Pasqualina Colella
Senior Research Scientist, Pediatrics - Genetics
Bio
Dr. Pasqualina Colella is a distinguished scientist with expertise in gene and cell therapy for inherited diseases. She is currently a Senior Research Scientist at Stanford University, where she pioneers innovative cell therapy approaches for neuropathic lysosomal storage diseases (LSDs). Her research investigates hematopoietic stem cell transplantation (HSCT) and genome editing to target untreatable diseases caused by progranulin deficiency.
Dr. Colella earned her PhD at TIGEM (Italy), where she developed therapeutic strategies for inherited blindness due to retinal photoreceptor degeneration. Her work included pioneering dual AAV-mediated large gene delivery to the retina for Usher syndrome type 1B (USH1B) and Stargardt disease (STGD).
During her postdoctoral studies at Genethon (France), Dr. Colella focused on gene therapy for Pompe disease, an LSD with neuromuscular manifestations. She addressed immune responses and efficacy limitations of preclinical in vivo AAV gene therapy through transgene engineering, including the development of highly secretable GAA and tandem promoters.
Dr. Colella’s research has been foundational for ongoing clinical trials for USH1B and Pompe disease.
With 30 published manuscripts—16 as the first author and 6 as a co-corresponding author—and 5 patents, Dr. Colella has a robust track record in the field. She is dedicated to driving innovations in gene and cell therapy for untreatable diseases and mentoring the next generation of scientists.
Dr. Colella has been awarded the prestigious Marie Skłodowska-Curie Individual Fellowship and was selected as a finalist for the ERC Starting Grant 2019.
Contact
https://orcid.org/0000-0002-4268-2391All Publications
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Advances in Pompe Disease Treatment: From Enzyme Replacement to Gene Therapy.
Molecular diagnosis & therapy
2024
Abstract
Pompe disease is a neuromuscular disorder caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA), hydrolyzing glycogen to glucose. Pathological glycogen storage, the hallmark of the disease, disrupts the metabolism and function of various cell types, especially muscle cells, leading to cardiac, motor, and respiratory dysfunctions. The spectrum of Pompe disease manifestations spans two main forms: classical infantile-onset (IOPD) and late-onset (LOPD). IOPD, caused by almost complete GAA deficiency, presents at birth and leads to premature death by the age of 2 years without treatment. LOPD, less severe due to partial GAA activity, appears in childhood, adolescence, or adulthood with muscle weakness and respiratory problems. Since 2006, enzyme replacement therapy (ERT) has been approved for Pompe disease, offering clinical benefits but not a cure. However, advances in early diagnosis through newborn screening, recognizing disease manifestations, and developing improved treatments are set to enhance Pompe disease care. This article reviews recent progress in ERT and ongoing translational research, including the approval of second-generation ERTs, a clinical trial of in utero ERT, and preclinical development of gene and substrate reduction therapies. Notably, gene therapy using intravenous delivery of adeno-associated virus (AAV) vectors is in phase I/II clinical trials for both LOPD and IOPD. Promising data from LOPD trials indicate that most participants met the criteria to discontinue ERT several months after gene therapy. The advantages and challenges of this approach are discussed. Overall, significant progress is being made towards curative therapies for Pompe disease. While several challenges remain, emerging data are promising and suggest the potential for a once-in-a-lifetime treatment.
View details for DOI 10.1007/s40291-024-00733-x
View details for PubMedID 39134822
View details for PubMedCentralID 7933537
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CNS-wide repopulation by hematopoietic-derived microglia-like cells corrects progranulin deficiency in mice.
Nature communications
2024; 15 (1): 5654
Abstract
Hematopoietic stem cell transplantation can deliver therapeutic proteins to the central nervous system (CNS) through transplant-derived microglia-like cells. However, current conditioning approaches result in low and slow engraftment of transplanted cells in the CNS. Here we optimized a brain conditioning regimen that leads to rapid, robust, and persistent microglia replacement without adverse effects on neurobehavior or hematopoiesis. This regimen combines busulfan myeloablation and six days of Colony-stimulating factor 1 receptor inhibitor PLX3397. Single-cell analyses revealed unappreciated heterogeneity of microglia-like cells with most cells expressing genes characteristic of homeostatic microglia, brain-border-associated macrophages, and unique markers. Cytokine analysis in the CNS showed transient inductions of myeloproliferative and chemoattractant cytokines that help repopulate the microglia niche. Bone marrow transplant of progranulin-deficient mice conditioned with busulfan and PLX3397 restored progranulin in the brain and eyes and normalized brain lipofuscin storage, proteostasis, and lipid metabolism. This study advances our understanding of CNS repopulation by hematopoietic-derived cells and demonstrates its therapeutic potential for treating progranulin-dependent neurodegeneration.
View details for DOI 10.1038/s41467-024-49908-4
View details for PubMedID 38969669
View details for PubMedCentralID PMC11226701
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CNS Repopulation by Hematopoietic-Derived Microglia-Like Cells Corrects Progranulin deficiency.
Research square
2023
Abstract
Hematopoietic stem cell transplantation can deliver therapeutic proteins to the CNS through donor-derived hematopoietic cells that become microglia-like cells. However, using standard conditioning approaches, hematopoietic stem cell transplantation is currently limited by low and slow engraftment of microglia-like cells. We report an efficient conditioning regimen based on Busulfan and a six-day course of microglia depletion using the colony-stimulating factor receptor 1 inhibitor PLX3397. Combining Busulfan-myeloablation and transient microglia depletion results in robust, rapid, and persistent microglia replacement by bone marrow-derived microglia-like cells throughout the CNS. Adding PLX3397 does not affect neurobehavior or has adverse effects on hematopoietic reconstitution. Through single-cell RNA sequencing and high-dimensional CyTOF mass cytometry, we show that microglia-like cells are a heterogeneous population and describe six distinct subpopulations. Though most bone-marrow-derived microglia-like cells can be classified as homeostatic microglia, their gene signature is a hybrid of homeostatic/embryonic microglia and border associated-macrophages. Busulfan-myeloablation and transient microglia depletion induce specific cytokines in the brain, ultimately combining myeloid proliferative and chemo-attractive signals that act locally to repopulate microglia from outside the niche. Importantly, this conditioning approach demonstrates therapeutic efficacy in a mouse model of GRN deficiency. Transplanting wild-type bone marrow into Grn-/- mice conditioned with Busulfan plus PLX3397 results in high engraftment of microglia-like cells in the brain and retina, restoring GRN levels and normalizing lipid metabolism.
View details for DOI 10.21203/rs.3.rs-3263412/v1
View details for PubMedID 37790525
View details for PubMedCentralID PMC10543302
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Editorial: Ex-vivo and in-vivo genome engineering for metabolic and neurometabolic diseases.
Frontiers in genome editing
2023; 5: 1248904
View details for DOI 10.3389/fgeed.2023.1248904
View details for PubMedID 37484653
View details for PubMedCentralID PMC10359423
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Successful treatment of severe MSUD in Bckdhb-/- mice with neonatal AAV gene therapy.
Journal of inherited metabolic disease
2023
Abstract
Maple syrup urine disease (MSUD) is rare autosomal recessive metabolic disorder caused by the dysfunction of the mitochondrial branched-chain 2-ketoacid dehydrogenase (BCKD) enzyme complex leading to massive accumulation of branched-chain amino acids and 2-keto acids. MSUD management, based on a life-long strict protein restriction with nontoxic amino acids oral supplementation represents an unmet need as it is associated with a poor quality of life, and does not fully protect from acute life-threatening decompensations or long-term neuropsychiatric complications. Orthotopic liver transplantation is a beneficial therapeutic option, which shows that restoration of only a fraction of whole-body BCKD enzyme activity is therapeutic. MSUD is thus an ideal target for gene therapy. We and others have tested AAV gene therapy in mice for two of the three genes involved in MSUD, BCKDHA and DBT. In this study, we developed a similar approach for the third MSUD gene, BCKDHB. We performed the first characterization of a Bckdhb-/- mouse model, which recapitulates the severe human phenotype of MSUD with early-neonatal symptoms leading to death during the first week of life with massive accumulation of MSUD biomarkers. Based on our previous experience in Bckdha-/- mice, we designed a transgene carrying the human BCKDHB gene under the control of a ubiquitous EF1α promoter, encapsidated in an AAV8 capsid. Injection in neonatal Bckdhb-/- mice at 1014 vg/kg achieved long-term rescue of the severe MSUD phenotype of Bckdhb-/- mice. These data further validate the efficacy of gene therapy for MSUD opening perspectives towards clinical translation.
View details for DOI 10.1002/jimd.12604
View details for PubMedID 36880392
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Improved engraftment and therapeutic efficacy by human genome-edited hematopoietic stem cells with Busulfan-based myeloablation.
Molecular therapy. Methods & clinical development
2022; 25: 392-409
Abstract
Autologous hematopoietic stem cell transplantation using genome-edited cells can become a definitive therapy for hematological and non-hematological disorders with neurological involvement. Proof-of-concept studies using human genome-edited hematopoietic stem cells have been hindered by the low efficiency of engraftment of the edited cells in the bone marrow and their modest efficacy in the CNS. To address these challenges, we tested a myeloablative conditioning regimen based on Busulfan in an immunocompromised model of mucopolysaccharidosis type 1. Compared with sub-lethal irradiation, Busulfan conditioning enhanced the engraftment of edited CD34+ cells in the bone marrow, as well the long-term homing and survival of bone-marrow-derived cells in viscera, and in the CNS, resulting in higher transgene expression and biochemical correction in these organs. Edited cell selection using a clinically compatible marker resulted in a population withlow engraftment potential. We conclude that conditioning can impact the engraftment of edited hematopoietic stem cells.Furthermore, Busulfan-conditioned recipients have a higher expression of therapeutic proteins in target organs, particularly in the CNS, constituting a better conditioning approach for non-hematological diseases with neurological involvement.
View details for DOI 10.1016/j.omtm.2022.04.009
View details for PubMedID 35573043
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Neonatal gene therapy achieves sustained disease rescue of maple syrup urine disease in mice.
Nature communications
2022; 13 (1): 3278
Abstract
Maple syrup urine disease (MSUD) is a rare recessively inherited metabolic disorder causing accumulation of branched chain amino acids leading to neonatal death, if untreated. Treatment for MSUD represents an unmet need because the current treatment with life-long low-protein diet is challenging to maintain, and despite treatment the risk of acute decompensations and neuropsychiatric symptoms remains. Here, based on significant liver contribution to the catabolism of the branched chain amino acid leucine, we develop a liver-directed adeno-associated virus (AAV8) gene therapy for MSUD. We establish and characterize the Bckdha (branched chain keto acid dehydrogenase a)-/- mouse that exhibits a lethal neonatal phenotype mimicking human MSUD. Animals were treated at P0 with intravenous human BCKDHA AAV8 vectors under the control of either a ubiquitous or a liver-specific promoter. BCKDHA gene transfer rescued the lethal phenotype. While the use of a ubiquitous promoter fully and sustainably rescued the disease (long-term survival, normal phenotype and correction of biochemical abnormalities), liver-specific expression of BCKDHA led to partial, though sustained rescue. Here we show efficacy of gene therapy for MSUD demonstrating its potential for clinical translation.
View details for DOI 10.1038/s41467-022-30880-w
View details for PubMedID 35672312
View details for PubMedCentralID PMC9174284
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Hepatic expression of GAA results in enhanced enzyme bioavailability in mice and non-human primates.
Nature communications
2021; 12 (1): 6393
Abstract
Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach.
View details for DOI 10.1038/s41467-021-26744-4
View details for PubMedID 34737297
View details for PubMedCentralID PMC8568898
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Gene therapy with secreted acid alpha-glucosidase rescues Pompe disease in a novel mouse model with early-onset spinal cord and respiratory defects.
EBioMedicine
2020; 61: 103052
Abstract
Pompe disease (PD) is a neuromuscular disorder caused by deficiency of acidalpha-glucosidase (GAA), leading to motor and respiratory dysfunctions. Available Gaa knock-out (KO) mouse models do not accurately mimic PD, particularly its highly impaired respiratory phenotype.Here we developed a new mouse model of PD crossing Gaa KOB6;129 with DBA2/J mice. We subsequently treated Gaa KODBA2/J mice with adeno-associated virus (AAV) vectors expressing a secretable form of GAA (secGAA).Male Gaa KODBA2/J mice present most of the key features of the human disease, including early lethality, severe respiratory impairment, cardiac hypertrophy and muscle weakness. Transcriptome analyses of Gaa KODBA2/J, compared to the parental Gaa KOB6;129 mice, revealed a profoundly impaired gene signature in the spinal cord and a similarly deregulated gene expression in skeletal muscle. Muscle and spinal cord transcriptome changes, biochemical defects, respiratory and muscle function in the Gaa KODBA2/J model were significantly improved upon gene therapy with AAV vectors expressing secGAA.These data show that the genetic background impacts on the severity of respiratory function and neuroglial spinal cord defects in the Gaa KO mouse model of PD. Our findings have implications for PD prognosis and treatment, show novel molecular pathophysiology mechanisms of the disease and provide a unique model to study PD respiratory defects, which majorly affect patients.This work was supported by Genethon, the French Muscular Dystrophy Association (AFM), the European Commission (grant nos. 667751, 617432, and 797144), and Spark Therapeutics.
View details for DOI 10.1016/j.ebiom.2020.103052
View details for PubMedID 33039711
View details for PubMedCentralID PMC7553357
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Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase.
Molecular therapy : the journal of the American Society of Gene Therapy
2020; 28 (9): 2056-2072
Abstract
Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa-/-) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa-/- mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa-/- mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa-/- mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa-/- mouse model via liver gene transfer of secGAA.
View details for DOI 10.1016/j.ymthe.2020.05.025
View details for PubMedID 32526204
View details for PubMedCentralID PMC7474269
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Engineering monocyte/macrophage−specific glucocerebrosidase expression in human hematopoietic stem cells using genome editing
Nature Communications
2020; 11: 1-14
View details for DOI 10.1038/s41467-020-17148-x
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Role of Regulatory T Cell and Effector T Cell Exhaustion in Liver-Mediated Transgene Tolerance in Muscle.
Molecular therapy. Methods & clinical development
2019; 15: 83-100
Abstract
The pro-tolerogenic environment of the liver makes this tissue an ideal target for gene replacement strategies. In other peripheral tissues such as the skeletal muscle, anti-transgene immune response can result in partial or complete clearance of the transduced fibers. Here, we characterized liver-induced transgene tolerance after simultaneous transduction of liver and muscle. A clinically relevant transgene, α-sarcoglycan, mutated in limb-girdle muscular dystrophy type 2D, was fused with the SIINFEKL epitope (hSGCA-SIIN) and expressed with adeno-associated virus vectors (AAV-hSGCA-SIIN). Intramuscular delivery of AAV-hSGCA-SIIN resulted in a strong inflammatory response, which could be prevented and reversed by concomitant liver expression of the same antigen. Regulatory T cells and upregulation of checkpoint inhibitor receptors were required to establish and maintain liver-mediated peripheral tolerance. This study identifies the fundamental role of the synergy between Tregs and upregulation of checkpoint inhibitor receptors in the liver-mediated control of anti-transgene immunity triggered by muscle-directed gene transfer.
View details for DOI 10.1016/j.omtm.2019.08.012
View details for PubMedID 31649958
View details for PubMedCentralID PMC6804827
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Gene Therapy for Pompe Disease: The Time is now.
Human gene therapy
2019; 30 (10): 1245-1262
Abstract
Pompe disease (PD) is caused by the deficiency of the lysosomal enzyme acid α-glucosidase (GAA), resulting in systemic pathological glycogen accumulation. PD can present with cardiac, skeletal muscle, and central nervous system manifestations, as a continuum of phenotypes among two main forms: classical infantile-onset PD (IOPD) and late-onset PD (LOPD). IOPD is caused by severe GAA deficiency and presents at birth with cardiac hypertrophy, muscle hypotonia, and severe respiratory impairment, leading to premature death, if not treated. LOPD is characterized by levels of residual GAA activity up to ∼20% of normal and presents both in children and adults with a varied severity of muscle weakness and motor and respiratory deficit. Enzyme replacement therapy (ERT), based on repeated intravenous (i.v.) infusions of recombinant human GAA (rhGAA), represents the only available treatment for PD. Upon more than 10 years from its launch, it is becoming evident that ERT can extend the life span of IOPD and stabilize disease progression in LOPD; however, it does not represent a cure for PD. The limited uptake of the enzyme in key affected tissues and the high immunogenicity of rhGAA are some of the hurdles that limit ERT efficacy. GAA gene transfer with adeno-associated virus (AAV) vectors has been shown to reduce glycogen storage and improve the PD phenotype in preclinical studies following different approaches. Here, we present an overview of the different gene therapy approaches for PD, focusing on in vivo gene transfer with AAV vectors and discussing the potential opportunities and challenges in developing safe and effective gene therapies for the disease. Based on emerging safety and efficacy data from clinical trials for other protein deficiencies, in vivo gene therapy with AAV vectors appears to have the potential to provide a therapeutically relevant, stable source of GAA enzyme, which could be highly beneficial in PD.
View details for DOI 10.1089/hum.2019.109
View details for PubMedID 31298581
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Dual muscle-liver transduction imposes immune tolerance for muscle transgene engraftment despite preexisting immunity.
JCI insight
2019; 4 (11)
Abstract
Immune responses to therapeutic transgenes are a potential hurdle to treat monogenic muscle disorders. These responses result from the neutralizing activity of transgene-specific B cells and cytotoxic T cells recruited upon gene transfer. We explored here how dual muscle-liver expression of a foreign transgene allows muscle transgene engraftment after adenoassociated viral vector delivery. We found in particular that induction of transgene-specific tolerance is imposed by concurrent muscle and liver targeting, resulting in the absence of CD8+ T cell responses to the transgene. This tolerance can be temporally decoupled, because transgene engraftment can be achieved in muscle weeks after liver transduction. Importantly, transgene-specific CD8+ T cell tolerance can be established despite preexisting immunity to the transgene. Whenever preexisting, transgene-specific CD4+ and CD8+ memory T cell responses are present, dual muscle-liver transduction turns polyclonal, transgene-specific CD8+ T cells into typically exhausted T cells with high programmed cell death 1 (PD-1) expression and lack of IFN-γ production. Our results demonstrate that successful transduction of muscle tissue can be achieved through liver-mediated control of humoral and cytotoxic T cell responses, even in the presence of preexisting immunity to the muscle-associated transgene.
View details for DOI 10.1172/jci.insight.127008
View details for PubMedID 31167976
View details for PubMedCentralID PMC6629094
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AAV Gene Transfer with Tandem Promoter Design Prevents Anti-transgene Immunity and Provides Persistent Efficacy in Neonate Pompe Mice
MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT
2019; 12: 85–101
Abstract
Hepatocyte-restricted, AAV-mediated gene transfer is being used to provide sustained, tolerogenic transgene expression in gene therapy. However, given the episomal status of the AAV genome, this approach cannot be applied to pediatric disorders when hepatocyte proliferation may result in significant loss of therapeutic efficacy over time. In addition, many multi-systemic diseases require widespread expression of the therapeutic transgene that, when provided with ubiquitous or tissue-specific non-hepatic promoters, often results in anti-transgene immunity. Here we have developed tandem promoter monocistronic expression cassettes that, packaged in a single AAV, provide combined hepatic and extra-hepatic tissue-specific transgene expression and prevent anti-transgene immunity. We validated our approach in infantile Pompe disease, a prototype disease caused by lack of the ubiquitous enzyme acid-alpha-glucosidase (GAA), presenting multi-systemic manifestations and detrimental anti-GAA immunity. We showed that the use of efficient tandem promoters prevents immune responses to GAA following systemic AAV gene transfer in immunocompetent Gaa-/- mice. Then we demonstrated that neonatal gene therapy with either AAV8 or AAV9 in Gaa-/- mice resulted in persistent therapeutic efficacy when using a tandem liver-muscle promoter (LiMP) that provided high and persistent transgene expression in non-dividing extra-hepatic tissues. In conclusion, the tandem promoter design overcomes important limitations of AAV-mediated gene transfer and can be beneficial when treating pediatric conditions requiring persistent multi-systemic transgene expression and prevention of anti-transgene immunity.
View details for DOI 10.1016/j.omtm.2018.11.002
View details for Web of Science ID 000461055600007
View details for PubMedID 30581888
View details for PubMedCentralID PMC6299151
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Emerging Issues in AAV-Mediated In Vivo Gene Therapy.
Molecular therapy. Methods & clinical development
2018; 8: 87-104
Abstract
In recent years, the number of clinical trials in which adeno-associated virus (AAV) vectors have been used for in vivo gene transfer has steadily increased. The excellent safety profile, together with the high efficiency of transduction of a broad range of target tissues, has established AAV vectors as the platform of choice for in vivo gene therapy. Successful application of the AAV technology has also been achieved in the clinic for a variety of conditions, including coagulation disorders, inherited blindness, and neurodegenerative diseases, among others. Clinical translation of novel and effective "therapeutic products" is, however, a long process that involves several cycles of iterations from bench to bedside that are required to address issues encountered during drug development. For the AAV vector gene transfer technology, several hurdles have emerged in both preclinical studies and clinical trials; addressing these issues will allow in the future to expand the scope of AAV gene transfer as a therapeutic modality for a variety of human diseases. In this review, we will give an overview on the biology of AAV vector, discuss the design of AAV-based gene therapy strategies for in vivo applications, and present key achievements and emerging issues in the field. We will use the liver as a model target tissue for gene transfer based on the large amount of data available from preclinical and clinical studies.
View details for DOI 10.1016/j.omtm.2017.11.007
View details for PubMedID 29326962
View details for PubMedCentralID PMC5758940
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Rescue of GSDIII Phenotype with Gene Transfer Requires Liver- and Muscle-Targeted GDE Expression
MOLECULAR THERAPY
2018; 26 (3): 890–901
Abstract
Glycogen storage disease type III (GSDIII) is an autosomal recessive disorder caused by a deficiency of glycogen-debranching enzyme (GDE), which results in profound liver metabolism impairment and muscle weakness. To date, no cure is available for GSDIII and current treatments are mostly based on diet. Here we describe the development of a mouse model of GSDIII, which faithfully recapitulates the main features of the human condition. We used this model to develop and test novel therapies based on adeno-associated virus (AAV) vector-mediated gene transfer. First, we showed that overexpression of the lysosomal enzyme alpha-acid glucosidase (GAA) with an AAV vector led to a decrease in liver glycogen content but failed to reverse the disease phenotype. Using dual overlapping AAV vectors expressing the GDE transgene in muscle, we showed functional rescue with no impact on glucose metabolism. Liver expression of GDE, conversely, had a direct impact on blood glucose levels. These results provide proof of concept of correction of GSDIII with AAV vectors, and they indicate that restoration of the enzyme deficiency in muscle and liver is necessary to address both the metabolic and neuromuscular manifestations of the disease.
View details for DOI 10.1016/j.ymthe.2017.12.019
View details for Web of Science ID 000427911800024
View details for PubMedID 29396266
View details for PubMedCentralID PMC5910667
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Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid alpha-glucosidase
SCIENCE TRANSLATIONAL MEDICINE
2017; 9 (418)
Abstract
Glycogen storage disease type II or Pompe disease is a severe neuromuscular disorder caused by mutations in the lysosomal enzyme, acid α-glucosidase (GAA), which result in pathological accumulation of glycogen throughout the body. Enzyme replacement therapy is available for Pompe disease; however, it has limited efficacy, has high immunogenicity, and fails to correct pathological glycogen accumulation in nervous tissue and skeletal muscle. Using bioinformatics analysis and protein engineering, we developed transgenes encoding GAA that could be expressed and secreted by hepatocytes. Then, we used adeno-associated virus (AAV) vectors optimized for hepatic expression to deliver the GAA transgenes to Gaa knockout (Gaa-/-) mice, a model of Pompe disease. Therapeutic gene transfer to the liver rescued glycogen accumulation in muscle and the central nervous system, and ameliorated cardiac hypertrophy as well as muscle and respiratory dysfunction in the Gaa-/- mice; mouse survival was also increased. Secretable GAA showed improved therapeutic efficacy and lower immunogenicity compared to nonengineered GAA. Scale-up to nonhuman primates, and modeling of GAA expression in primary human hepatocytes using hepatotropic AAV vectors, demonstrated the therapeutic potential of AAV vector-mediated liver expression of secretable GAA for treating pathological glycogen accumulation in multiple tissues in Pompe disease.
View details for PubMedID 29187643
View details for PubMedCentralID PMC5826611
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Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics.
Molecular therapy. Nucleic acids
2016; 5 (11): e392
Abstract
Disease-causing splicing mutations can be rescued by variants of the U1 small nuclear RNA (U1snRNAs). However, the evaluation of the efficacy and safety of modified U1snRNAs as therapeutic tools is limited by the availability of cellular and animal models specific for a given mutation. Hence, we exploited the hyperactive Sleeping Beauty transposon system (SB100X) to integrate human factor IX (hFIX) minigenes into genomic DNA in vitro and in vivo. We generated stable HEK293 cell lines and C57BL/6 mice harboring splicing-competent hFIX minigenes either wild type (SChFIX-wt) or mutated (SChFIXex5-2C). In both models the SChFIXex5-2C variant, found in patients affected by Hemophilia B, displayed an aberrant splicing pattern characterized by exon 5 skipping. This allowed us to test, for the first time in a genomic DNA context, the efficacy of the snRNA U1-fix9, delivered with an adeno-associated virus (AAV) vector. With this approach, we showed rescue of the correct splicing pattern of hFIX mRNA, leading to hFIX protein expression. These data validate the SB100X as a versatile tool to quickly generate models of human genetic mutations, to study their effect in a stable DNA context and to assess mutation-targeted therapeutic strategies.
View details for DOI 10.1038/mtna.2016.97
View details for PubMedID 27898092
View details for PubMedCentralID PMC5155329
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Long-term exposure to Myozyme results in a decrease of anti-drug antibodies in late-onset Pompe disease patients.
Scientific reports
2016; 6: 36182
Abstract
Immunogenicity of recombinant human acid-alpha glucosidase (rhGAA) in enzyme replacement therapy (ERT) is a safety and efficacy concern in the management of late-onset Pompe disease (LOPD). However, long-term effects of ERT on humoral and cellular responses to rhGAA are still poorly understood. To better understand the impact of immunogenicity of rhGAA on the efficacy of ERT, clinical data and blood samples from LOPD patients undergoing ERT for >4 years (n = 28) or untreated (n = 10) were collected and analyzed. In treated LOPD patients, anti-rhGAA antibodies peaked within the first 1000 days of ERT, while long-term exposure to rhGAA resulted in clearance of antibodies with residual production of non-neutralizing IgG. Analysis of T cell responses to rhGAA showed detectable T cell reactivity only after in vitro restimulation. Upregulation of several cytokines and chemokines was detectable in both treated and untreated LOPD subjects, while IL2 secretion was detectable only in subjects who received ERT. These results indicate that long-term ERT in LOPD patients results in a decrease in antibody titers and residual production of non-inhibitory IgGs. Immune responses to GAA following long-term ERT do not seem to affect efficacy of ERT and are consistent with an immunomodulatory effect possibly mediated by regulatory T cells.
View details for DOI 10.1038/srep36182
View details for PubMedID 27812025
View details for PubMedCentralID PMC5096052
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Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease.
Human molecular genetics
2015; 24 (23): 6811-25
Abstract
Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4-/- mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5'-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4-/- mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1.
View details for DOI 10.1093/hmg/ddv386
View details for PubMedID 26420842
View details for PubMedCentralID PMC4634381
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Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors.
Gene therapy
2014; 21 (4): 450-6
Abstract
Gene therapy with adeno-associated viral (AAV) vectors is limited by AAV cargo capacity that prevents their application to the inherited retinal diseases (IRDs), such as Stargardt disease (STGD) or Usher syndrome type IB (USH1B), which are due to mutations in genes larger than 5 kb. Trans-splicing or hybrid dual AAV vectors have been successfully exploited to reconstitute large gene expression in the mouse retina. Here, we tested them in the large cone-enriched pig retina that closely mimics the human retina. We found that dual AAV trans-splicing and hybrid vectors transduce pig photoreceptors, the major cell targets for treatment of IRDs, to levels that were about two- to threefold lower than those obtained with a single AAV vector of normal size. This efficiency is significantly higher than that in mice, and is potentially due to the high levels of dual AAV co-transduction we observe in pigs. We also show that subretinal delivery in pigs of dual AAV trans-splicing and hybrid vectors successfully reconstitute, albeit at variable levels, the expression of the large genes ABCA4 and MYO7A mutated in STGD and USH1B, respectively. Our data support the potential of dual AAV vectors for large gene reconstitution in the cone-enriched pig retina that is a relevant preclinical model.
View details for DOI 10.1038/gt.2014.8
View details for PubMedID 24572793
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Effective delivery of large genes to the retina by dual AAV vectors.
EMBO molecular medicine
2014; 6 (2): 194-211
Abstract
Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on 'forced' packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.
View details for DOI 10.1002/emmm.201302948
View details for PubMedID 24150896
View details for PubMedCentralID PMC3927955
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Myosin7a deficiency results in reduced retinal activity which is improved by gene therapy.
PloS one
2013; 8 (8): e72027
Abstract
Mutations in MYO7A cause autosomal recessive Usher syndrome type IB (USH1B), one of the most frequent conditions that combine severe congenital hearing impairment and retinitis pigmentosa. A promising therapeutic strategy for retinitis pigmentosa is gene therapy, however its pre-clinical development is limited by the mild retinal phenotype of the shaker1 (sh1(-/-)) murine model of USH1B which lacks both retinal functional abnormalities and degeneration. Here we report a significant, early-onset delay of sh1(-/-) photoreceptor ability to recover from light desensitization as well as a progressive reduction of both b-wave electroretinogram amplitude and light sensitivity, in the absence of significant loss of photoreceptors up to 12 months of age. We additionally show that subretinal delivery to the sh1(-/-) retina of AAV vectors encoding the large MYO7A protein results in significant improvement of sh1(-/-) photoreceptor and retinal pigment epithelium ultrastructural anomalies which is associated with improvement of recovery from light desensitization. These findings provide new tools to evaluate the efficacy of experimental therapies for USH1B. In addition, although AAV vectors expressing large genes might have limited clinical applications due to their genome heterogeneity, our data show that AAV-mediated MYO7A gene transfer to the sh1(-/-) retina is effective.
View details for DOI 10.1371/journal.pone.0072027
View details for PubMedID 23991031
View details for PubMedCentralID PMC3753344
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Photoreceptor degeneration in mice: adeno-associated viral vector-mediated delivery of erythropoietin.
Methods in molecular biology (Clifton, N.J.)
2013; 982: 237-63
Abstract
The exogenous delivery of erythropoietin (EPO) and EPO derivatives (EPO-Ds) represents a valuable strategy to protect the retina from degeneration. In this chapter we describe a method to deliver EPO and the EPO derivative S100E in the light-damage model of induced retinal degeneration using adeno--associated viral (AAV) vectors and to evaluate the functional and morphological protection of the retina from light damage.
View details for DOI 10.1007/978-1-62703-308-4_16
View details for PubMedID 23456874
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Gene therapy of inherited retinopathies: a long and successful road from viral vectors to patients.
Human gene therapy
2012; 23 (8): 796-807
Abstract
Inherited retinopathies (IRs) are common and untreatable blinding conditions inherited mostly as monogenic due to mutations in genes expressed in retinal photoreceptors (PRs) and in retinal pigment epithelium (RPE). Over the last two decades, the retina has emerged as one of the most favorable target tissues for gene therapy given its small size and its enclosed and immune-privileged environment. Different types of viral vectors have been developed, especially those based on the adeno-associated virus (AAV), which efficiently deliver therapeutic genes to PRs or RPE upon subretinal injections. Dozens of successful proofs of concept of the efficacy of gene therapy for recessive and dominant IRs have been generated in small and large models that have paved the way to the first clinical trials using AAV in patients with Leber congenital amaurosis, a severe form of childhood blindness. The results from these initial trials suggest that retinal gene therapy with AAV is safe in humans, that vision can be improved in patients that have suffered from severe impairment of visual function, in some cases for decades, and that readministration of AAV to the subretinal space is feasible, effective, and safe. However, none of the trials could match the levels of efficacy of gene therapy observed in a dog model of the disease, suggesting that there is room for improvement. In conclusion, these results bode well for further testing of AAV-mediated retinal gene therapy in patients with other monogenic and complex forms of blindness.
View details for DOI 10.1089/hum.2012.123
View details for PubMedID 22734691
View details for PubMedCentralID PMC3413895
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TFEB links autophagy to lysosomal biogenesis.
Science (New York, N.Y.)
2011; 332 (6036): 1429-33
Abstract
Autophagy is a cellular catabolic process that relies on the cooperation of autophagosomes and lysosomes. During starvation, the cell expands both compartments to enhance degradation processes. We found that starvation activates a transcriptional program that controls major steps of the autophagic pathway, including autophagosome formation, autophagosome-lysosome fusion, and substrate degradation. The transcription factor EB (TFEB), a master gene for lysosomal biogenesis, coordinated this program by driving expression of autophagy and lysosomal genes. Nuclear localization and activity of TFEB were regulated by serine phosphorylation mediated by the extracellular signal-regulated kinase 2, whose activity was tuned by the levels of extracellular nutrients. Thus, a mitogen-activated protein kinase-dependent mechanism regulates autophagy by controlling the biogenesis and partnership of two distinct cellular organelles.
View details for DOI 10.1126/science.1204592
View details for PubMedID 21617040
View details for PubMedCentralID PMC3638014
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Non-erythropoietic erythropoietin derivatives protect from light-induced and genetic photoreceptor degeneration.
Human molecular genetics
2011; 20 (11): 2251-62
Abstract
Given the high genetic heterogeneity of inherited retinal degenerations (IRDs), a wide applicable treatment would be desirable to halt/slow progressive photoreceptor (PR) cell loss in a mutation-independent manner. In addition to its erythropoietic activity, erythropoietin (EPO) presents neurotrophic characteristics. We have previously shown that adeno-associated viral (AAV) vector-mediated systemic EPO delivery protects from PR degeneration. However, this is associated with an undesired hematocrit increase that could contribute to PR protection. Non-erythropoietic EPO derivatives (EPO-D) are available which allow us to dissect erythropoiesis's role in PR preservation and may be more versatile and safe than EPO as anti-apoptotic agents. We delivered in animal models of light-induced or genetic retinal degeneration either intramuscularly or subretinally AAV vectors encoding EPO or one of the three selected EPO-D: the mutant S100E, the helix A- and B-derived EPO-mimetic peptides. We observed that (i) systemic expression of S100E induces a significantly lower hematocrit increase than EPO and provides similar protection from PR degeneration, and (ii) intraocular expression of EPO-D protects PR from degeneration in the absence of significant hematocrit increase. On the basis of this, we conclude that erythropoiesis is not required for EPO-mediated PR protection. However, the lower efficacy observed when EPO or S100E is expressed intraocularly rather than systemically suggests that hormone systemic effects contribute to PR protection. Unlike S100E, EPO-mimetic peptides preserve PR only when given locally, suggesting that different EPO-D have a different potency or mode of action. In conclusion, our data show that subretinal delivery of AAV vectors encoding EPO-D protects from light-induced and genetic PR degeneration.
View details for DOI 10.1093/hmg/ddr115
View details for PubMedID 21421996
View details for PubMedCentralID PMC3090200
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AAV-mediated gene supply for treatment of degenerative and neovascular retinal diseases.
Current gene therapy
2010; 10 (5): 371-80
Abstract
Common blinding diseases that are currently untreatable include conditions characterized by progressive neuronal degeneration, such as retinitis pigmentosa, leber congenital amaurosis or glaucoma, or characterized by ocular neovascularization, like wet age-related macular degeneration, proliferative diabetic retinopathy and retinopathy of prematurity. The pathogenic mechanisms underlying either neuronal degeneration or new vessel formation may be similar and independent of the mutation underlying the disease, thus allowing to test therapeutic strategies acting downstream of the primary causative event. Gene supply is the delivery of a gene that can prevent or arrest disease progression without being directly implicated in the disease pathogenesis. To this end, one of the most efficient and safe retinal gene delivery vehicles derives from the small adeno-associated virus (AAV). We review studies on AAV-mediated gene supply of: neurotrophic/antiapoptotic factors to prevent retinal neurons degeneration, and anti-angiogenic molecules to inhibit retinal neovascularization. Successful gene supply may represent a one-fit-all treatment for inherited and acquired blinding diseases.
View details for DOI 10.2174/156652310793180670
View details for PubMedID 20712580
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Ocular gene therapy: current progress and future prospects.
Trends in molecular medicine
2009; 15 (1): 23-31
Abstract
As gene therapy begins to produce its first clinical successes, interest in ocular gene transfer has grown owing to the favorable safety and efficacy characteristics of the eye as a target organ for drug delivery. Important advances also include the availability of viral and non-viral vectors that are able to efficiently transduce various ocular cell types, the use of intraocular delivery routes and the development of transcriptional regulatory elements that allow sustained levels of gene transfer in small and large animal models after a single administration. Here, we review recent progress in the field of ocular gene therapy. The first experiments in humans with severe inherited forms of blindness seem to confirm the good safety and efficacy profiles observed in animal models and suggest that gene transfer has the potential to become a valuable therapeutic strategy for otherwise untreatable blinding diseases.
View details for DOI 10.1016/j.molmed.2008.11.003
View details for PubMedID 19097940
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AP20187-mediated activation of a chimeric insulin receptor results in insulin-like actions in skeletal muscle and liver of diabetic mice.
Human gene therapy
2007; 18 (2): 106-17
Abstract
Diabetes mellitus (DM) derives from either insulin deficiency (type 1) or resistance (type 2). Insulin regulates glucose metabolism and homeostasis by binding to a specific membrane receptor (IR) with tyrosine kinase activity, expressed by its canonical target tissues. General or tissue-specific IR ablation in mice results in complex metabolic abnormalities, which give partial insights into the role of IR signaling in glucose homeostasis and diabetes development. We generated a chimeric IR (LFv2IRE) inducible on administration of the small molecule drug AP20187. This represents a powerful tool to induce insulin receptor signaling in the hormone target tissues in DM animal models. Here we use adeno-associated viral (AAV) vectors to transduce muscle and liver of nonobese diabetic (NOD) mice with LFv2IRE. Systemic AP20187 administration results in time-dependent LFv2IRE tyrosine phosphorylation and activation of the insulin signaling pathway in both liver and muscle of AAV-treated NOD mice. AP20187 stimulation significantly increases hepatic glycogen content and muscular glucose uptake similarly to insulin. The LFv2IRE-AP20187 system represents a useful tool for regulated and rapid tissue-specific restoration of IR signaling and for dissection of insulin signaling and function in the hormone canonical and noncanonical target tissues.
View details for DOI 10.1089/hum.2006.116
View details for PubMedID 17328681