Papers by Assumpcio Bosch
Frontiers in Cellular Neuroscience, 2021
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare genetic disorder belon... more Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare genetic disorder belonging to the group of vacuolating leukodystrophies. It is characterized by megalencephaly, loss of motor functions, epilepsy, and mild mental decline. In brain biopsies of MLC patients, vacuoles were observed in myelin and in astrocytes surrounding blood vessels. It is mainly caused by recessive mutations in MLC1 and HEPACAM (also called GLIALCAM) genes. These disease variants are called MLC1 and MLC2A with both types of patients sharing the same clinical phenotype. Besides, dominant mutations in HEPACAM were also identified in a subtype of MLC patients (MLC2B) with a remitting phenotype. MLC1 and GlialCAM proteins form a complex mainly expressed in brain astrocytes at the gliovascular interface and in Bergmann glia at the cerebellum. Both proteins regulate several ion channels and transporters involved in the control of ion and water fluxes in glial cells, either directly influencing the...
Journal of Virology, 1999
Group C adenovirus-mediated gene transfer to central nervous system cells is inefficient. We foun... more Group C adenovirus-mediated gene transfer to central nervous system cells is inefficient. We found that wild-type group D viruses, or recombinant adenovirus type 2 (Ad2) (group C) modified to contain Ad17 (group D) fiber, were more efficient in infecting primary cultures of neurons. Together with studies on primary vascular endothelial cells and tissue culture cell lines, our results indicate that there is not a universally applicable adenovirus serotype for use as a gene transfer vector.
Human Molecular Genetics, 2019
Mucopolysaccharidosis (MPS) type VII is a lysosomal storage disease caused by ß-glucuronidase def... more Mucopolysaccharidosis (MPS) type VII is a lysosomal storage disease caused by ß-glucuronidase deficiency, prompting glycosaminoglycan accumulation in enlarged vesicles, leading to peripheral and neuronal dysfunction. Here, we present a gene therapy strategy using lumbar puncture of AAVrh10 encoding human β-glucuronidase (AAVrh10-GUSB) to adult MPS VII mice. This minimally invasive technique efficiently delivers the recombinant vector to the cerebrospinal fluid (CSF) with a single intrathecal injection. We show that AAVrh10 delivery to the CSF allows global, stable transduction of CNS structures. In addition, drainage of AAVrh10-GUSB from the CSF to the bloodstream resulted in the transduction of somatic organs such as liver, which provided a systemic β-glucuronidase source sufficient to achieve serum enzyme activity comparable to wild type mice. ß-glucuronidase levels were enough to correct biochemical and histopathological hallmarks of the disease in the CNS and somatic organs at s...
Scientific reports, Jan 30, 2018
Here we used a systems biology approach and artificial intelligence to identify a neuroprotective... more Here we used a systems biology approach and artificial intelligence to identify a neuroprotective agent for the treatment of peripheral nerve root avulsion. Based on accumulated knowledge of the neurodegenerative and neuroprotective processes that occur in motoneurons after root avulsion, we built up protein networks and converted them into mathematical models. Unbiased proteomic data from our preclinical models were used for machine learning algorithms and for restrictions to be imposed on mathematical solutions. Solutions allowed us to identify combinations of repurposed drugs as potential neuroprotective agents and we validated them in our preclinical models. The best one, NeuroHeal, neuroprotected motoneurons, exerted anti-inflammatory properties and promoted functional locomotor recovery. NeuroHeal endorsed the activation of Sirtuin 1, which was essential for its neuroprotective effect. These results support the value of network-centric approaches for drug discovery and demonst...
Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics, Jan 7, 2017
The role of the T helper (Th)17 pathway has been clearly demonstrated in the onset and progressio... more The role of the T helper (Th)17 pathway has been clearly demonstrated in the onset and progression of autoimmune diseases, where interleukin (IL)-23 is a key molecule in maintaining the response mediated by Th17 cells. As a consequence, recent strategies based on blocking the interaction between IL-23 and its receptor (IL-23R), for example the anti-p19 antibody tildrakizumab, have been developed to regulate the Th17 pathway from the initial stages of the disease. Here, a soluble (s)IL-23R cDNA was cloned in expression plasmids and viral vectors. The clinical efficacy of sIL-23R was evaluated in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis mice intravenously injected with a single dose of adeno-associated virus AAV8-sIL-23R vectors. Cytokine secretion was determined by multiplex assay, while histopathological analysis of the central nervous system was performed to study demyelination, inflammatory infiltration, and microglia and astroglia acti...
Neurobiology of Disease, 2016
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive d... more Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motoneurons, which is preceded by loss of neuromuscular connections in a "dying back" process. Neuregulin-1 (Nrg1) is a neurotrophic factor essential for the development and maintenance of neuromuscular junctions, and Nrg1 receptor ErbB4 loss-of-function mutations have been reported as causative for ALS. Our main goal was to investigate the role of Nrg1 type I (Nrg1-I) in SOD1 G93A mice muscles. We overexpressed Nrg1-I by means of an adeno-associated viral (AAV) vector, and investigated its effect by means of neurophysiological techniques assessing neuromuscular function, as well as molecular approaches (RT-PCR, western blot, immunohistochemetry, ELISA) to determine the mechanisms underlying Nrg1-I action. AAV-Nrg1-I intramuscular administration promoted motor axon collateral sprouting by acting on terminal Schwann cells, preventing denervation of the injected muscles through Akt and ERK1/2 pathways. We further used a model of muscle partial denervation by transecting the L4 spinal nerve. AAV-Nrg1-I intramuscular injection enhanced muscle reinnervation by collateral sprouting, whereas administration of lapatinib (ErbB receptor inhibitor) completely blocked it. We demonstrated that Nrg1-I plays a crucial role in the collateral reinnervation process, opening a new window for developing novel ALS therapies for functional recovery rather than preservation.
PLOS ONE, 2015
The Klotho protein is a β-glucuronidase, and its overexpression is associated with life extension... more The Klotho protein is a β-glucuronidase, and its overexpression is associated with life extension. Its mechanism of action is not fully understood, although it has been recently reported that αKlotho improves synaptic and cognitive functions, and it may also influence a variety of structures and functions during CNS maturation and aging. The αKlotho gene has two transcripts, one encoding a transmembrane isoform (m-KL), and the other a putative secreted isoform (s-KL). Unfortunately, little is known about the secreted αKlotho isoform, since available antibodies cannot discriminate s-KL from the KL1 domain cleaved from the transmembrane isoform. This study shows, for the first time, that the klotho transcript produced by alternative splicing generates a stable protein (70 kDa), and that in contrast to the transmembrane Klotho isoform, it is ten times more abundant in the brain than in the kidney suggesting that the two isoforms may have different functions. We also studied whether klotho expression in the CNS was influenced by aging, Alzheimer's disease (AD), or a healthy lifestyle, such as voluntary moderate continuous exercise. We observed a strong correlation between high expression levels of the two klotho transcripts and the healthy status of the animals. Expression of Klotho in brain areas decayed more rapidly in the 3xTg-AD model of AD than in healthy animals, whilst moderate continuous exercise in adulthood prevents the decline in expression of both klotho transcripts.
Medicina clínica, Jan 31, 1990
The development of polymerase chain reaction (PCR) which allows the specific amplification of DNA... more The development of polymerase chain reaction (PCR) which allows the specific amplification of DNA sequences has improved considerably the genetic analysis of hereditary diseases. We present here the application of this new technique to the genetic analysis of cystic fibrosis (CF), the most frequent severe genetic disease in caucasians. We have amplified four sequences containing polymorphisms linked to the CF gene (CS.7, KM.19, MP6d-9 and J3.11), and analysed the amplified products with restriction enzymes. Complete concordance was found with classical Southern methods, allowing the application of PCR to routine CF family studies.
Gene Therapy, 2014
Simian adeno-associated virus (AAV) serotype rh.10 is a promising gene therapy tool, achieving sa... more Simian adeno-associated virus (AAV) serotype rh.10 is a promising gene therapy tool, achieving safe, sustained transgene expression in the nervous system, lung, liver and heart in animal models. To date, preexisting immunity in humans has not been confirmed, though exposure is unexpected. We compared the humoral immune response with serotypes AAVrh.10 and AAV9 in mice, and AAVrh.10, AAV9 and AAV2 in 100 healthy humans. Mice, injected-intravenously, raised significantly more anti-AAV9 than anti-AAVrh.10 IgG (immunoglobulins), and sera demonstrated greater neutralizing capacity, correspondingly. Antibody crossbinding studies in mice showed negligible cross-recognition between AAVrh.10, AAV9 and AAV2. In humans, IgG prevalence against the most common human serotype, AAV2, was 72%; AAV9, 47% and AAVrh.10, a surprising, 59%. Yet, neutralizing-antibody seroprevalences were 71% for AAV2, 18% for AAV9 and 21% for AAVrh.10. Thus, most anti-AAV9 and anti-AAVrh.10 IgG were nonneutralizing. Indeed, sera generally neutralized AAV2 more strongly than AAVrh.10. Further, all samples neutralizing AAVrh.10 or AAV9 also neutralized AAV2, suggesting antibody cross-recognition. This contrasts with the results in mice, and highlights the complexity of tailoring gene therapy to minimize the immune response in humans, when multiple-mixed infections during a lifetime evoke a broad repertoire of preexisting antibodies capable of cross reacting with non-human serotypes.
Virology, Jan 10, 2007
Current strategies to amplify helper-dependent adenovirus, based on excision of the packaging sig... more Current strategies to amplify helper-dependent adenovirus, based on excision of the packaging signal, do not routinely reduce helper adenovirus contamination below 1%. Here, we have tested if reducing the efficiency of the packaging process of the helper adenovirus could impair its packaging without affecting helper-dependent adenovirus production. Interestingly, insertion of attB/attP-PhiC31 sequences flanking the packaging signal significantly lengthens adenovirus cycle up to 60 h without reducing virus viability or production yield. This delay occurs in the absence of PhiC31 recombinase indicating that other mechanisms different from excision of packaging signal must be involved. In addition, at 36 h post-coinfection helper-dependent adenovirus are efficiently produced, while production levels of helper attB/attP-modified adenovirus are 100-1000 times lower than controls. Therefore, these results suggest that attB/attP-mediated packaging impairment of the adenovirus genome is an ...
PLoS ONE, 2013
DNA transposons are mobile elements with the ability to mobilize and transport genetic informatio... more DNA transposons are mobile elements with the ability to mobilize and transport genetic information between different chromosomal loci. Unfortunately, most transposons copies are currently inactivated, little is known about mariner elements in humans despite their role in the evolution of the human genome, even though the Hsmar2 transposon is associated to hotspots for homologous recombination involved in human genetic disorders as Charcot-Marie-Tooth, Prader-Willi/Angelman, and Williams syndromes. This manuscript describes the functional characterization of the human HSMAR2 transposase generated from fossil sequences and shows that the native HSMAR2 is active in human cells, but also in bacteria, with an efficiency similar to other mariner elements. We observe that the subcellular localization of HSMAR2 is dependent on the host cell type, and is cytotoxic when overexpressed in HeLa cells. Finally, we also demonstrate that the binding of HSMAR2 to its own ITRs is specific, and that the excision reaction leaves non-canonical footprints both in bacteria and eukaryotic cells.
PLoS ONE, 2011
We have previously described a new family of mutant adenoviruses carrying different combinations ... more We have previously described a new family of mutant adenoviruses carrying different combinations of attB/attP sequences from bacteriophage PhiC31 flanking the Ad5 packaging domain. These novel helper viruses have a significantly delayed viral life cycle and a severe packaging impairment, regardless of the presence of PhiC31 recombinase. Their infectious viral titers are significantly lower (100-1000 fold) than those of control adenovirus at 36 hours post-infection, but allow for efficient packaging of helper-dependent adenovirus. In the present work, we have analyzed which steps of the adenovirus life cycle are altered in attB-helper adenoviruses and investigated whether these viruses can provide the necessary viral proteins in trans. The entry of attB-adenoviral genomes into the cell nucleus early at early timepoints post-infection was not impaired and viral protein expression levels were found to be similar to those of control adenovirus. However, electron microscopy and capsid protein composition analyses revealed that attB-adenoviruses remain at an intermediate state of maturation 36 hours post-infection in comparison to control adenovirus which were fully mature and infective at this time point. Therefore, an additional 20-24 hours were found to be required for the appearance of mature attB-adenovirus. Interestingly, attB-adenovirus assembly and infectivity was restored by inserting a second packaging signal close to the right-end ITR, thus discarding the possibility that the attB-adenovirus genome was retained in a nuclear compartment deleterious for virus assembly. The present study may have substantive implications for helper-dependent adenovirus technology since helper attB-adenovirus allows for preferential packaging of helper-dependent adenovirus genomes.
PLoS ONE, 2012
The human adenovirus 40 (Ad40) is a promising tool for gene therapy of intestinal diseases. Since... more The human adenovirus 40 (Ad40) is a promising tool for gene therapy of intestinal diseases. Since the production of Ad40 in vitro is extremely inefficient, chimeric Adenovirus 5/40S vectors carrying the Ad40 short fiber on the Ad5 capsid have been developed. However, Ad5/40S productivity is low. We hypothesized that low productivity was a result of inefficient viral entry into producer cells during amplification. To this end, we have developed a production strategy based on using 211B cells (expressing Ad5 fiber) during amplification steps, while Ad5/40S infectivity is further improved by adding polybrene during infections. In addition, the optimal harvesting time was determined by evaluating the Ad5/40S viral cycle. The developed production strategy significantly reduces the number of amplification cycles and duration of the process. Finally, to further facilitate Ad5/40S production, 211B cells were adapted to suspension thus allowing to easily upscale the production process in bioreactors.
Nucleic Acids Research, 1991
Source/Description: YM40 (D2S75) is a 3.7 Kb single copy EcoRI fragment isolated from a Charon 21... more Source/Description: YM40 (D2S75) is a 3.7 Kb single copy EcoRI fragment isolated from a Charon 21A human chromosome 2 library (1). Polym01phism: Pstl detects a two allele system.
Molecular Therapy, 2006
efficiency of apical transduction with these three serotypes in the human airway, we tested the e... more efficiency of apical transduction with these three serotypes in the human airway, we tested the effectiveness of proteasome inhibitors (LLnL and doxorubicin, known to augment intracellular processing of rAAV2 and rAAV5 to the nucleus) to augment transduction with these three serotypes. Augmentation of rAAV2/1 apical transduction of human polarized airway epithelia was 10-fold lower than that for rAAV2/2 and rAAV2/5. Cellular fractionation assays demonstrated that proteasome inhibitors significantly increased virion nuclear uptake for rAAV2/2 and rAAV2/5, but not for rAAV2/1. Cumulatively, these findings suggest that the ubiquitin/proteasome pathway is less of a barrier for rAAV2/1 as compared to the other AAV serotypes. They also demonstrate that AAV1 serotype transduction biology is quite different from AAV2 and AAV5 in human airway epithelia and is potentially a more efficient vector for gene therapy to the airway.
Journal of Clinical Investigation, 1996
Retroviral gene transfer to liver without prior injury has not yet been accomplished. We hypothes... more Retroviral gene transfer to liver without prior injury has not yet been accomplished. We hypothesized that recombinant human keratinocyte growth factor would stimulate proliferation of hepatocytes and allow for efficient in vivo gene transfer with high titer murine Moloney retroviral vectors. This report shows that 48 h after intravenous injection of keratinocyte growth factor, hepatocyte proliferation increased approximately 40-fold compared to non-stimulated livers. When keratinocyte growth factor treatment was followed by intravenous injection of high titer (1 ϫ 10 8 colony forming units/ml) retrovirus coding for the Escherichia Coli -galactosidase gene, there was a 600-fold increase in -galactosidase expression, with 2% of hepatocytes transduced. Thus, by exploiting the mitogenic properties of keratinocyte growth factor, retrovirus-mediated gene transfer to liver may be accomplished in vivo without the use of partial hepatectomy or pretreatment with other toxins to induce hepatocyte cell division.
Journal of Clinical Investigation, 2004
IGF-1 has been associated with the pathogenesis of diabetic retinopathy, although its role is not... more IGF-1 has been associated with the pathogenesis of diabetic retinopathy, although its role is not fully understood. Here we show that normoglycemic/normoinsulinemic transgenic mice overexpressing IGF-1 in the retina developed most alterations seen in human diabetic eye disease. A paracrine effect of IGF-1 in the retina initiated vascular alterations that progressed from nonproliferative to proliferative retinopathy and retinal detachment. Eyes from 2-month-old transgenic mice showed loss of pericytes and thickening of basement membrane of retinal capillaries. In mice 6 months and older, venule dilatation, intraretinal microvascular abnormalities, and neovascularization of the retina and vitreous cavity were observed. Neovascularization was consistent with increased IGF-1 induction of VEGF expression in retinal glial cells. In addition, IGF-1 accumulated in aqueous humor, which may have caused rubeosis iridis and subsequently adhesions between the cornea and iris that hampered aqueous humor drainage and led to neovascular glaucoma. Furthermore, all transgenic mice developed cataracts. These findings suggest a role of IGF-1 in the development of ocular complications in long-term diabetes. Thus, these transgenic mice may be used to study the mechanisms that lead to diabetes eye disease and constitute an appropriate model in which to assay new therapies. Nonstandard abbreviations used: glial fibrillar acidic protein (GFAP); intraocular pressure (IOP); intraretinal microvascular abnormalities (IRMAs); rat insulin promoter-I (RIP-I); streptozotocin (STZ); von Willebrand factor (vWF).
Human Gene Therapy, 2014
Canine adenovirus type 2 vectors (CAV-2) are promising tools to treat global central nervous syst... more Canine adenovirus type 2 vectors (CAV-2) are promising tools to treat global central nervous system (CNS) disorders because of their preferential transduction of neurons and efficient retrograde axonal transport. Here we tested the potential of a helper-dependent CAV-2 vector expressing b-glucuronidase (HD-RIGIE) in a mouse model of mucopolysaccharidosis type VII (MPS VII), a lysosomal storage disease caused by deficiency in bglucuronidase activity. MPS VII leads to glycosaminoglycan accumulation into enlarged vesicles in peripheral tissues and the CNS, resulting in peripheral and neuronal dysfunction. After intracranial administration of HD-RIGIE, we show long-term expression of b-glucuronidase that led to correction of neuropathology around the injection site and in distal areas. This phenotypic correction correlated with a decrease in secondary-elevated lysosomal enzyme activity and glycosaminoglycan levels, consistent with global biochemical correction. Moreover, HD-RIGIE-treated mice show significant cognitive improvement. Thus, injections of HD-CAV-2 vectors in the brain allow a global and sustained expression and may have implications for brain therapy in patients with lysosomal storage disease.
Human Gene Therapy, 1998
We have previously shown that intravenous administration of keratinocyte growth factor (KGF) indu... more We have previously shown that intravenous administration of keratinocyte growth factor (KGF) induces hepatocyte proliferation, allowing for efficient and noninvasive in vivo gene transfer with high-titer retroviral vectors in mice. The distinctive periportal distribution of transduced cells led us to investigate the ability of virus-sized particles to perfuse the liver adequately after growth factor treatment. We found that perfusion was adequate, and that transduction was limited to the periportal region because only those cells were stimulated to divide. Cells in this region also showed increased expression of Ram-1, the receptor for the murine Moloney leukemia virus (MoMLV) amphotropic envelope, after KGF treatment. In further studies we found that recombinant hepatocyte growth factor (HGF) induces a different population of hepatocytes to divide and upregulate Ram-1. The differential pattern of induction suggested that combining KGF and HGF would improve gene transfer efficiency further. Indeed, simultaneous delivery of both growth factors leads to an overall increase in the number of proliferating cells. Importantly, when coupled with MoMLV delivery, efficiency of gene transfer increased. These results confirm the utility of growth factors for noninvasive hepatic gene transfer in mice, and demonstrate how experiments to define the mechanism of transduction can be taken advantage of to develop improved gene transfer protocols.
Genome Research, 1999
The short arm of human chromosome 21 (21p) contains many different types of repetitive sequences ... more The short arm of human chromosome 21 (21p) contains many different types of repetitive sequences and is highly homologous to the short arms of other acrocentric chromosomes. Owing to its repetitive nature and the lack of chromosome 21p-specific molecular markers, most physical maps of chromosome 21 exclude this region. We constructed a physical map of chromosome 21p using sequence tagged site (STS) content mapping of yeast artificial chromosomes (YACs). To this end, 39 STSs located on the short arm or near the centromere of chromosome 21 were constructed, including four polymorphic simple tandem repeats (STRs) and two expressed sequence tags (ESTs). Thirty YACs were selected from the St. Louis YAC library, the chromosome 21-enriched ICRF YAC library, and the CEPH YAC and megaYAC libraries. These were assembled in a YAC contig map ranging from the centromere to the rDNA gene cluster at 21p12. The total size of the region covered by YACs is estimated between 2.9 and 5 Mb. The integrit...
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Papers by Assumpcio Bosch