Pre-clinical development of AP4B1 gene replacement therapy for hereditary spastic paraplegia type 47.

Spastic paraplegia 47 (SPG47) is a neurological disorder caused by mutations in the adaptor protein complex 4 ß1 subunit (AP4B1) gene leading to AP-4 complex deficiency. SPG47 is characterised by progressive spastic paraplegia, global developmental delay, intellectual disability and epilepsy. Gene therapy aimed at restoring functional AP4B1 protein levels is a rational therapeutic strategy to ameliorate the disease phenotype. Here we report that a single delivery of adeno-associated virus serotype 9 expressing hAP4B1 (AAV9/hAP4B1) into the cisterna magna leads to widespread gene transfer and restoration of various hallmarks of disease, including AP-4 cargo (ATG9A) mislocalisation, calbindin-positive spheroids in the deep cerebellar nuclei, anatomical brain defects and motor dysfunction, in an SPG47 mouse model. Furthermore, AAV9/hAP4B1-based gene therapy demonstrated a restoration of plasma neurofilament light (NfL) levels of treated mice. Encouraged by these preclinical proof-of-concept data, we conducted IND-enabling studies, including immunogenicity and GLP non-human primate (NHP) toxicology studies. Importantly, NHP safety and biodistribution study revealed no significant adverse events associated with the therapeutic intervention. These findings provide evidence of both therapeutic efficacy and safety, establishing a robust basis for the pursuit of an IND application for clinical trials targeting SPG47 patients.

Pneumolysin Is Responsible for Differential Gene Expression and Modifications in the Epigenetic Landscape of Primary Monocyte Derived Macrophages.

Epigenetic modifications regulate gene expression in the host response to a diverse range of pathogens. The extent and consequences of epigenetic modification during macrophage responses to Streptococcus pneumoniae, and the role of pneumolysin, a key Streptococcus pneumoniae virulence factor, in influencing these responses, are currently unknown. To investigate this, we infected human monocyte derived macrophages (MDMs) with Streptococcus pneumoniae and addressed whether pneumolysin altered the epigenetic landscape and the associated acute macrophage transcriptional response using a combined transcriptomic and proteomic approach. Transcriptomic analysis identified 503 genes that were differentially expressed in a pneumolysin-dependent manner in these samples. Pathway analysis highlighted the involvement of transcriptional responses to core innate responses to pneumococci including modules associated with metabolic pathways activated in response to infection, oxidative stress responses and NFκB, NOD-like receptor and TNF signalling pathways. Quantitative proteomic analysis confirmed pneumolysin-regulated protein expression, early after bacterial challenge, in representative transcriptional modules associated with innate immune responses. In parallel, quantitative mass spectrometry identified global changes in the relative abundance of histone post translational modifications (PTMs) upon pneumococcal challenge. We identified an increase in the relative abundance of H3K4me1, H4K16ac and a decrease in H3K9me2 and H3K79me2 in a PLY-dependent fashion. We confirmed that pneumolysin blunted early transcriptional responses involving TNF-α and IL-6 expression. Vorinostat, a histone deacetylase inhibitor, similarly downregulated TNF-α production, reprising the pattern observed with pneumolysin. In conclusion, widespread changes in the macrophage transcriptional response are regulated by pneumolysin and are associated with global changes in histone PTMs. Modulating histone PTMs can reverse pneumolysin-associated transcriptional changes influencing innate immune responses, suggesting that epigenetic modification by pneumolysin plays a role in dampening the innate responses to pneumococci.

T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses.

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations.

TMEM203 is a binding partner and regulator of STING-mediated inflammatory signaling in macrophages.

Regulation of IFN signaling is critical in host recognition and response to pathogens while its dysregulation underlies the pathogenesis of several chronic diseases. STimulator of IFN Genes (STING) has been identified as a critical mediator of IFN inducing innate immune pathways, but little is known about direct coregulators of this protein. We report here that TMEM203, a conserved putative transmembrane protein, is an intracellular regulator of STING-mediated signaling. We show that TMEM203 interacts, functionally cooperates, and comigrates with STING following cell stimulation, which in turn leads to the activation of the kinase TBK1, and the IRF3 transcription factor. This induces target genes in macrophages, including IFN-ß. Using Tmem203 knockout bone marrow-derived macrophages and transient knockdown of TMEM203 in human monocyte-derived macrophages, we show that TMEM203 protein is required for cGAMP-induced STING activation. Unlike STING, TMEM203 mRNA levels are elevated in T cells from patients with systemic lupus erythematosus, a disease characterized by the overexpression of type I interferons. Moreover, TMEM203 mRNA levels are associated with disease activity, as assessed by serum levels of the complement protein C3. Identification of TMEM203 sheds light into the control of STING-mediated innate immune responses, providing a potential novel mechanism for therapeutic interventions in STING-associated inflammatory diseases.

CD16/32-specific biotinylated 2.4G2 single-chain Fv complexed with avidin-FITC enhances FITC-specific humoral immune response in vivo in a CD16-dependent manner.

Fcgamma receptors (FcgammaRs) play an essential role in the regulation of immune response due to their ability to bind immune complexes. Activating FcgammaRs may facilitate antigen presentation and dendritic-cell maturation, while in the late phase of the immune response, the inhibitory FcgammaRIIb may down-regulate B-cell activation upon cross-linking with activating receptors. In this study, we investigated the in vivo role of FcgammaRs on the modulation of humoral immune response. In order to get well-defined immune complexes that can bind to both the activating and the inhibitory FcgammaRs, we designed a mono-biotinylated single-chain fragment variable construct from the rat anti-mouse CD16/32 clone 2.4G2, linked to avidin-FITC, and tested its effect on the FITC-hapten-specific T-independent type 2 (TI-2) and T-dependent (TD) immune response. When injected intravenously in mice, the complex bound to a small portion of B220+, CD11b(high) and CD11c(high) cells and was localized in the spleen on marginal zone macrophages 15 min after treatment. When applied as a booster following primary immunization with TI-2 (FITC-dextran) or TD (FITC-keyhole limpet haemocyanin) antigens, the complex elevated the number of hapten-specific IgM/IgG-producing B cells. This effect was diminished in CD16KO mice, suggesting that the activating-type FcgammaRIII might be a key mediator of this mechanism.

Grb2-associated binder 1 (Gab1) adaptor/scaffolding protein regulates Erk signal in human B cells.

RNA silencing experiments showed that knocking down Gab1 adaptor protein in BL41 human Burkitt lymphoma cells significantly reduced B cell receptor (BCR)-induced Erk phosphorylation, indicating that Gab1 plays a pivotal role in regulating Erk activity in B cells.