Emerging Issues in AAV-Mediated In Vivo Gene Therapy Colella, Pasqualina; Ronzitti, Giuseppe; Mingozzi, Federico
Molecular therapy. Methods & clinical development,
03/2018, Letnik:
8, Številka:
C
Journal Article
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In recent years, the number of clinical trials in which adeno-associated virus (AAV) vectors have been used for
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
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
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.
Gaucher disease is a lysosomal storage disorder caused by insufficient glucocerebrosidase activity. Its hallmark manifestations are attributed to infiltration and inflammation by macrophages. Current ...therapies for Gaucher disease include life-long intravenous administration of recombinant glucocerebrosidase and orally-available glucosylceramide synthase inhibitors. An alternative approach is to engineer the patient's own hematopoietic system to restore glucocerebrosidase expression, thereby replacing the affected cells, and constituting a potential one-time therapy for this disease. Here, we report an efficient CRISPR/Cas9-based approach that targets glucocerebrosidase expression cassettes with a monocyte/macrophage-specific element to the CCR5 safe-harbor locus in human hematopoietic stem and progenitor cells. The targeted cells generate glucocerebrosidase-expressing macrophages and maintain long-term repopulation and multi-lineage differentiation potential with serial transplantation. The combination of a safe-harbor and a lineage-specific promoter establishes a universal correction strategy and circumvents potential toxicity of ectopic glucocerebrosidase in the stem cells. Furthermore, it constitutes an adaptable platform for other lysosomal enzyme deficiencies.
TFEB Links Autophagy to Lysosomal Biogenesis Settembre, Carmine; Di Malta, Chiara; Polito, Vinicia Assunta ...
Science (American Association for the Advancement of Science),
06/2011, Letnik:
332, Številka:
6036
Journal Article
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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.
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.
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.
Synopsis
The Authors provide proof‐of‐concept of gene therapy for two inherited retinal degeneration conditions by using dual AAV vectors. This allows the expansion of AAV cargo capacity for gene therapy of syndromes that require the transfer of large genes.
Dual AAV vectors significantly expand AAV cargo capacity in the retina.
Unlike dual AAV overlapping vectors, trans‐splicing and hybrid vectors efficiently transduce photoreceptors.
Subretinal delivery of dual AAV vectors improves the retinal phenotype of murine models of inherited blinding conditions due to mutations in large genes.
The Authors provide proof‐of‐concept of gene therapy for two inherited retinal degeneration conditions by using dual AAV vectors. This allows the expansion of AAV cargo capacity for gene therapy of syndromes that require the transfer of large genes.
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.
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.
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.
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