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Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID‐19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF‐A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF‐A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP‐mRNAs between cells is functionally extended by cells’ own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF‐A mRNA delivery to cells via LNPs, a fraction of internalized VEGF‐A mRNA is secreted via EVs. The overexpressed VEGF‐A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA‐Seq analysis reveals that as cells’ response to LNP‐VEGF‐A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF‐A mRNA in vitro and in vivo. Upon equal amount of VEGF‐A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF‐A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF‐A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF‐A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this mRNA differently than LNPs. The study shows that a fraction of LNP‐mRNA that is cell‐endocytosed can be sent to other cells via the secretion of extracellular vesicles (EVs). LNPs transform these EVs  as functional extensions to distribute therapeutic mRNA between cells.Importantly, EVs can be isolated such as from cardiac progenitor cells (CPC‐EVs), and thus utilized for mRNA delivery in vivo. Upon mRNA delivery to cardiac tissue, CPC‐EVs cause less expression of inflammatory cytokines, compared to other vehicles used.

Many diseases are linked to dysregulation of the striatum. Striatal function depends on neuronal compartmentation into striosomes and matrix. Striatal projection neurons are GABAergic medium spiny neurons (MSNs), subtyped by selective expression of receptors, neuropeptides, and other gene families. Neurogenesis of the striosome and matrix occurs in separate waves, but the factors regulating compartmentation and neuronal differentiation are largely unidentified. We performed RNA- and ATAC-seq on sorted striosome and matrix cells at postnatal day 3, using the Nr4a1 -EGFP striosome reporter mouse. Focusing on the striosome, we validated the localization and/or role of Irx1 , Foxf2 , Olig2 , and Stat1/2 in the developing striosome and the in vivo enhancer function of a striosome-specific open chromatin region 4.4 Kb downstream of Olig2 . These data provide novel tools to dissect and manipulate the networks regulating MSN compartmentation and differentiation, including in human iPSC-derived striatal neurons for disease modeling and drug discovery.

The aim of this study was to identify immune factors that distinguish patients with herpes simplex virus type 2 (HSV-2) meningitis from patients with HSV-2 genital herpes by analyzing demographic data, in vitro production of cytokines and other immune factors secreted by patient peripheral blood mononuclear cells (PBMC), and existing antibody responses. PBMC and plasma were collected from patients previously diagnosed with HSV-2 meningitis (n=49) and HSV-2 genital herpes (n=38). PBMC were cultured in the presence or absence of HSV-2 and followed by multiplex analyses of culture supernatants for a panel of immune factors including Th1 and inflammatory cytokines, interferons, and chemokines. Plasma was analyzed for type-specific HSV antibodies and HSV-2 DNA. The multivariate method OPLS-DA was used to identify immune response patterns that differentiate the two patient groups. The multivariate analysis showed that the immune profile differed significantly between the two different HSV-2 disease manifestations. Meningitis patients were distinguished by the spontaneous production of several anti-viral immune factors by PBMC including type I and type III IFNs. PBMC from HSV-2 meningitis patients also secreted significantly higher levels of IFN-γ in response to HSV-2 compared to PBMC from HSV-2 genital herpes patients. Blocking the type I IFN receptor reduced the production of HSV-2-induced IFN-γ by PBMC suggesting that enhanced production of type I IFNs could promote IFN-γ recall responses. The levels of HSV-2 type-specific antibodies did not differ between the patient groups. In conclusion, we show that HSV-2 meningitis leads to a more profound activation of both innate and acquired PBMC immune responses, compared to that of HSV-2 genital herpes. Whether these differences are the cause, or the consequence, of the different disease manifestations remains to be determined.

The mechanisms underlying susceptibility to recurrent herpes simplex virus type 2 (HSV-2) meningitis remain incompletely understood. In a patient experiencing multiple episodes of HSV-2 meningitis, we identified a monoallelic variant in the IKBKE gene, which encodes the IKKε kinase involved in induction of antiviral IFN genes. Patient cells displayed impaired induction of IFN-β1 ( IFNB1 ) expression upon infection with HSV-2 or stimulation with double-stranded DNA (dsDNA) and failed to induce phosphorylation of STING, an activation marker of the DNA-sensing cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway. The patient allele encoded a truncated IKKε protein with loss of kinase activity and also capable of exerting dominant-negative activity. In stem cell–derived microglia, HSV-2–induced expression of IFNB1 was dependent on cGAS , TANK binding kinase 1 ( TBK1 ), and IKBKE , but not TLR3 , and supernatants from HSV-2–treated microglia exerted IKBKE -dependent type I IFN–mediated antiviral activity upon neurons. Reintroducing wild-type IKBKE into patient cells rescued IFNB1 induction following treatment with HSV-2 or dsDNA and restored antiviral activity. Collectively, we identify IKKε to be important for protection against HSV-2 meningitis and suggest a nonredundant role for the cGAS/STING pathway in human antiviral immunity.

Genetic determinants of stroke, the leading neurological cause of death and disability, are poorly understood and have seldom been explored in the general population. Our aim was to identify additional loci for stroke by doing a meta-analysis of genome-wide association studies. For the discovery sample, we did a genome-wide analysis of common genetic variants associated with incident stroke risk in 18 population-based cohorts comprising 84 961 participants, of whom 4348 had stroke. Stroke diagnosis was ascertained and validated by the study investigators. Mean age at stroke ranged from 45·8 years to 76·4 years, and data collection in the studies took place between 1948 and 2013. We did validation analyses for variants yielding a significant association (at p<5 × 10−6) with all-stroke, ischaemic stroke, cardioembolic ischaemic stroke, or non-cardioembolic ischaemic stroke in the largest available cross-sectional studies (70 804 participants, of whom 19 816 had stroke). Summary-level results of discovery and follow-up stages were combined using inverse-variance weighted fixed-effects meta-analysis, and in-silico lookups were done in stroke subtypes. For genome-wide significant findings (at p<5 × 10−8), we explored associations with additional cerebrovascular phenotypes and did functional experiments using conditional (inducible) deletion of the probable causal gene in mice. We also studied the expression of orthologs of this probable causal gene and its effects on cerebral vasculature in zebrafish mutants. We replicated seven of eight known loci associated with risk for ischaemic stroke, and identified a novel locus at chromosome 6p25 (rs12204590, near FOXF2) associated with risk of all-stroke (odds ratio [OR] 1·08, 95% CI 1·05–1·12, p=1·48 × 10−8; minor allele frequency 21%). The rs12204590 stroke risk allele was also associated with increased MRI-defined burden of white matter hyperintensity—a marker of cerebral small vessel disease—in stroke-free adults (n=21 079; p=0·0025). Consistently, young patients (aged 2–32 years) with segmental deletions of FOXF2 showed an extensive burden of white matter hyperintensity. Deletion of Foxf2 in adult mice resulted in cerebral infarction, reactive gliosis, and microhaemorrhage. The orthologs of FOXF2 in zebrafish (foxf2b and foxf2a) are expressed in brain pericytes and mutant foxf2b−/− cerebral vessels show decreased smooth muscle cell and pericyte coverage. We identified common variants near FOXF2 that are associated with increased stroke susceptibility. Epidemiological and experimental data suggest that FOXF2 mediates this association, potentially via differentiation defects of cerebral vascular mural cells. Further expression studies in appropriate human tissues, and further functional experiments with long follow-up periods are needed to fully understand the underlying mechanisms. NIH, NINDS, NHMRC, CIHR, European national research institutions, Fondation Leducq.

Efficient and specific delivery of mRNA to target tissues is critical for maximising therapeutic benefits while minimising off-target effects and systemic toxicity. Systemic administration of mRNA using lipid nanoparticles (LNPs) or extracellular vesicles (EVs) typically leads to predominant accumulation in the liver. We hypothesised that cardiac-specific EVs could promote enhanced relative cardiac enrichment of delivered mRNA compared with non-cardiac EVs or LNPs. In mice, intravenous administration of cardiac progenitor cell-derived EVs (CPC-EVs) achieved the greatest relative cardiac selectivity of modified mRNA encoding vascular endothelial growth factor A (VEGF-A) to the heart, with reduced liver accumulation relative to non-cardiac EVs and LNPs. Cytokine profiling across seven organs revealed that LNP delivery triggered a widespread pro-inflammatory response, whereas CPC-EVs elicited only a localised and limited cytokine activation, suggesting a more favourable safety profile. Furthermore, direct intramyocardial injection of CPC-EVs not only led to efficient mRNA uptake by cardiac tissue and robust VEGF-A protein expression, but also minimal transcriptomic perturbation in the cardiac tissue, as confirmed by RNA-seq. In contrast, LNPs and non-cardiac EVs induced widespread perturbation in the transcriptome of cardiac tissue. Functionally, VEGF-A mRNA delivery via CPC-EVs markedly increased CD31 and α-SMA expression and vessel formation in ex vivo aortic ring assays, confirming enhanced angiogenic potential. Together, these findings support CPC-EVs as a promising platform for achieving enhanced cardiac delivery of mRNA, with reduced liver accumulation, limited off-target transcriptomic perturbation, a more selective cytokine response, and enhanced angiogenic activity in ex vivo assays.

Targeted mRNA transport plays a crucial role in enhancing the therapeutic efficacy of the molecule, reducing its side effects, and minimizing off-target effects. Systemic administration of mRNA through lipid nanoparticles (LNPs) or extracellular vesicles (EVs) predominantly results in mRNA accumulation in the liver. We hypothesized that cardiac-specific EVs could more effectively target the transport of mRNA to the heart, in comparison to non-cardiac-specific EVs or LNPs. In mice, after intravenous administration, EVs from cardiac progenitor cells (CPC-EVs) were the most efficient to transport the modified mRNA, encoding vascular endothelial growth factor A (VEGF-A), to mouse heart, with minimal liver accumulation compared to non-cardiac-specific EVs or LNPs. Additionally, intracardiac injections of CPC-EVs not only demonstrate that they are the most adapted vehicle for interacting with heart tissue, delivering the mRNA to cells, and inducing maximal VEGF-A protein production, but RNA-seq analyses also revealed their minimal impact on overall gene expression, compared to LNPs or non-cardiac-specific EVs. Furthermore, immunofluorescence staining of CD31 and α-SMA, markers of microvascular density, showed increased vessel density in mouse aortic rings following the delivery of VEGF-A mRNA via CPC-EVs. These findings suggest that CPC-EVs are superior in mRNA targeting to heart, communication with cardiac cells, and causing minimal transcriptomic changes during VEGF-A mRNA delivery. Therefore, CPC-EVs could be promising vectors for heart-targeted mRNA delivery, potentially reducing liver accumulation.Competing Interest StatementThe authors have declared no competing interest.Footnotes* https://www.ncbi.nlm.nih.gov/geo/