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BackgroundCurrent understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem and progenitor cells (HSPC) in these animal models is only partially understood. Using a new clonal tracking methodology designed for small sample volumes, we aim to reveal the underlying clonal dynamics of human cell repopulation in a mouse environment.MethodsHumanized bone marrow-liver-thymus (hu-BLT) mice were generated by transplanting lentiviral vector-transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25 µl of mouse blood for longitudinal and quantitative clonal analysis of human HSPC repopulation in mouse environment. We explored transcriptional and epigenetic features of human HSPC for possible VIS bias.ResultsA total of 897 HSPC clones were longitudinally tracked in hu-BLT mice-providing a first-ever demonstration of clonal dynamics and coordinated expansion of therapeutic and control vector-modified human cell populations simultaneously repopulating in the same humanized mice. The polyclonal repopulation stabilized at 19 weeks post-transplant and the contribution of the largest clone doubled within 4 weeks. Moreover, 550 (~ 60%) clones persisted over 6 weeks and were highly shared between different organs. The normal clonal profiles confirmed the safety of our gene therapy vectors. Multi-omics analysis of human FL-HSPC revealed that 54% of vector integrations in repopulating clones occurred within ± 1 kb of H3K36me3-enriched regions.ConclusionsHuman repopulation in mice is polyclonal and stabilizes more rapidly than that previously observed in humans. VIS preference for H3K36me3 has no apparent negative effects on HSPC repopulation. Our study provides a methodology to longitudinally track clonal repopulation in small animal models extensively used for stem cell and gene therapy research and with lentiviral vectors designed for clinical applications. Results of this study provide a framework for understanding the clonal behavior of human HPSC repopulating in a mouse environment, critical for translating results from humanized mice models to the human settings.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER-Golgi intermediate compartment. Here, we found that deficiency in the Ca2+ sensor stromal interaction molecule 1 (STIM1) caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient with combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and coexpression of full-length STIM1 or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1-STING circuit that maintains the resting state of the STING pathway.

A designed repeat scaffold protein (AnkGAG1D4) recognizing the human immunodeficiency virus-1 (HIV-1) capsid (CA) was formerly established with antiviral assembly. Here, we investigated the molecular mechanism of AnkGAG1D4 function during the late stages of the HIV-1 replication cycle. By applying stimulated emission-depletion (STED) microscopy, Gag polymerisation was interrupted at the plasma membrane. Disturbance of Gag polymerisation triggered Gag accumulation inside producer cells and trapping of the CD81 tetraspanin on the plasma membrane. Moreover, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) experiments were performed to validate the packaging efficiency of RNAs. Our results advocated that AnkGAG1D4 interfered with the Gag precursor protein from selecting HIV-1 and cellular RNAs for encapsidation into viral particles. These findings convey additional information on the antiviral activity of AnkGAG1D4 at late stages of the HIV-1 life cycle, which is potential for an alternative anti-HIV molecule. [ABSTRACT FROM AUTHOR]

Background: Assembly and budding in the late-stage of human immunodeficiency virus type 1 (HIV-1) production rely on Gag protein polymerization at the inner leaflet of the plasma membrane. We previously generated a monomeric ankyrin repeat protein (Ank1D4) that specifically interacts with capsid protein (CAp24) of HIV-1, however this protein had modest binding affinity., Objective: This study aimed to improve the avidity of Ank1D4 by generating two Ank1D4 dimers: (Ank1D4NC-NC) and its inverted form (Ank1D4NC-CN), with each domain connected by a flexible (G4S)4 linker peptide., Methods: Binding properties of monomeric and dimeric Ank1D4 was performed by capture enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA was used to examine bifunctional module of dimeric Ank1D4. Ank1D4NC-NC and Ank1D4NC-CN were evaluated using bio-layer interferometry (BLI), compared to monomeric Ank1D4., Results: Similar binding surfaces were observed in both dimers which was comparable with monomeric Ank1D4. The interaction of Ank1D4NC-CN with CAp24 was significantly greater than that of Ank1D4NC-NC and Ank1D4 by capture ELISA. Ank1D4NC-CN also exhibited bifunctionality using a sandwich ELISA. The KD of Ank1D4NC-CN, Ank1D4NC-NC and monomeric Ank1D4 was 3.5 nM, 53.7 nM, and 126.2 nM, respectively using bio-layer interferometry analysis., Conclusions: This study provides a strategy for increasing Ank1D4 avidity through the construction of novel inverted dimers with a flexible linker. Ank1D4NC-CN may provide an alternative treatment strategy for inhibiting HIV-1 replication.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER-Golgi intermediate compartment. Here, we found that deficiency in the Ca 2+ sensor stromal interaction molecule 1 (STIM1) caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient with combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and coexpression of full-length STIM1 or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1-STING circuit that maintains the resting state of the STING pathway.

Background: AnkGAG1D4 is an artificial ankyrin repeat protein which recognizes the capsid protein (CA) of the human immunodeficiency virus type 1 (HIV-1) and exhibits the intracellular antiviral activity on the viral assembly process. Improving the binding affinity of AnkGAG1D4 would potentially enhance the AnkGAG1D4-mediated antiviral activity., Objective: To augment the affinity of AnkGAG1D4 scaffold towards its CA target, through computational predictions and experimental designs., Method: Three dimensional structure of the binary complex formed by AnkGAG1D4 docked to the CA was used as a model for van der Waals (vdW) binding energy calculation. The results generated a simple guideline to select the amino acids for modifications. Following the predictions, modified AnkGAG1D4 proteins were produced and further evaluated for their CA-binding activity, using ELISA-modified method and bio-layer interferometry (BLI)., Results: Tyrosine at position 56 (Y56) in AnkGAG1D4 was experimentally identified as the most critical residue for CA binding. Rational substitutions of this residue diminished the binding affinity. However, vdW calculation preconized to substitute serine for tyrosine at position 45. Remarkably, the affinity for the viral CA was significantly enhanced in AnkGAG1D4-S45Y mutant, with no alteration of the target specificity., Conclusions: The S-to-Y mutation at position 45, based on the prediction of interacting amino acids and on vdW binding energy calculation, resulted in a significant enhancement of the affinity of AnkGAG1D4 ankyrin for its CA target. AnkGAG1D4-S45Y mutant represented the starting point for further construction of variants with even higher affinity towards the viral CA, and higher therapeutic potential in the future.

Designed molecular scaffolds have been proposed as alternative therapeutic agents against HIV-1. The ankyrin repeat protein (Ank(GAG)1D4) and the zinc finger protein (2LTRZFP) have recently been characterized as intracellular antivirals, but these molecules, used individually, do not completely block HIV-1 replication and propagation. The capsid-binder Ank(GAG)1D4, which inhibits HIV-1 assembly, does not prevent the genome integration of newly incoming viruses. 2LTRZFP, designed to target the 2-LTR-circle junction of HIV-1 cDNA and block HIV-1 integration, would have no antiviral effect on HIV-1-infected cells. However, simultaneous expression of these two molecules should combine the advantage of preventive and curative treatments. To test this hypothesis, the genes encoding the N-myristoylated Myr(+)Ank(GAG)1D4 protein and the 2LTRZFP were introduced into human T-cells, using a third-generation lentiviral vector. SupT1 cells stably expressing 2LTRZFP alone or with Myr(+)Ank(GAG)1D4 showed a complete resistance to HIV-1 in viral challenge. Administration of the Myr(+)Ank(GAG)1D4 vector to HIV-1-preinfected SupT1 cells resulted in a significant antiviral effect. Resistance to viral infection was also observed in primary human CD4+ T-cells stably expressing Myr(+)Ank(GAG)1D4, and challenged with HIV-1, SIVmac, or SHIV. Our data suggest that our two anti-HIV-1 molecular scaffold prototypes are promising antiviral agents for anti-HIV-1 gene therapy.