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1.
J Exp Med ; 221(9)2024 Sep 02.
Article in English | MEDLINE | ID: mdl-38953896

ABSTRACT

Gain-of-function mutations in STING cause STING-associated vasculopathy with onset in infancy (SAVI) characterized by early-onset systemic inflammation, skin vasculopathy, and interstitial lung disease. Here, we report and characterize a novel STING variant (F269S) identified in a SAVI patient. Single-cell transcriptomics of patient bone marrow revealed spontaneous activation of interferon (IFN) and inflammatory pathways across cell types and a striking prevalence of circulating naïve T cells was observed. Inducible STING F269S expression conferred enhanced signaling through ligand-independent translocation of the protein to the Golgi, protecting cells from viral infections but preventing their efficient immune priming. Additionally, endothelial cell activation was promoted and further exacerbated by cytokine secretion by SAVI immune cells, resulting in inflammation and endothelial damage. Our findings identify STING F269S mutation as a novel pathogenic variant causing SAVI, highlight the importance of the crosstalk between endothelial and immune cells in the context of lung disease, and contribute to a better understanding of how aberrant STING activation can cause pathology.


Subject(s)
Endothelial Cells , Membrane Proteins , Humans , Infant , Endothelial Cells/metabolism , Endothelial Cells/pathology , Gain of Function Mutation , Golgi Apparatus/metabolism , Interferons/metabolism , Interferons/genetics , Lung Diseases, Interstitial/genetics , Lung Diseases, Interstitial/pathology , Lung Diseases, Interstitial/immunology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutation , Signal Transduction , Vascular Diseases/genetics , Vascular Diseases/pathology , Infant, Newborn , Child, Preschool , Female
2.
Mol Ther ; 32(1): 124-139, 2024 Jan 03.
Article in English | MEDLINE | ID: mdl-37990494

ABSTRACT

Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in long-term repopulating quiescent HSC, allowing their efficient genetic manipulation. Mechanistically, lentiviral vector transduction of quiescent HSC was found to be restricted at the level of vector entry and by limited pyrimidine pools. These restrictions were overcome by the combined addition of cyclosporin H (CsH) and deoxynucleosides (dNs) during lentiviral vector transduction. Clinically relevant transduction levels were paired with higher polyclonal engraftment of long-term repopulating HSC as compared with standard ex vivo cultured controls. These findings identify the cell-intrinsic barriers that restrict the transduction of quiescent HSC and provide a means to overcome them, paving the way for the genetic engineering of unstimulated HSC.


Subject(s)
Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells , Humans , Transduction, Genetic , Lentivirus/genetics , Genetic Therapy/methods , Immunity, Innate , Genetic Vectors/genetics , Antigens, CD34
3.
Hum Gene Ther ; 34(17-18): 836-852, 2023 09.
Article in English | MEDLINE | ID: mdl-37672519

ABSTRACT

As the clinical experience in adeno-associated viral (AAV) vector-based gene therapies is expanding, the necessity to better understand and control the host immune responses is also increasing. Immunogenicity of AAV vectors in humans has been linked to several limitations of the platform, including lack of efficacy due to antibody-mediated neutralization, tissue inflammation, loss of transgene expression, and in some cases, complement activation and acute toxicities. Nevertheless, significant knowledge gaps remain in our understanding of the mechanisms of immune responses to AAV gene therapies, further hampered by the failure of preclinical animal models to recapitulate clinical findings. In this review, we focus on the current knowledge regarding immune responses, spanning from innate immunity to humoral and adaptive responses, triggered by AAV vectors and how they can be mitigated for safer, durable, and more effective gene therapies.


Subject(s)
Complement Activation , Immunity, Innate , Animals , Humans , Genetic Therapy , Inflammation , Models, Animal
4.
Cell Stem Cell ; 30(5): 549-570, 2023 05 04.
Article in English | MEDLINE | ID: mdl-37146580

ABSTRACT

The growing clinical success of hematopoietic stem/progenitor cell (HSPC) gene therapy (GT) relies on the development of viral vectors as portable "Trojan horses" for safe and efficient gene transfer. The recent advent of novel technologies enabling site-specific gene editing is broadening the scope and means of GT, paving the way to more precise genetic engineering and expanding the spectrum of diseases amenable to HSPC-GT. Here, we provide an overview of state-of-the-art and prospective developments of the HSPC-GT field, highlighting how advances in biological characterization and manipulation of HSPCs will enable the design of the next generation of these transforming therapeutics.


Subject(s)
CRISPR-Cas Systems , Hematopoietic Stem Cells , Prospective Studies , Gene Editing , Genetic Therapy , Biology
5.
EMBO J ; 42(10): e112234, 2023 05 15.
Article in English | MEDLINE | ID: mdl-36970857

ABSTRACT

The interferon-induced transmembrane proteins (IFITM) are implicated in several biological processes, including antiviral defense, but their modes of action remain debated. Here, taking advantage of pseudotyped viral entry assays and replicating viruses, we uncover the requirement of host co-factors for endosomal antiviral inhibition through high-throughput proteomics and lipidomics in cellular models of IFITM restriction. Unlike plasma membrane (PM)-localized IFITM restriction that targets infectious SARS-CoV2 and other PM-fusing viral envelopes, inhibition of endosomal viral entry depends on lysines within the conserved IFITM intracellular loop. These residues recruit Phosphatidylinositol 3,4,5-trisphosphate (PIP3) that we show here to be required for endosomal IFITM activity. We identify PIP3 as an interferon-inducible phospholipid that acts as a rheostat for endosomal antiviral immunity. PIP3 levels correlated with the potency of endosomal IFITM restriction and exogenous PIP3 enhanced inhibition of endocytic viruses, including the recent SARS-CoV2 Omicron variant. Together, our results identify PIP3 as a critical regulator of endosomal IFITM restriction linking it to the Pi3K/Akt/mTORC pathway and elucidate cell-compartment-specific antiviral mechanisms with potential relevance for the development of broadly acting antiviral strategies.


Subject(s)
Antiviral Agents , COVID-19 , Humans , Interferons/metabolism , Phospholipids , Phosphatidylinositol 3-Kinases/metabolism , RNA, Viral , RNA-Binding Proteins/metabolism , SARS-CoV-2/metabolism , Virus Internalization , Membrane Proteins/metabolism
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