Long-term Amelioration of Feline Mucopolysaccharidosis VI After AAV-mediated Liver Gene Transfer

Mucopolysaccharidosis VI (MPS VI) is caused by deficient arylsulfatase B (ARSB) activity resulting in lysosomal storage of glycosaminoglycans (GAGs). MPS VI is characterized by dysostosis multiplex, organomegaly, corneal clouding, and heart valve thickening. Gene transfer to a factory organ like liver may provide a lifetime source of secreted ARSB. We show that intravascular administration of adeno-associated viral vectors (AAV) 2/8-TBG-felineARSB in MPS VI cats resulted in ARSB expression up to 1 year, the last time point of the study. In newborn cats, normal circulating ARSB activity was achieved following delivery of high vector doses (6 × 1013 genome copies (gc)/kg) whereas delivery of AAV2/8 vector doses as low as 2 × 1012 gc/kg resulted in higher than normal serum ARSB levels in juvenile MPS VI cats. In MPS VI cats showing high serum ARSB levels, independent of the age at treatment, we observed: (i) clearance of GAG storage, (ii) improvement of long bone length, (iii) reduction of heart valve thickness, and (iv) improvement in spontaneous mobility. Thus, AAV2/ 8-mediated liver gene transfer represents a promising therapeutic strategy for MPS VI patients

Y chromosome sequence and epigenomic reconstruction across human populations

Recent advances in long-read sequencing technologies have allowed the generation and curation of more complete genome assemblies, enabling the analysis of traditionally neglected chromosomes, such as the human Y chromosome (chrY). Native DNA was sequenced on a MinION Oxford Nanopore Technologies sequencing device to generate genome assemblies for seven major chrY human haplogroups. We analyzed and compared the chrY enrichment of sequencing data obtained using two different selective sequencing approaches: adaptive sampling and flow cytometry chromosome sorting. We show that adaptive sampling can produce data to create assemblies comparable to chromosome sorting while being a less expensive and time-consuming technique. We also assessed haplogroup-specific structural variants, which would be otherwise difficult to study using short-read sequencing data only. Finally, we took advantage of this technology to detect and profile epigenetic modifications among the considered haplogroups. Altogether, we provide a framework to study complex genomic regions with a simple, fast, and affordable methodology that could be applied to larger population genomics datasets

Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease

Parkinson's disease (PD) is associated with the degeneration of ventral midbrain dopaminergic neurons (vmDAns) and the accumulation of toxic α-synuclein. A non-cell-autonomous contribution, in particular of astrocytes, during PD pathogenesis has been suggested by observational studies, but remains to be experimentally tested. Here, we generated induced pluripotent stem cell-derived astrocytes and neurons from familial mutant LRRK2 G2019S PD patients and healthy individuals. Upon co-culture on top of PD astrocytes, control vmDAns displayed morphological signs of neurodegeneration and abnormal, astrocyte-derived α-synuclein accumulation. Conversely, control astrocytes partially prevented the appearance of disease-related phenotypes in PD vmDAns. We additionally identified dysfunctional chaperone-mediated autophagy (CMA), impaired macroautophagy, and progressive α-synuclein accumulation in PD astrocytes. Finally, chemical enhancement of CMA protected PD astrocytes and vmDAns via the clearance of α-synuclein accumulation. Our findings unveil a crucial non-cell-autonomous contribution of astrocytes during PD pathogenesis, and open the path to exploring novel therapeutic strategies aimed at blocking the pathogenic cross talk between neurons and glial cells.We are thankful to Mark Cookson for the LRRK2 G2019S plasmid (Addgene Plasmid No. 29401). We are grateful to the Advanced Fluorescence Microscopy Unit of the Institute of Biomedicine of the University of Barcelona (especially to Elena Rebollo Arredondo). Research from the authors' laboratories is supported by the European Research Council (ERC) (2012-StG-311736-PD-HUMMODEL), the Spanish Ministry of Economy and Competitiveness (MINECO) (FIS2016-78507-C2-2-P, SAF2015-69706-R, and BFU2016-80870-P), Instituto de Salud Carlos III (ISCIII/FEDER) (Red de Terapia Celular [TerCel] RD16/0011/0024), AGAUR (2014-SGR-878 and 2017-SGR-899), and CERCA Program/Generalitat de Catalunya. A.D. is supported by the PD-HUMMODEL ERC-Ideas PhD fellowship. C.C. and G.C. are partially supported by predoctoral fellowships from the Spanish Ministry of Education (MEC) (FPU12/03332) and MINECO (BES-2014-069603), respectively

Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease

Parkinson's disease (PD) is associated with the degeneration of ventral midbrain dopaminergic neurons (vmDAns) and the accumulation of toxic α-synuclein. A non-cell-autonomous contribution, in particular of astrocytes, during PD pathogenesis has been suggested by observational studies, but remains to be experimentally tested. Here, we generated induced pluripotent stem cell-derived astrocytes and neurons from familial mutant LRRK2 G2019S PD patients and healthy individuals. Upon co-culture on top of PD astrocytes, control vmDAns displayed morphological signs of neurodegeneration and abnormal, astrocyte-derived α-synuclein accumulation. Conversely, control astrocytes partially prevented the appearance of disease-related phenotypes in PD vmDAns. We additionally identified dysfunctional chaperone-mediated autophagy (CMA), impaired macroautophagy, and progressive α-synuclein accumulation in PD astrocytes. Finally, chemical enhancement of CMA protected PD astrocytes and vmDAns via the clearance of α-synuclein accumulation. Our findings unveil a crucial non-cell-autonomous contribution of astrocytes during PD pathogenesis, and open the path to exploring novel therapeutic strategies aimed at blocking the pathogenic cross talk between neurons and glial cells

Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson's disease

Parkinson's disease (PD) is associated with the degeneration of ventral midbrain dopaminergic neurons (vmDAns) and the accumulation of toxic α-synuclein. A non-cell-autonomous contribution, in particular of astrocytes, during PD pathogenesis has been suggested by observational studies, but remains to be experimentally tested. Here, we generated induced pluripotent stem cell-derived astrocytes and neurons from familial mutant LRRK2 G2019S PD patients and healthy individuals. Upon co-culture on top of PD astrocytes, control vmDAns displayed morphological signs of neurodegeneration and abnormal, astrocyte-derived α-synuclein accumulation. Conversely, control astrocytes partially prevented the appearance of disease-related phenotypes in PD vmDAns. We additionally identified dysfunctional chaperone-mediated autophagy (CMA), impaired macroautophagy, and progressive α-synuclein accumulation in PD astrocytes. Finally, chemical enhancement of CMA protected PD astrocytes and vmDAns via the clearance of α-synuclein accumulation. Our findings unveil a crucial non-cell-autonomous contribution of astrocytes during PD pathogenesis, and open the path to exploring novel therapeutic strategies aimed at blocking the pathogenic cross talk between neurons and glial cells

Y chromosome sequence and epigenomic reconstruction across human populations

Recent advances in long-read sequencing technologies have allowed the generation and curation of more complete genome assemblies, enabling the analysis of traditionally neglected chromosomes, such as the human Y chromosome (chrY). Native DNA was sequenced on a MinION Oxford Nanopore Technologies sequencing device to generate genome assemblies for seven major chrY human haplogroups. We analyzed and compared the chrY enrichment of sequencing data obtained using two different selective sequencing approaches: adaptive sampling and flow cytometry chromosome sorting. We show that adaptive sampling can produce data to create assemblies comparable to chromosome sorting while being a less expensive and time-consuming technique. We also assessed haplogroup-specific structural variants, which would be otherwise difficult to study using short-read sequencing data only. Finally, we took advantage of this technology to detect and profile epigenetic modifications among the considered haplogroups. Altogether, we provide a framework to study complex genomic regions with a simple, fast, and affordable methodology that could be applied to larger population genomics datasets.M.P.-F. has the support of an INPhINIT Retaining Fellowship from “La Caixa” Foundation (ID 100010434) with code LCF/BQ/DR20/11790032. T.M.-B. is supported by funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 864203), PID2021-126004NB-100 (MICIIN/FEDER, UE) and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2021 SGR 00177)

Catalytic domain modification and viral gene delivery of activated factor VII confers hemostasis at reduced expression levels and vector doses in vivo

Catalytic domain variants of activated factor VII (FVIIa) with enhanced hemostatic properties are highly attractive for the treatment of bleeding disorders via gene-based therapy. To explore this in a hemophilic mouse model, we characterized 2 variants of murine activated FVII (mFVIIa-VEAY and mFVIIa-DVQ) with modified catalytic domains, based on recombinant human FVIIa (rhFVIIa) variants. Using purified recombinant proteins, we showed that murine FVIIa (mFVIIa) and variants had comparable binding to human and murine tissue factor (TF) and exhibited similar extrinsic coagulant activity. In vitro in the absence of TF, the variants showed a 6- to 17-fold enhanced proteolytic and coagulant activity relative to mFVIIa, but increased inactivation by antithrombin. Gene delivery of mFVIIa-VEAY resulted in long-term, effective hemostasis at 5-fold lower expression levels relative to mFVIIa in hemophilia A mice or in hemophilia B mice with inhibitors to factor IX. However, expression of mFVIIa-VEAY at 14-fold higher than therapeutic levels resulted in a progressive mortality to 70% within 6 weeks after gene delivery. These results are the first demonstration of the hemostatic efficacy of continuous expression, in the presence or absence of inhibitors, of a high-activity gene-based FVIIa variant in an animal model of hemophilia