All Publications

  • Gene regulation in inborn errors of immunity: Implications for gene therapy design and efficacy. Immunological reviews Ghanim, H. Y., Porteus, M. H. 2024


    Inborn errors of immunity (IEI) present a unique paradigm in the realm of gene therapy, emphasizing the need for precision in therapeutic design. As gene therapy transitions from broad-spectrum gene addition to careful modification of specific genes, the enduring safety and effectiveness of these therapies in clinical settings have become crucial. This review discusses the significance of IEIs as foundational models for pioneering and refining precision medicine. We explore the capabilities of gene addition and gene correction platforms in modifying the DNA sequence of primary cells tailored for IEIs. The review uses four specific IEIs to highlight key issues in gene therapy strategies: X-linked agammaglobulinemia (XLA), X-linked chronic granulomatous disease (X-CGD), X-linked hyper IgM syndrome (XHIGM), and immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX). We detail the regulatory intricacies and therapeutic innovations for each disorder, incorporating insights from relevant clinical trials. For most IEIs, regulated expression is a vital aspect of the underlying biology, and we discuss the importance of endogenous regulation in developing gene therapy strategies.

    View details for DOI 10.1111/imr.13305

    View details for PubMedID 38233996

  • Genetically Corrected RAG2-SCID Human Hematopoietic Stem Cells Restore V(D)J-Recombinase and Rescue Lymphoid Deficiency. Blood advances Pavel-Dinu, M., Gardner, C. L., Nakauchi, Y., Kawai, T., Delmonte, O. M., Palterer, B., Bosticardo, M., Pala, F., Viel, S., Malech, H. L., Ghanim, H. Y., Bode, N. M., Kurgan, G. L., Detweiler, A. M., Vakulskas, C. A., Neff, N. F., Sheikali, A., Menezes, S. T., Chrobok, J., Hernandez Gonzalez, E. M., Majeti, R., Notarangelo, L. D., Porteus, M. H. 2023


    Recombination-activating genes (RAG1 and RAG2) are critical in lymphoid cell development and function by initiating the V(D)J-recombination process to generate polyclonal lymphocytes with broad antigen-specificity. Clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCID), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phases of the cell cycle at the early stages of T and B cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based "universal gene-correction" approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and one RAG2-SCID patient. V(D)J recombinase activity was restored following gene correction of RAG2-SCID-derived HSPCs, resulting in the development of TCR ab and gd CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of the CDR3 length and preserved usage of distal TRAV genes. We confirmed in vivo rescue of B-cell development, with normal IgM surface expression and a significant decrease in CD56bright NK cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.

    View details for DOI 10.1182/bloodadvances.2023011766

    View details for PubMedID 38096800

  • Engineering a Potential Curative Treatment for Hemophilia A Using an AAV Dual Targeting Strategy Johnston, N., Amorin, A., Nguygen, L., Ghanim, H., Porteus, M. H. CELL PRESS. 2023: 267
  • Gibbin mesodermal regulation patterns epithelial development. Nature Collier, A., Liu, A., Torkelson, J., Pattison, J., Gaddam, S., Zhen, H., Patel, T., McCarthy, K., Ghanim, H., Oro, A. E. 2022


    Proper ectodermal patterning during human development requires previously identified transcription factors such as GATA3 and p63, as well as positional signalling from regional mesoderm1-6. However, the mechanism by which ectoderm and mesoderm factors act to stably pattern gene expression and lineage commitment remains unclear. Here we identify the protein Gibbin,encoded by the Xia-Gibbs AT-hook DNA-binding-motif-containing 1 (AHDC1) disease gene7-9, as a key regulator of early epithelial morphogenesis. We find that enhancer- or promoter-bound Gibbin interacts with dozens of sequence-specific zinc-finger transcription factors and methyl-CpG-binding proteins to regulate the expression of mesoderm genes. The loss of Gibbin causes an increase in DNA methylation at GATA3-dependent mesodermal genes, resulting in a loss of signalling between developing dermal and epidermal cell types. Notably, Gibbin-mutant human embryonic stem-cell-derived skin organoids lack dermal maturation, resulting in p63-expressing basal cells that possess defective keratinocyte stratification. In vivo chimeric CRISPR mouse mutants reveal a spectrum of Gibbin-dependent developmental patterning defects affecting craniofacial structure, abdominal wall closure and epidermal stratification that mirror patient phenotypes. Our results indicate that the patterning phenotypes seen in Xia-Gibbs and related syndromes derive from abnormal mesoderm maturation as a result of gene-specific DNA methylation decisions.

    View details for DOI 10.1038/s41586-022-04727-9

    View details for PubMedID 35585237

  • Allele-Specific Gene Editing Rescues Pathology in a Human Model of Charcot-Marie-Tooth Disease Type 2E FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY Feliciano, C. M., Wu, K., Watry, H. L., Marley, C. E., Ramadoss, G. N., Ghanim, H. Y., Liu, A. Z., Zholudeva, L., McDevitt, T. C., Saporta, M. A., Conklin, B. R., Judge, L. M. 2021; 9: 723023


    Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death. The targeted inactivation of dominant disease alleles by gene editing is a promising approach with the potential to completely remove the cause of pathology with a single treatment. Here, we demonstrate that allele-specific CRISPR gene editing in a human model of axonal Charcot-Marie-Tooth (CMT) disease rescues pathology caused by a dominant missense mutation in the neurofilament light chain gene (NEFL, CMT type 2E). We utilized a rapid and efficient method for generating spinal motor neurons from human induced pluripotent stem cells (iPSCs) derived from a patient with CMT2E. Diseased motor neurons recapitulated known pathologic phenotypes at early time points of differentiation, including aberrant accumulation of neurofilament light chain protein in neuronal cell bodies. We selectively inactivated the disease NEFL allele in patient iPSCs using Cas9 enzymes to introduce a frameshift at the pathogenic N98S mutation. Motor neurons carrying this allele-specific frameshift demonstrated an amelioration of the disease phenotype comparable to that seen in an isogenic control with precise correction of the mutation. Our results validate allele-specific gene editing as a therapeutic approach for CMT2E and as a promising strategy to silence dominant mutations in any gene for which heterozygous loss-of-function is well tolerated. This highlights the potential for gene editing as a therapy for currently untreatable dominant neurologic diseases.

    View details for DOI 10.3389/fcell.2021.723023

    View details for Web of Science ID 000692925500001

    View details for PubMedID 34485306

    View details for PubMedCentralID PMC8415563

  • Blood-brain barrier dysfunction in aging induces hyperactivation of TGF beta signaling and chronic yet reversible neural dysfunction SCIENCE TRANSLATIONAL MEDICINE Senatorov, V. V., Friedman, A. R., Milikovsky, D. Z., Ofer, J., Saar-Ashkenazy, R., Charbash, A., Jahan, N., Chin, G., Mihaly, E., Lin, J. M., Ramsay, H. J., Moghbel, A., Preininger, M. K., Eddings, C. R., Harrison, H. V., Patel, R., Shen, Y., Ghanim, H., Sheng, H., Veksler, R., Sudmant, P. H., Becker, A., Hart, B., Rogawski, M. A., Dillin, A., Friedman, A., Kaufer, D. 2019; 11 (521)


    Aging involves a decline in neural function that contributes to cognitive impairment and disease. However, the mechanisms underlying the transition from a young-and-healthy to aged-and-dysfunctional brain are not well understood. Here, we report breakdown of the vascular blood-brain barrier (BBB) in aging humans and rodents, which begins as early as middle age and progresses to the end of the life span. Gain-of-function and loss-of-function manipulations show that this BBB dysfunction triggers hyperactivation of transforming growth factor-β (TGFβ) signaling in astrocytes, which is necessary and sufficient to cause neural dysfunction and age-related pathology in rodents. Specifically, infusion of the serum protein albumin into the young rodent brain (mimicking BBB leakiness) induced astrocytic TGFβ signaling and an aged brain phenotype including aberrant electrocorticographic activity, vulnerability to seizures, and cognitive impairment. Furthermore, conditional genetic knockdown of astrocytic TGFβ receptors or pharmacological inhibition of TGFβ signaling reversed these symptomatic outcomes in aged mice. Last, we found that this same signaling pathway is activated in aging human subjects with BBB dysfunction. Our study identifies dysfunction in the neurovascular unit as one of the earliest triggers of neurological aging and demonstrates that the aging brain may retain considerable latent capacity, which can be revitalized by therapeutic inhibition of TGFβ signaling.

    View details for DOI 10.1126/scitranslmed.aaw8283

    View details for Web of Science ID 000500927700002

    View details for PubMedID 31801886