies, R., Dickinson, M., Pedroza-Pacheco, I., Schilling, M., Vaughan-Jackson, A., Magri, A., James, W., Balfe, P., Borrow, P., McKeating, J. A., Zhuang, X. 2020; 10 (1): 13271

  Abstract

  Human immunodeficiency virus 1 (HIV-1) is a life-threatening pathogen that still lacks a curative therapy or vaccine. Despite the reduction in AIDS-related deaths achieved by current antiretroviral therapies, drawbacks including drug resistance and the failure to eradicate infection highlight the need to identify new pathways to target the infection. Circadian rhythms are endogenous 24-h oscillations which regulate physiological processes including immune responses to infection, and there is an emerging role for the circadian components in regulating viral replication. The molecular clock consists of transcriptional/translational feedback loops that generate rhythms. In mammals, BMAL1 and CLOCK activate rhythmic transcription of genes including the nuclear receptor REV-ERBα, which represses BMAL1 and plays an essential role in sustaining a functional clock. We investigated whether REV-ERB activity regulates HIV-1 replication and found REV-ERB agonists inhibited HIV-1 promoter activity in cell lines, primary human CD4 T cells and macrophages, whilst antagonism or genetic disruption of REV-ERB increased promoter activity. The REV-ERB agonist SR9009 inhibited promoter activity of diverse HIV-subtypes and HIV-1 replication in primary T cells. This study shows a role for REV-ERB synthetic agonists to inhibit HIV-1 LTR promoter activity and viral replication, supporting a role for circadian clock components in regulating HIV-1 replication.

  View details for DOI 10.1038/s41598-020-70170-3

  View details for PubMedID 32764708

  View details for PubMedCentralID PMC7413328

• Alpha kinase 1 controls intestinal inflammation by suppressing the IL-12/Th1 axis. Nature communications Ryzhakov, G., West, N. R., Franchini, F., Clare, S., Ilott, N. E., Sansom, S. N., Bullers, S. J., Pearson, C., Costain, A., Vaughan-Jackson, A., Goettel, J. A., Ermann, J., Horwitz, B. H., Buti, L., Lu, X., Mukhopadhyay, S., Snapper, S. B., Powrie, F. 2018; 9 (1): 3797

  Abstract

  Inflammatory bowel disease (IBD) are heterogenous disorders of the gastrointestinal tract caused by a spectrum of genetic and environmental factors. In mice, overlapping regions of chromosome 3 have been associated with susceptibility to IBD-like pathology, including a locus called Hiccs. However, the specific gene that controls disease susceptibility remains unknown. Here we identify a Hiccs locus gene, Alpk1 (encoding alpha kinase 1), as a potent regulator of intestinal inflammation. In response to infection with the commensal pathobiont Helicobacter hepaticus (Hh), Alpk1-deficient mice display exacerbated interleukin (IL)-12/IL-23 dependent colitis characterized by an enhanced Th1/interferon(IFN)-γ response. Alpk1 controls intestinal immunity via the hematopoietic system and is highly expressed by mononuclear phagocytes. In response to Hh, Alpk1-/- macrophages produce abnormally high amounts of IL-12, but not IL-23. This study demonstrates that Alpk1 promotes intestinal homoeostasis by regulating the balance of type 1/type 17 immunity following microbial challenge.

  View details for DOI 10.1038/s41467-018-06085-5

  View details for PubMedID 30228258

  View details for PubMedCentralID PMC6143560