The authors regret that the original legends for Figure 3, Supplementary Figure 2 and Supplementary Figure 5 contained errors. The correct legends should read: Figure 3. Cell-type specific rescue of function. (A) Schematic representations of the misPiggy CRISPR/Cas9 plasmid containing the sgRNA targeting exon 2 of Xenopus tropicalis Dcc and the rescue plasmid containing a CRISPR resistant human DCC sequence fused to a hemagglutinin (HA) tag. (B) Schematic representation of the experimental strategy. The eye primordium was co-injected with the misPiggy CRISPR/Cas9 plasmid and the rescue plasmid expressing human DCC. Injections and electroporation were performed at Nieuwkoop and Faber (NF) stage 28; analysis of RGC axon arborization was performed at NF stage 46. RGC soma (magenta) are located in the eye and axons project contralaterally to their target regions in the tectum (black square), where they develop a highly branched terminal arborization. (C–K) Analysis of RGCs at the tectum in NF stage 46 animals, showing axonal branches from a single RGC expressing TdTomato (TdT) (magenta; C, F, I) and HA-tagged DCC (green; D, G, J); colocalized staining is shown in the merged image (white; E, H, K). The tectum is delineated with a white dotted line. (L-N) Quantification of the length of axon branches (L), total branch number (M) and total branch area (N) in control and rescue conditions. **p < 0.005, ***p < 0.001, ****p < 0.0001. Supplementary Figure 2. Cell-type specific loss of function of Dcc using misPiggy CRISPR/Cas9 in Xenopus tropicalis. (A) Schematic illustrating the single guide RNA (sgRNA) targeting exon 2, exon 3 or exon 4 of the Deleted in Colorectal Cancer (Dcc) gene in Xenopus tropicalis. (B) Multiple bicistronic misPiggy plasmids were produced, each carrying a specific sgRNA targeting independent exons of Xenopus tropicalis Dcc, or a non-target sgRNA which was used as a control. In all cases, sgRNA expression was driven by the ubiquitously active U6 promoter: To achieve LoF in RGC neurons, Cas9 expression was driven by the cell-type specific Crest3 enhancer, with simultaneous expression of TdTomato. (C-J) misPiggy CRISPR/Cas9 plasmids were injected in the eye primordium at NF stage 28 followed by electroporation, resulting in labelling of single RGC. Axon arborization was analyzed at NF stage 46 (see Figure 2). Images of single RGC axon branches at the tectum (dotted white lines) of animals injected with misPiggy CRISPR/Cas9 expressing non-targeting sgRNA (C, D), or sgRNAs targeting X. tropicalis Dcc at exon 2 (E, F), exon 3 (G, H), or exon 4 (I, J). Scale bars: 50 μm. Quantifications are shown in Figure 2. (K) misPiggy CRISPR/Cas9 plasmids containing sgRNAs targeting Dcc exon 2, exon 3 or exon 4 were co-injected with additional Cas9 protein at the 1 cell stage. Protein lysates were then prepared at NF stage 25 to test knock-down of DCC using Western blotting. Injected embryos show a decrease of DCC for each of the three gRNAs used, compared to wild type (WT) embryos. (L) Graph of DCC relative intensity normalized to beta-tubulin for each condition in (K). Supplementary Figure 5. Clonal analysis experiments using misPiggy. (Top) Schematic of the misPiggy plasmids used for transposon-based lineage tracing. Plasmids share a common design with use of a GFAP-promoter to drive fluorescent protein expression in astrocytes. Constructs expressed one of the following fluorescent proteins, mCherry, EYFP, EGFP or mCerulean in either a membrane labelling (kmyr-tag) or nuclear labelling (nls-) form. (Bottom) Specific fluorescent fingerprints label populations of clonally related astrocytes. A mixture of misPiggy plasmids encoding various fluorescent proteins was injected into mice and electroporated at E16. Samples were collected at P21. Due to the stochastic nature of plasmid uptake, progenitors acquire a unique combination of plasmids during electroporation, which is stably inherited by their daughter cells. Hence, clonally related astrocytes can be determined by a unique shared spectral fingerprint: (left) merge image showing the complex spectral fingerprints defining small clusters of clonally related astrocytes, (right) individual color channels used to make the merged image, showing differential fluorescent protein expression between individual cells. Scale bar 50 μm (left), 100 μm (right). Data are representative images seen in a minimum of 3 slices from 3 independent animals for each experiment. The authors would like to apologise for any inconvenience caused. A New Technical Approach for Cross-species Examination of Neuronal Wiring and Adult Neuron-glia FunctionsNeuroscienceVol. 508PreviewComparative analysis across model systems has been extremely powerful in elucidating the principles of CNS development and function. For example, studies on the development of the retino-tectal system in frogs and chickens have been instrumental in developing the concept of topographic maps (Cowan et al., 1961; De Long and Coulombre, 1965; Holt and Harris, 1983), and established important concepts regarding chemotactic guidance and the role of complementary gradients of Eph receptors and ephrin ligands across multiple cell types (Cheng et al., 1995; Yates et al., 2001; Mann et al., 2002). Full-Text PDF Open Access

Neutralizing antibody (NAb) activity against the viral capsid of adeno-associated viral (AAV) vectors decreases transduction efficiency, thus limiting transgene expression. Several reports have mentioned a variation in NAb prevalence according to age, AAV serotype, and, most importantly, geographic location. There are currently no reports specifically describing the anti-AAV NAb prevalence in Latin America. Here, we describe the prevalence of NAb against different serotypes of AAV vectors (AAV1, AAV2, and AAV9) in Colombian patients with heart failure (HF) (referred to as cases) and healthy individuals (referred to as controls). The levels of NAb were evaluated in serum samples of 60 subjects from each group using an in vitro inhibitory assay. The neutralizing titer was reported as the first dilution inhibiting ≥50% of the transgene signal, and the samples with neutralizing titers at ≥1:50 dilution were considered positive. The prevalence of NAb in the case and control groups were similar (AAV2: 43% and 45%, respectively; AAV1 33.3% in each group; AAV9: 20% and 23.2%, respectively). The presence of NAb for two or more of the serotypes analyzed was observed in 25% of the studied samples, with the largest amount in the positive samples for AAV1 (55–75%) and AAV9 (93%), suggesting serial exposures, cross-reactivity, or coinfection. Moreover, patients in the HF group exhibited more common combined seropositivity for NAb against AAV1 d AAV9 than those in the control group (91.6% vs. 35.7%, respectively; p = 0.003). Finally, exposure to toxins was significantly associated with the presence of NAb in all regression models. These results constitute the first report of the prevalence of NAb against AAV in Latin America, being the first step to implementing therapeutic strategies based on AAV vectors in this population in our region.

Advances in single cell sequencing have enabled the identification of a large number of genes, expressed in many different cell types, and across a variety of model organisms. In particular, the nervous system harbors an immense number of interacting cell types, which are poorly characterized. Future loss- and gain-of-function experiments will be essential in determining how novel genes play critical roles in diverse cellular, as well as evolutionarily adapted, contexts. However, functional analysis across species is often hampered by technical limitations, in non-genetic animal systems. Here, we describe a new single plasmid system, misPiggy. The system is based around the hyperactive piggyBac transposon system, which combines stable genomic integration of transgenes (for long-term expression) with large cargo capacity. Taking full advantage of these characteristics, we engineered novel expression modules into misPiggy that allow for cell-type specific loss- and gain-of-gene function. These modules work widely across species from frog to ferret. As a proof of principle, we present a loss-of-function analysis of the neuronal receptor Deleted in Colorectal Cancer (DCC) in retinal ganglion cells (RGCs) of Xenopus tropicalis tadpoles. Single axon tracings of mosaic knock-out cells reveal a specific cell-intrinsic requirement of DCC, specifically in axonal arborization within the frog tectum, rather than retina-to-brain axon guidance. Furthermore, we report additional technical advances that enable temporal control of knock-down or gain-of-function analysis. We applied this to visualize and manipulate labeled neurons, astrocytes and other glial cells in the central nervous system (CNS) of mouse, rat and ferret. We propose that misPiggy will be a valuable tool for rapid, flexible and cost-effective screening of gene function across a variety of animal models.

Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood–brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB‐crossing adeno‐associated virus (AAV)‐based vectors to deliver VHH directly into the CNS. As a proof‐of‐concept, we explored the potential of AAV‐delivered VHH to inhibit BACE1, a well‐characterized target in Alzheimer’s disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH‐B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV‐VHH‐B9 produces positive long‐term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the AppNL‐G‐F Alzheimer’s mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets. VHH and blood‐brain‐barrier (BBB)‐crossing AAV‐based vectors are combined to achieve highly‐specific, long‐term BACE1 inhibition in a mouse model of Alzheimer's disease (AD). VHH and blood‐brain‐barrier (BBB)‐crossing AAV‐based vectors are combined to achieve highly‐specific, long‐term BACE1 inhibition in a mouse model of Alzheimer's disease (AD).

Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells1–4. Macrophages and satellite cells communicate in different ways1–5, but their metabolic interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1-knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles. Mouse models of muscle injuries and ageing characterized by low levels of intra-tissue glutamine are ameliorated by macrophage-specific deletion or systemic pharmacological inhibition of glutamate dehydrogenase 1, which results in constitutively high activity of glutamine synthetase.

Chagas disease (CD) is one of the most important neglected tropical diseases in the American continent. Host-derived nitroxidative stress in response to Trypanosoma cruzi infection can induce tissue damage contributing to the progression of Chagas disease. Antioxidant supplementation has been suggested as adjuvant therapy to current treatment. In this article, we synthesize and discuss the current evidence regarding the use of antioxidants as adjunctive compounds to fight harmful reactive oxygen species and lower the tissue oxidative damage during progression of chronic Chagas disease. Several antioxidants evaluated in recent studies have shown potential benefits for the control of oxidative stress in the host's tissues. Melatonin, resveratrol, the combination of vitamin C/vitamin E (vitC/vitE) or curcumin/benznidazole, and mitochondria-targeted antioxidants seem to be beneficial in reducing plasma and cardiac levels of lipid peroxidation products. Nevertheless, further research is needed to validate beneficial effects of antioxidant therapies in Chagas disease.

There is an urgent need to develop the next-generation vectors for gene therapy of muscle disorders, given the relatively modest advances in clinical trials. These vectors should express substantially higher levels of the therapeutic transgene, enabling the use of lower and safer vector doses. In the current study, we identify potent muscle-specific transcriptional cis -regulatory modules ( CRMs ), containing clusters of transcription factor binding sites, using a genome-wide data-mining strategy. These novel muscle-specific CRMs result in a substantial increase in muscle-specific gene transcription (up to 400-fold) when delivered using adeno-associated viral vectors in mice. Significantly higher and sustained human micro-dystrophin and follistatin expression levels are attained than when conventional promoters are used. This results in robust phenotypic correction in dystrophic mice, without triggering apoptosis or evoking an immune response. This multidisciplinary approach has potentially broad implications for augmenting the efficacy and safety of muscle-directed gene therapy.

Single domain antibodies (VHH) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHH have not been widely used in the central nervous system (CNS), as it is hard to reach therapeutic levels, both because of their restricted blood-brain-barrier penetration and their apparent rapid clearance from the parenchyma. Here, we propose a gene transfer strategy based on adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS, ensuring continuous production at therapeutic levels. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimer’s disease. First, we generated a panel of VHHs targeting BACE1. One of them, VHH-B9, showed high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We then went on to demonstrate significant reductions in amyloid beta (Aβ) levels after AAV-based delivery of VHH-B9 into the CNS of a mouse model of cerebral amyloidosis. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

In recent years, gene therapy has been positioned as a real and safe option in the development of therapeutic alternatives for the cure and prevention of different diseases. It consists in the insertion of genetic material in a defective tissue or cell, through the use of a vector. There are several considerations for selecting the most appropriate vector, including the potential for binding and entry to the target cell, the ability of the genetic material to transfer to the nucleus, the ability to express the insert, and the absence of toxicity. In the current scenario, the most commonly used viral vectors are those derived from adeno-associated viruses (AAV). Characteristics such as biosafety, low toxicity and selective tropism have enabled its evaluation as a therapeutic option in many monogenic or complex diseases. Despite their advantages, AAV vectors have drawbacks, the most important being the patient's immune response to the vector, especially the response mediated by neutralizing antibodies (NAb). NAbs decrease the transduction of the vector and prevent the expression of the gene it transports, limiting its clinical application. Therefore, identifying and quantifying the presence and activity of NAbs is the first step in any gene therapy protocol with AAV vectors. The presence of NAbs depends mainly on exposure to the virus in nature and varies drastically according to age, geographic location and health status of the person evaluated.

Infection with Trypanosoma cruzi Chagas, 1909 is reported to increase the production of reactive oxygen species in patients with Chagas disease.Mitochondria dysfunction, host inflammatory response and inadequate antioxidant response are described as the main factors leading to oxidative stress during acute and chronic stages of the disease.The Seahorse XFe24 extracellular flux platform allows energy metabolism determination through mitochondrial respiration and glycolysis measurements.XFe24 platform can be used in in vitro models of T. cruzi-infected cells, which allow the assessment and even modulation of endogenous conditions of infected cells, generating readouts of real-time cellular bioenergetics changes.In this protocol, we standardised the use of XFe24 technology in T. cruzi infected AC16 cardiomyocytes and SGHPL-5 trophoblasts.In addition, we provide a list of optimised assay specifications, advantages and critical steps to be considered during the process.Cardiomyocytes and trophoblasts are attractive target cells to evaluate the metabolic environment in acute, chronic and congenital Chagas transmission scenarios.

In utero electroporation is increasingly used for manipulation of gene expression in the mammalian central nervous system. Here we created a transposon-based single plasmid system, that allows long-term conditional loss- and gain-of-function experiments across major CNS cell types. The system is widely applicable across species, allowing fast, flexible and cheap cell manipulation in previously non-transgenic species.

In vivo tissue-specific genome editing at the desired loci is still a challenge. Here, we report that AAV9-delivery of truncated guide RNAs (gRNAs) and Cas9 under the control of a computationally designed hepatocyte-specific promoter lead to liver-specific and sequence-specific targeting in the mouse factor IX (F9) gene. The efficiency of in vivo targeting was assessed by T7E1 assays, site-specific Sanger sequencing, and deep sequencing of on-target and putative off-target sites. Though AAV9 transduction was apparent in multiple tissues and organs, Cas9 expression was restricted mainly to the liver, with only minimal or no expression in other non-hepatic tissues. Consequently, the insertions and deletion (indel) frequency was robust in the liver (up to 50%) in the desired target loci of the F9 gene, with no evidence of targeting in other organs or other putative off-target sites. This resulted in a substantial loss of FIX activity and the emergence of a bleeding phenotype, consistent with hemophilia B. The in vivo efficacy of the truncated gRNA was as high as that of full-length gRNA. Cas9 expression was transient in neonates, representing an attractive "hit-and-run" paradigm. Our findings have potentially broad implications for somatic gene targeting in the liver using the CRISPR/Cas9 platform.

Until recently, adeno-associated virus 9 (AAV9) was considered the AAV serotype most effective in crossing the blood-brain barrier (BBB) and transducing cells of the central nervous system (CNS), following systemic injection. However, a newly engineered capsid, AAV-PHP.B, is reported to cross the BBB at even higher efficiency. We investigated how much we could boost CNS transgene expression by using AAV-PHP.B carrying a self-complementary (sc) genome. To allow comparison, 6 weeks old C57BL/6 mice received intravenous injections of scAAV2/9-GFP or scAAV2/PHP.B-GFP at equivalent doses. Three weeks postinjection, transgene expression was assessed in brain and spinal cord. We consistently observed more widespread CNS transduction and higher levels of transgene expression when using the scAAV2/PHP.B-GFP vector. In particular, we observed an unprecedented level of astrocyte transduction in the cortex, when using a ubiquitous CBA promoter. In comparison, neuronal transduction was much lower than previously reported. However, strong neuronal expression (including spinal motor neurons) was observed when the human synapsin promoter was used. These findings constitute the first reported use of an AAV-PHP.B capsid, encapsulating a scAAV genome, for gene transfer in adult mice. Our results underscore the potential of this AAV construct as a platform for safer and more efficacious gene therapy vectors for the CNS.

Clustered, regularly interspersed, short palindromic repeat RNA-guided nucleases have emerged as highly efficient genome editing tools. However, in vivo tissue-specific genome editing at the desired loci is still a challenge. Here, we report that truncated guide RNAs (gRNAs) and Cas9 under the control of a computationally designed hepatocyte-specific promoter lead to liver-specific and target site-specific indel formation in the mouse factor IX (FIX) gene. The truncated gRNAs targeting unique sites in exon 1 and exon 6 of the mouse FIX gene were designed using a computational CRISPR design tool and the target sites overlap with mutations known to cause hemophilia B in patients. The gRNA and Cas9-expressing constructs were delivered in vivo using AAV9 vectors. The efficiency of in vivo targeting was assessed by T7E1 assays, site-specific Sanger sequencing and deep sequencing of on-target and putative off-target sites. Though AAV9 transduction was apparent in multiple tissues and organs, Cas9 expression was restricted mainly to the liver, with only minimal or no expression in other non-hepatic tissues. Consequently, the indel frequency was robust in the liver (up to 50%) in the desired target locus of the FIX gene, with no evidence of targeting in other organs. This resulted in a substantial loss of FIX activity and the emergence of a bleeding phenotype, consistent with hemophilia B. Deep sequencing of putative off-target sites revealed no off-target editing. Our findings have potentially broad implications for somatic gene editing in the liver using the CRISPR/Cas9 platform.

BACKGROUND:Recent progress in the pathophysiology of heart failure (HF) has led to the development of new therapeutic options such as gene therapy and the use of adeno-associated viral (AAV) vectors. Despite the promising results in early clinical trials of gene therapy for HF, various obstacles have been faced, such as the presence of neutralizing antibodies (NAbs) against the capsid vectors. NAb activity limits vector transduction levels and therefore diminishes the final therapeutic response. Recent studies evaluating the prevalence of NAbs in various populations found considerable geographic variability for each AAV serotype. However, the levels of NAbs in Latin American populations are unknown, becoming a limiting factor to conducting AAV vector therapeutic trials in this population.OBJECTIVE:The goal of this study is to determine for the first time, the prevalence of anti-AAV NAbs for the serotypes 1, 2, and 9 in HF patients from the city of Bucaramanga, Colombia, using the in vitro transduction inhibition assay.METHODS:We will conduct a cross-sectional study with patients who periodically attend the HF clinic of the Cardiovascular Foundation of Colombia and healthy volunteers matched for age and sex. For all participants, we will evaluate the NAb levels against serotypes AAV1, AAV2, and AAV9. We will determine NAb levels using the in vitro transduction inhibition assay. In addition, participants will answer a survey to evaluate their epidemiological and socioeconomic variables. Participation in the study will be voluntary and all participants will sign an informed consent document before any intervention.RESULTS:The project is in the first phase: elaboration of case report forms and the informed consent form, and design of the recruitment strategy. Patient recruitment is expected to begin in the spring of 2016. We expect to have preliminary results, including the titer of the viral vectors, multiplicity of infections that we will use for each serotype, and the general validation of the assay, at the end of 2016. The final results are expected mid-2017.CONCLUSIONS:This project is the first effort to evaluate NAb levels against AAV1, AAV2, and AAV9 serotypes in patients with HF in Latin America. Our results will allow us to check the cross-reactivity response between the serotypes assessed, to describe the epidemiological characteristics of the participant population, and to set up a link with earlier reports of NAb prevalence in the literature.