IKAROS (encoded by IKZF1) is an important hematopoietic transcription factor critical for early B cell differentiation, with major defects known to lead to low B cell numbers and hypogammaglobulinemia. More perplexing is the link between IKZF1 variants and autoimmunity, including polymorphisms associated with susceptibility to SLE, and recently, rare variants driving monogenic autoimmunity. We identified a novel p.L188V mutation in IKZF1 in the index patient and her father and found this mutation to lead to loss of DNA binding. Peripheral B cells lacking a full complement of IKAROS function show upregulation of molecules accentuating B cell activation, while CD22, a key negative feedback circuit, is suppressed. The resulting hyperresponsiveness of peripheral B cells, in combination with elevated follicular helper T cell (Tfh) numbers, provides a putative mechanistic explanation for the association of IKZF1 variants with the emergence of autoimmune manifestations in this kindred.

The use of stably integrated reporter gene imaging provides a manner to monitor the in vivo fate of engrafted cells over time in a non-invasive manner. Here, we optimized multimodal imaging (small-animal PET, Cerenkov luminescence imaging (CLI) and bioluminescence imaging (BLI)) of mesenchymal stem cells (MSCs), by means of the human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) as reporters.

Transportin-SR2 (TRN-SR2, TNPO3, transportin 3) was previously identified as an interaction partner of human immunodeficiency virus type 1 (HIV-1) integrase and functions as a nuclear import factor of HIV-1. A possible role of capsid in transportin-SR2-mediated nuclear import was recently suggested by the findings that a chimeric HIV virus, carrying the murine leukemia virus (MLV) capsid and matrix proteins, displayed a transportin-SR2 independent phenotype, and that the HIV-1 N74D capsid mutant proved insensitive to transportin-SR2 knockdown.

LEDGINs are novel allosteric HIV integrase (IN) inhibitors that target the lens epithelium-derived growth factor (LEDGF)/p75 binding pocket of IN. They block HIV-1 integration by abrogating the interaction between LEDGF/p75 and IN as well as by allosterically inhibiting the catalytic activity of IN.

Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a transcriptional coactivator involved in stress response, autoimmune disease, cancer and HIV replication. A fusion between the nuclear pore protein NUP98 and LEDGF/p75 has been found in human acute and chronic myeloid leukemia and association of LEDGF/p75 with mixed-lineage leukemia (MLL)/menin is critical for leukemic transformation. During lentiviral replication, LEDGF/p75 tethers the pre-integration complex to the host chromatin resulting in a bias of integration into active transcription units (TUs). The consensus function of LEDGF/p75 is tethering of cargos to chromatin. In this regard, we determined the LEDGF/p75 chromatin binding profile. To this purpose, we used DamID technology and focused on the highly annotated ENCODE (Encyclopedia of DNA Elements) regions. LEDGF/p75 primarily binds downstream of the transcription start site of active TUs in agreement with the enrichment of HIV-1 integration sites at these locations. We show that LEDGF/p75 binding is not restricted to stress response elements in the genome, and correlation analysis with more than 200 genomic features revealed an association with active chromatin markers, such as H3 and H4 acetylation, H3K4 monomethylation and RNA polymerase II binding. Interestingly, some associations did not correlate with HIV-1 integration indicating that not all LEDGF/p75 complexes on the chromosome are amenable to HIV-1 integration.

The capacity to integrate transgenes into the host cell genome makes retroviral vectors an interesting tool for gene therapy. Although stable insertion resulted in successful correction of several monogenic disorders, it also accounts for insertional mutagenesis, a major setback in otherwise successful clinical gene therapy trials due to leukemia development in a subset of treated patients. Despite improvements in vector design, their use is still not risk-free. Lentiviral vector (LV) integration is directed into active transcription units by LEDGF/p75, a host-cell protein co-opted by the viral integrase. We engineered LEDGF/p75-based hybrid tethers in an effort to elicit a more random integration pattern to increase biosafety, and potentially reduce proto-oncogene activation. We therefore truncated LEDGF/p75 by deleting the N-terminal chromatin-reading PWWP-domain, and replaced this domain with alternative pan-chromatin binding peptides. Expression of these LEDGF-hybrids in LEDGF-depleted cells efficiently rescued LV transduction and resulted in LV integrations that distributed more randomly throughout the host-cell genome. In addition, when considering safe harbor criteria, LV integration sites for these LEDGF-hybrids distributed more safely compared to LEDGF/p75-mediated integration in wild-type cells. This approach should be broadly applicable to introduce therapeutic or suicide genes for cell therapy, such as patient-specific iPS cells.

Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy.

The bone morphogenetic protein receptor II (BMPRII) signaling pathway is impaired in pulmonary arterial hypertension and mutations in the BMPR2 gene have been observed in both heritable and idiopathic pulmonary arterial hypertension. However, all BMPR2 mutation carriers do not develop pulmonary arterial hypertension, and inflammation could trigger the development of the disease in BMPR2 mutation carriers. Circulating levels and/or lung tissue expression of cytokines such as tumor necrosis factor-α or interleukin-18 are elevated in patients with pulmonary arterial hypertension and could be involved in the pathogenesis of pulmonary arterial hypertension. We consequently hypothesized that cytokines could trigger endothelial dysfunction in addition to impaired BMPRII signaling. Our aim was to determine whether impairment of BMPRII signaling might affect endothelium barrier function and adhesiveness to monocytes, in response to cytokines. BMPR2 was silenced in human lung microvascular endothelial cells (HLMVECs) using lentiviral vectors encoding microRNA-based hairpins. Effects of tumor necrosis factor-α and interleukin-18 on HLMVEC adhesiveness to the human monocyte cell line THP-1, adhesion molecule expression, endothelial barrier function and activation of P38MAPK were investigated in vitro. Stable BMPR2 silencing in HLMVECs resulted in impaired endothelial barrier function and constitutive activation of P38MAPK. Adhesiveness of BMPR2-silenced HLMVECs to THP-1 cells was enhanced by tumor necrosis factor-α and interleukin-18 through ICAM-1 adhesion molecule. Interestingly, tumor necrosis factor-α induced activation of P38MAPK and disrupted endothelial barrier function in BMPR2-silenced HLMVECs. Altogether, our findings showed that stable BMPR2 silencing resulted in impaired endothelial barrier function and activation of P38MAPK in HLMVECs. In BMPR2-silenced HLMVECs, cytokines enhanced adhesiveness capacities, activation of P38MAPK and impaired endothelial barrier function suggesting that cytokines could trigger the development of pulmonary arterial hypertension in a context of impaired BMPRII signaling pathway.

Vaccination against measles, mumps, and rubella (MMR) and yellow fever (YF) with live attenuated viruses can rarely cause life-threatening disease. Severe illness by MMR vaccines can be caused by inborn errors of type I and/or III interferon (IFN) immunity (mutations in IFNAR2, STAT1, or STAT2). Adverse reactions to the YF vaccine have remained unexplained. We report two otherwise healthy patients, a 9-yr-old boy in Iran with severe measles vaccine disease at 1 yr and a 14-yr-old girl in Brazil with viscerotropic disease caused by the YF vaccine at 12 yr. The Iranian patient is homozygous and the Brazilian patient compound heterozygous for loss-of-function IFNAR1 variations. Patient-derived fibroblasts are susceptible to viruses, including the YF and measles virus vaccine strains, in the absence or presence of exogenous type I IFN. The patients' fibroblast phenotypes are rescued with WT IFNAR1 Autosomal recessive, complete IFNAR1 deficiency can result in life-threatening complications of vaccination with live attenuated measles and YF viruses in previously healthy individuals.

STAT1 gain-of-function (GOF) is a primary immunodeficiency typically characterized by chronic mucocutaneous candidiasis (CMC), recurrent respiratory infections, and autoimmunity. Less commonly, also immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX)-like syndromes with CMC, and combined immunodeficiency without CMC have been described. Recently, our group and others have shown that different mutation-specific mechanisms underlie STAT1 GOF in vitro, including faster nuclear accumulation (R274W), and reduced mobility (R321, N574I) to near immobility in the nucleus (T419R) upon IFNγ stimulation. In this work, we evaluated the transcriptomic fingerprint of the aforementioned STAT1 GOF mutants (R274W, R321S, T419R, and N574I) relative to STAT1 wild-type upon IFNγ stimulation in an otherwise isogenic cell model. The majority of genes up-regulated in wild-type STAT1 cells were significantly more up-regulated in cells expressing GOF mutants, except for T419R. In addition to the common interferon regulated genes (IRG), STAT1 GOF mutants up-regulated an additional set of genes, that were in part shared with other GOF mutants or mutation-specific. Overall, R274W and R321S transcriptomes clustered with STAT1 WT, while T419R and N574I had a more distinct fingerprint. We observed reduced frequency of canonical IFNγ activation site (GAS) sequences in promoters of genes up-regulated by all the STAT1 GOF mutants, suggesting loss of DNA binding specificity for the canonical GAS consensus. Interestingly, the T419R mutation, expected to directly increase the affinity for DNA, showed the most pronounced effects on the transcriptome. T419R STAT1 dysregulated more non-IRG than the other GOF mutants and fewer GAS or degenerate GAS promotor sequences could be found in the promoter regions of these genes. In conclusion, our work confirms hyperactivation of common sets of IFNγ-induced genes in STAT1 GOF with additional dysregulation of mutation-specific genes, in line with the earlier observed mutation-specific mechanisms. Binding to more degenerate GAS sequences is proposed as a mechanism toward transcriptional dysregulation in R274W, R321S, and N574I. For T419R, an increased interaction with the DNA is suggested to result in a broader and less GAS-specific response. Our work indicates that multiple routes leading to STAT1 GOF are associated with common and private transcriptomic fingerprints, which may contribute to the phenotypic variation observed in vivo.

To develop safer retroviral murine leukemia virus (MLV)-based vectors, we previously mutated and re-engineered the MLV integrase: the W390A mutation abolished the interaction with its cellular tethering factors, BET proteins, and a retargeting peptide (the chromodomain of the CBX1 protein) was fused C-terminally. The resulting BET-independent MLVW390A-CBX was shown to integrate efficiently and more randomly, away from typical retroviral markers. In this study, we assessed the functionality and stability of expression of the redistributed MLVW390A-CBX vector in more depth, and evaluated safety using a clinically more relevant vector design encompassing a self-inactivated (SIN) LTR and a weak internal elongation factor 1α short (EFS) promoter. MLVW390A-CBX-EFS produced like MLVWT and efficiently transduced laboratory cells and primary human CD34+ hematopoetic stem cells (HSC) without transgene silencing over time, while displaying a more preferred, redistributed, and safer integration pattern. In a human mesoangioblast (MAB) stem cell model, the myogenic fusion capacity was hindered following MLVWT transduction, while this remained unaffected when applying MLVW390A-CBX. Likewise, smooth muscle cell differentiation of MABs was unaltered by MLVW390A-CBX-EFS. Taken together, our results underscore the potential of MLVW390A-CBX-EFS as a clinically relevant viral vector for ex-vivo gene therapy, combining efficient production with a preferable integration site distribution profile and stable expression over time.

Vaginal birth causes pelvic floor injury which may lead to urinary incontinence. Cell therapy has been proposed to assist in functional recovery. We aim to assess if intra-arterial injection of rat mesoangioblasts (MABs) and stable Vascular Endothelial Growth Factor (VEGF)-expressing MABs, improve recovery of urethral and vaginal function following simulated vaginal delivery (SVD). Female rats (n = 86) were assigned to either injection of saline (control), allogeneic-MABs (MABsallo), autologous-MABs (MABsauto) or allogeneic-MABs transduced to stably expressed VEGF (MABsallo-VEGF). One hour after SVD, 0.5 × 106 MABs or saline were injected into the aorta. Primary outcome was urethral (7d and 14d) and vaginal (14d) function; others were bioluminescent imaging for cell tracking (1, 3 and 7d), morphometry (7, 14 and 60d) and mRNAseq (3 and 7d). All MABs injected rats had external urethral sphincter and vaginal function recovery within 14d, as compared to only half of saline controls. Functional recovery was paralleled by improved muscle regeneration and microvascularization. Recovery rate was not different between MABsallo and MABsauto. MABsallo-VEGF accelerated functional recovery and increased GAP-43 expression at 7d. At 3d we detected major transcriptional changes in the urethra of both MABsallo and MABsallo-VEGF-injected animals, with upregulation of Rho/GTPase activity, epigenetic factors and dendrite development. MABSallo also upregulated transcripts that encode proteins involved in myogenesis and downregulated pro-inflammatory processes. MABsallo-VEGF also upregulated transcripts that encode proteins involved in neuron development and downregulated genes involved in hypoxia and oxidative stress. At 7d, urethras of MABsallo-VEGF-injected rats showed downregulation of oxidative and inflammatory response compared to MABSallo. Intra-arterial injection of MABsallo-VEGF enhances neuromuscular regeneration induced by untransduced MABs and accelerates the functional urethral and vaginal recovery after SVD.

Fetal gene therapy is one of the possible new therapeutic strategies for congenital or perinatal diseases with high mortality or morbidity. We developed a novel delivery strategy to inject directly into the fetal mouse trachea. Intratracheal (i.t.) injection at embryonic day 18 (E18) was more efficient in targeting the fetal lung than conventional intra-amniotic (i.a.) delivery. Viral vectors derived from adeno-associated virus serotype 6.2, with tropism for the airway epithelium and not earlier tested in the fetal mouse lung, were injected into the fetal trachea. Bioluminescence (BL) imaging (BLI) was combined with magnetic resonance (MR) imaging (MRI) for noninvasive and accurate localization of transgene expression in vivo. Histological analysis for β-galactosidase (β-gal) revealed 17.5% of epithelial cells transduced in the conducting airways and 1.5% in the alveolar cells. Stable gene expression was observed up to 1 month after injection. This study demonstrates that direct injection of rAAV2/6.2 in the fetal mouse trachea is superior to i.a. delivery for transducing the lung. Second, as stable gene transfer was detected up to 1 postnatal month, this approach may be useful to evaluate fetal gene therapy for pulmonary diseases such as cystic fibrosis, requiring both substantial numbers of transduced cells as well as prolonged gene expression to obtain a stable phenotypic effect.

Employing viruses as nanoscopic lipid-enveloped test tubes allows the miniaturization of protein-protein interaction (PPI) assays while preserving the physiological environment necessary for particular biological processes. Applied to the study of the human immunodeficiency virus type 1 (HIV-1), viral biology and pathology can also be investigated in novel ways, both in vitro as well as in infected cells. In this work we report on an experimental strategy that makes use of engineered HIV-1 viral particles, to allow for probing PPIs of the HIV-1 integrase (IN) inside viruses with single-molecule Förster resonance energy transfer (FRET) using fluorescent proteins (FP). We show that infectious fluorescently labeled viruses can be obtained and that the quantity of labels can be accurately measured and controlled inside individual viral particles. We demonstrate, with proper control experiments, the formation of IN oligomers in single viral particles and inside viral complexes in infected cells. Finally, we show a clear effect on IN oligomerization of small molecule inhibitors of interactions of IN with its natural human cofactor LEDGF/p75, corroborating that IN oligomer enhancing drugs are active already at the level of the virus and strongly suggesting the presence of a dynamic, enhanceable equilibrium between the IN dimer and tetramer in viral particles. Although applied to the HIV-1 IN enzyme, our methodology for utilizing HIV virions as nanoscopic test tubes for probing PPIs is generic, i.e., other PPIs targeted into the HIV-1, or PPIs targeted into other viruses, can potentially be studied with a similar strategy.

Testing of new therapeutic strategies for Parkinson's disease (PD) is currently hampered by the lack of relevant and reproducible animal models. Here, we developed a robust rat model for PD by injection of adeno-associated viral vectors (rAAV2/7) encoding α-synuclein into the substantia nigra, resulting in reproducible nigrostriatal pathology and behavioral deficits in a 4-week time period. Progressive dopaminergic dysfunction was corroborated by histopathologic and biochemical analysis, motor behavior testing and in vivo microdialysis. L-DOPA treatment was found to reverse the behavioral phenotype. Non-invasive positron emission tomography imaging and magnetic resonance spectroscopy allowed longitudinal monitoring of neurodegeneration. In addition, insoluble α-synuclein aggregates were formed in this model. This α-synuclein rat model shows improved face and predictive validity, and therefore offers the possibility to reliably test novel therapeutics. Furthermore, it will be of great value for further research into the molecular pathogenesis of PD and the importance of α-synuclein aggregation in the disease process.

Lens epithelium-derived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase (IN) that interacts with IN through its IN binding domain (IBD) and tethers the viral pre-integration complex to the host cell chromatin. Here we report the generation of a human somatic LEDGF/p75 knockout cell line that allows the study of spreading HIV-1 infection in the absence of LEDGF/p75. By homologous recombination the exons encoding the LEDGF/p75 IBD (exons 11 to 14) were knocked out. In the absence of LEDGF/p75 replication of laboratory HIV-1 strains was severely delayed while clinical HIV-1 isolates were replication-defective. The residual replication was predominantly mediated by the Hepatoma-derived growth factor related protein 2 (HRP-2), the only cellular protein besides LEDGF/p75 that contains an IBD. Importantly, the recently described IN-LEDGF/p75 inhibitors (LEDGINs) remained active even in the absence of LEDGF/p75 by blocking the interaction with the IBD of HRP-2. These results further support the potential of LEDGINs as allosteric integrase inhibitors.

The interaction between the human immunodeficiency virus (HIV) integrase (IN) and its cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is crucial for HIV replication. While recently discovered LEDGINs inhibit HIV-1 replication by occupying the LEDGF/p75 pocket in IN, it remained to be demonstrated whether LEDGF/p75 by itself can be targeted. By phage display we identified cyclic peptides (CPs) as the first LEDGF/p75 ligands that inhibit the LEDGF/p75-IN interaction. The CPs inhibit HIV replication in different cell lines without overt toxicity. In accord with the role of LEDGF/p75 in HIV integration and its inhibition by LEDGINs, CP64, and CP65 block HIV replication primarily by inhibiting the integration step. The CPs retained activity against HIV strains resistant to raltegravir or LEDGINs. Saturation transfer difference (STD) NMR showed residues in CP64 that strongly interact with LEDGF/p75 but not with HIV IN. Mutational analysis identified tryptophan as an important residue responsible for the activity of the peptides. Serial passaging of virus in the presence of CPs did not yield resistant strains. Our work provides proof-of-concept for direct targeting of LEDGF/p75 as novel therapeutic strategy and the CPs thereby serve as scaffold for future development of new HIV therapeutics.

Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations result in a primary immunodeficiency (PID) characterized typically by chronic mucocutaneous candidiasis (CMC), but a wider phenotypic range is reported and remains unexplained from a pathophysiological point-of-view. We hypothesized that different STAT1 GOF mutations may result in distinct molecular mechanisms, possibly explaining the variable phenotypes observed in patients. We selected STAT1 GOF mutants (R274W, R321S, T419R, and N574I) that are spread over the protein and studied their dynamic behavior in vitro in U3A and HeLa cell lines. All GOF mutants showed increased STAT1 phosphorylation compared to STAT1 WT. Real-time imaging demonstrated three underlying mechanisms for STAT1 GOF: (i) R274W showed a faster nuclear accumulation, (ii) both R321S and N574I showed a reduced nuclear mobility and slower dephosphorylation, whereas (iii) T419R was near-immobile in the nucleus, potentially due to enhanced binding to chromatin.

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9-Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.

Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of IFN-γ immunity. The most frequent genetic defects are found in IL12 or a subunit of its receptor. IL23R deficiency in MSMD has only been reported once, in two pediatric patients from the same kindred with isolated disseminated Bacille Calmette-Guérin disease. We evaluated the impact of a homozygous stop mutation in IL23R (R381X), identified by whole exome sequencing, in an adult patient with disseminated non-tuberculous mycobacterial disease.