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Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice. Synopsis: Lentiviral vectors bearing the baculovirus GP64 surface protein and high content of the phagocytosis inhibitor CD47 efficiently transduce liver endothelial cells in vivo and allow stable long-term correction of mice with hemophilia A, both when treated as adults and as newborns. Development of an in vitro phagocytosis assay, exploiting fluorescent virions and advanced imaging techniques, suitable to assess the susceptibility to phagocytosis of different LV versions in different experimental settings. Efficient tropism switch of LV from hepatocytes to liver endothelial cells in vitro and in vivo and shielding from phagocytosis. Reconstitution of FVIII activity in the normal range by a single intravenous administration of GP64-LV equipped with the endogenous endothelial-specific FVIII promoter. Proof-of-concept of stable long-term gene therapy for hemophilia A in both adult and newborn mice. Lentiviral vectors bearing the baculovirus GP64 surface protein and high content of the phagocytosis inhibitor CD47 efficiently transduce liver endothelial cells in vivo and allow stable long-term correction of mice with hemophilia A, both when treated as adults and as newborns. [ABSTRACT FROM AUTHOR]

Fragile X syndrome (FXS) is the most frequent form of familial intellectual disability. FXS results from the lack of the RNA‐binding protein FMRP and is associated with the deregulation of signaling pathways downstream of mGluRI receptors and upstream of mRNA translation. We previously found that diacylglycerol kinase kappa (DGKk), a main mRNA target of FMRP in cortical neurons and a master regulator of lipid signaling, is downregulated in the absence of FMRP in the brain of Fmr1‐KO mouse model. Here we show that adeno‐associated viral vector delivery of a modified and FMRP‐independent form of DGKk corrects abnormal cerebral diacylglycerol/phosphatidic acid homeostasis and FXS‐relevant behavioral phenotypes in the Fmr1‐KO mouse. Our data suggest that DGKk is an important factor in FXS pathogenesis and provide preclinical proof of concept that its replacement could be a viable therapeutic strategy in FXS. Synopsis: Brain neuron‐restricted expression of an FMRP‐independent DGKk enzyme via intra‐cerebral adeno‐associated virus (AAV) gene therapy corrects phosphatidic acid deficiency and rescues disease relevant behavioral phenotypic alterations in a mouse model of Fragile X syndrome (FXS). The expression of diacylglycerol kinase kappa (DGKk), a favored mRNA target of FMRP, is reduced in the FXS brain in both humans and mice.∆N‐DGKk is a truncated active enzyme whose synthesis bypasses the need for FMRP.AAV‐Rh10‐∆N‐DGKk enables the synthesis of active DGKk enzyme in neurons lacking FMRP.∆N‐DGKk neuronal expression has no adverse effect in wild type control mice and is associated with normal behavioral functions of the rescued Fmr1‐null mice. [ABSTRACT FROM AUTHOR]

Direct reprogramming based on genetic factors resembles a promising strategy to replace lost cells in degenerative diseases such as Parkinson's disease. For this, we developed a knock‐in mouse line carrying a dual dCas9 transactivator system (dCAM) allowing the conditional in vivo activation of endogenous genes. To enable a translational application, we additionally established an AAV‐based strategy carrying intein‐split‐dCas9 in combination with activators (AAV‐dCAS). Both approaches were successful in reprogramming striatal astrocytes into induced GABAergic neurons confirmed by single‐cell transcriptome analysis of reprogrammed neurons in vivo. These GABAergic neurons functionally integrate into striatal circuits, alleviating voluntary motor behavior aspects in a 6‐OHDA Parkinson's disease model. Our results suggest a novel intervention strategy beyond the restoration of dopamine levels. Thus, the AAV‐dCAS approach might enable an alternative route for clinical therapies of Parkinson's disease. Synopsis: GABAergic neurons generated by CRISPR‐mediated direct reprogramming of striatal astrocytes rescue voluntary motor behavior in a toxin‐induced murine model for Parkinson's disease, suggesting a novel intervention strategy beyond the restoration of dopamine levels. A novel CRISPRa mouse line dCAM is developed for the conditional induction of endogenous target genes.An AAV‐based split‐dCas9‐activator system is established for translational applications of CRISPRa.Direct reprogramming of murine striatal astrocytes using the factor combination Ascl1, Lmx1a, and Nr4a2 results in induced GABAergic neurons in vivo.Induced GABAergic neurons are capable of ameliorating specific motor symptoms of Parkinson's disease. [ABSTRACT FROM AUTHOR]

Pompe disease is a severe disorder caused by loss of acid α‐glucosidase (GAA), leading to glycogen accumulation in tissues and neuromuscular and cardiac dysfunction. Enzyme replacement therapy is the only available treatment. AT845 is an adeno‐associated viral vector designed to express human GAA specifically in skeletal muscle and heart. Systemic administration of AT845 in Gaa−/− mice led to a dose‐dependent increase in GAA activity, glycogen clearance in muscles and heart, and functional improvement. AT845 was tolerated in cynomolgus macaques at low doses, while high doses caused anti‐GAA immune response, inflammation, and cardiac abnormalities resulting in unscheduled euthanasia of two animals. Conversely, a vector expressing the macaque GAA caused no detectable pathology, indicating that the toxicity observed with AT845 was an anti‐GAA xenogeneic immune response. Western blot analysis showed abnormal processing of human GAA in cynomolgus muscle, adding to the species‐specific effects of enzyme expression. Overall, these studies show that AAV‐mediated GAA delivery to muscle is efficacious in Gaa−/− mice and highlight limitations in predicting the toxicity of AAV vectors encoding human proteins in non‐human species. Synopsis: Muscle‐restricted expression of GAA via systemic AAV gene therapy clears glycogen accumulation and improves function in a mouse model of Pompe disease. Xenogeneic immune responses in non‐human primates highlight limitations of animal studies to assess toxicity of vectors encoding human proteins. AT845 is an adeno‐associated viral vector designed to express human GAA specifically in skeletal muscle and heart.Systemic administration of AT845 in Gaa−/− mice led to a dose‐dependent increase in GAA expression and activity, glycogen clearance in skeletal and cardiac muscles, and functional improvement.In non‐human primates, AT845 caused a dose‐dependent increase of GAA activity in skeletal muscle and heart.Our results suggest that adverse reactions observed in non‐human primates with human GAA were due to an anti‐GAA xenogeneic immune response rather than GAA overexpression. [ABSTRACT FROM AUTHOR]

Optogenetic stimulation of spiral ganglion neurons (SGNs) in the ear provides a future alternative to electrical stimulation used in current cochlear implants. Here, we employed fast and very fast variants of the red‐light‐activated channelrhodopsin (ChR) Chrimson (f‐Chrimson and vf‐Chrimson) to study their utility for optogenetic stimulation of SGNs in mice. The light requirements were higher for vf‐Chrimson than for f‐Chrimson, even when optimizing membrane expression of vf‐Chrimson by adding potassium channel trafficking sequences. Optogenetic time and intensity coding by single putative SGNs were compared with coding of acoustic clicks. vf‐Chrimson enabled putative SGNs to fire at near‐physiological rates with good temporal precision up to 250 Hz of stimulation. The dynamic range of SGN spike rate coding upon optogenetic stimulation was narrower than for acoustic clicks but larger than reported for electrical stimulation. The dynamic range of spike timing, on the other hand, was more comparable for optogenetic and acoustic stimulation. In conclusion, f‐Chrimson and vf‐Chrimson are promising candidates for optogenetic stimulation of SGNs in auditory research and future cochlear implants. Synopsis: Identifying suitable channelrhodopsins is crucial for future optogenetic restoration of sound encoding by optical cochlear implants. Here, fast and very fast light‐activated Chrimsons were compared for their utility to optogenetically encode timing and intensity information in the auditory nerve. Very fast Chrimson increases temporal fidelity but confers lower light sensitivity of optogenetic auditory nerve fiber stimulation compared with fast Chrimson.Adding trafficking sequences of the inwardly rectifying potassium channel 2.1 improved plasma membrane expression of very fast Chrimson enabling shorter stimulus durationsThe dynamic range, based on the discharge rate, of optogenetic auditory nerve fiber stimulation was narrower than that of acoustic stimulation.The dynamic range, based on temporal precision of spiking, of optogenetic auditory nerve fiber stimulation was broader than that based on discharge rate. [ABSTRACT FROM AUTHOR]

Novel therapies for hemophilia, including non‐factor replacement and in vivo gene therapy, are showing promising results in the clinic, including for patients having a history of inhibitor development. Here, we propose a novel therapeutic approach for hemophilia based on llama‐derived single‐domain antibody fragments (sdAbs) able to restore hemostasis by inhibiting the antithrombin (AT) anticoagulant pathway. We demonstrated that sdAbs engineered in multivalent conformations were able to block efficiently AT activity in vitro, restoring the thrombin generation potential in FVIII‐deficient plasma. When delivered as a protein to hemophilia A mice, a selected bi‐paratopic sdAb significantly reduced the blood loss in a model of acute bleeding injury. We then packaged this sdAb in a hepatotropic AAV8 vector and tested its safety and efficacy profile in hemophilic mouse models. We show that the long‐term expression of the bi‐paratopic sdAb in the liver is safe and poorly immunogenic, and results in sustained correction of the bleeding phenotype in hemophilia A and B mice, even in the presence of inhibitory antibodies to the therapeutic clotting factor. Synopsis: This study presents a novel therapeutic approach for hemophilia A and B, with and without inhibitors. By using single‐domain antibodies targeting the anticoagulant antithrombin, a sustained correction of the in vivo bleeding phenotype was achieved following protein or AAV8‐based gene therapy. Bi‐paratopic single domain antibodies (sdAbs) have been generated that neutralized the anticoagulant protein antithrombin.Neutralization of antithrombin normalized thrombin generation in hemophilic plasma, irrespective of the presence of inhibitory antibodies.Liver expression of a bivalent sdAb restored clotting in hemophilia A and in hemophilia B mice, even in the presence of anti‐FIX antibodies. [ABSTRACT FROM AUTHOR]

Gene therapy critically relies on vectors that combine high transduction efficiency with a high degree of target specificity and that can be administered through a safe intravenous route. The lack of suitable vectors, especially for gene therapy of brain disorders, represents a major obstacle. Therefore, we applied an in vivo screening system of random ligand libraries displayed on adeno-associated viral capsids to select brain-targeted vectors for the treatment of neurovascular diseases. We identified a capsid variant showing an unprecedented degree of specificity and long-lasting transduction efficiency for brain microvasculature endothelial cells as the primary target of selection. A therapeutic vector based on this selected viral capsid was used to markedly attenuate the severe cerebrovascular pathology of mice with incontinentia pigmenti after a single intravenous injection. Furthermore, the versatility of this selection system will make it possible to select ligands for additional in vivo targets without requiring previous identification of potential target-specific receptors. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor 2 (IGF2) was recently found to play a critical role in memory consolidation in rats and mice, and hippocampal or systemic administration of recombinant IGF2 enhances memory. Here, using a gene therapy-based approach with adeno-associated virus ( AAV), we show that IGF2 overexpression in the hippocampus of aged wild-type mice enhances memory and promotes dendritic spine formation. Furthermore, we report that IGF2 expression decreases in the hippocampus of patients with Alzheimer's disease, and this leads us to hypothesize that increased IGF2 levels may be beneficial for treating the disease. Thus, we used the AAV system to deliver IGF2 or IGF1 into the hippocampus of the APP mouse model Tg2576 and demonstrate that IGF2 and insulin-like growth factor 1 (IGF1) rescue behavioural deficits, promote dendritic spine formation and restore normal hippocampal excitatory synaptic transmission. The brains of Tg2576 mice that overexpress IGF2 but not IGF1 also show a significant reduction in amyloid levels. This reduction probably occurs through an interaction with the IGF2 receptor (IGF2R). Hence, IGF2 and, to a lesser extent, IGF1 may be effective treatments for Alzheimer's disease. [ABSTRACT FROM AUTHOR]

The function of nuclear receptor corepressor 1 (NCoR1) in cardiomyocytes is unclear, and its physiological and pathological implications are unknown. Here, we found that cardiomyocyte‐specific NCoR1 knockout (CMNKO) mice manifested cardiac hypertrophy at baseline and had more severe cardiac hypertrophy and dysfunction after pressure overload. Knockdown of NCoR1 exacerbated whereas overexpression mitigated phenylephrine‐induced cardiomyocyte hypertrophy. Mechanistic studies revealed that myocyte enhancer factor 2a (MEF2a) and MEF2d mediated the effects of NCoR1 on cardiomyocyte hypertrophy. The receptor interaction domains (RIDs) of NCoR1 interacted with MEF2a to repress its transcriptional activity. Furthermore, NCoR1 formed a complex with MEF2a and class IIa histone deacetylases (HDACs) to suppress hypertrophy‐related genes. Finally, overexpression of RIDs of NCoR1 in the heart attenuated cardiac hypertrophy and dysfunction induced by pressure overload. In conclusion, NCoR1 cooperates with MEF2 and HDACs to repress cardiac hypertrophy. Targeting NCoR1 and the MEF2/HDACs complex may be an attractive therapeutic strategy to tackle pathological cardiac hypertrophy. Synopsis: Cardiac hypertrophy constitutes an important risk for heart failure. In this study, NCoR1 was identified as a critical suppressor of cardiac hypertrophy via interaction with MEF2/HDACs. NCoR1 deficiency in cardiomyocytes led to spontaneous cardiac hypertrophy in mice, and exacerbated pressure overload‐induced cardiac hypertrophy and heart failure.NCoR1 was recruited to the promoter region of hypertrophy‐related genes to suppress their expression though interacting with MEF2/Class IIa HDACs.The NCoR1 receptor interaction domain mediated NCoR1/MEF2 interaction and was sufficient to repress cardiomyocyte hypertrophy both in vitro and in vivo. [ABSTRACT FROM AUTHOR]