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1.
N Engl J Med ; 383(2): 151-158, 2020 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-32640133

RESUMEN

Two patients with familial amyotrophic lateral sclerosis (ALS) and mutations in the gene encoding superoxide dismutase 1 (SOD1) were treated with a single intrathecal infusion of adeno-associated virus encoding a microRNA targeting SOD1. In Patient 1, SOD1 levels in spinal cord tissue as analyzed on autopsy were lower than corresponding levels in untreated patients with SOD1-mediated ALS and in healthy controls. Levels of SOD1 in cerebrospinal fluid were transiently and only slightly lower in Patient 1 but were not affected in Patient 2. In Patient 1, meningoradiculitis developed after the infusion; Patient 2 was pretreated with immunosuppressive drugs and did not have this complication. Patient 1 had transient improvement in the strength of his right leg, a measure that had been relatively stable throughout his disease course, but there was no change in his vital capacity. Patient 2 had stable scores on a composite measure of ALS function and a stable vital capacity during a 12-month period. This study showed that intrathecal microRNA can be used as a potential treatment for SOD1-mediated ALS.


Asunto(s)
Esclerosis Amiotrófica Lateral/terapia , MicroARNs/uso terapéutico , Superóxido Dismutasa-1/líquido cefalorraquídeo , Esclerosis Amiotrófica Lateral/líquido cefalorraquídeo , Esclerosis Amiotrófica Lateral/genética , Dependovirus , Resultado Fatal , Silenciador del Gen , Terapia Genética , Vectores Genéticos , Humanos , Inyecciones Espinales , Masculino , Meningoencefalitis , Persona de Mediana Edad , Mutación , Prueba de Estudio Conceptual , Médula Espinal/química , Médula Espinal/patología , Superóxido Dismutasa-1/análisis , Superóxido Dismutasa-1/genética , Capacidad Vital , Adulto Joven
2.
Int J Mol Sci ; 24(13)2023 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-37445621

RESUMEN

Recombinant Adeno-Associated Virus (rAAV) is considered as one of the most successful and widely used viral vectors for in vivo gene therapy. However, host immune responses to the vector and/or the transgene product remain a major hurdle to successful AAV gene transfer. In contrast to antivector adaptive immunity, the initiation of the innate immunity towards rAAV is still poorly understood but is directly dependent on the interaction between the viral vector and innate immune cells. Here, we used a quantitative transcriptomic-based approach to determine the activation of inflammatory and anti-viral pathways after rAAV8-based infection of monocyte-derived dendritic cells (moDCs) obtained from 12 healthy human donors. We have shown that rAAV8 particles are efficiently internalized, but that this uptake does not induce any detectable transcriptomic change in moDCs in contrast to an adenoviral infection, which upregulates anti-viral pathways. These findings suggest an immunologically favorable profile for rAAV8 serotype with regard to in vitro activation of moDC model. Transcriptomic analysis of rAAV-infected innate immune cells is a powerful method to determine the ability of the viral vector to be seen by these sensor cells, which remains of great importance to better understand the immunogenicity of rAAV vectors and to design immune-stealth products.


Asunto(s)
Monocitos , Transcriptoma , Humanos , Vectores Genéticos/genética , Inmunidad Adaptativa , Células Dendríticas , Dependovirus/genética
3.
J Biol Chem ; 295(38): 13213-13223, 2020 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-32723872

RESUMEN

α1-Antitrypsin (AAT) encoded by the SERPINA1 gene is an acute-phase protein synthesized in the liver and secreted into the circulation. Its primary role is to protect lung tissue by inhibiting neutrophil elastase. The Z allele of SERPINA1 encodes a mutant AAT, named ATZ, that changes the protein structure and leads to its misfolding and polymerization, which cause endoplasmic reticulum (ER) stress and liver disease through a gain-of-function toxic mechanism. Hepatic retention of ATZ results in deficiency of one of the most important circulating proteinase inhibitors and predisposes to early-onset emphysema through a loss-of-function mechanism. The pathogenetic mechanisms underlying the liver disease are not completely understood. C/EBP-homologous protein (CHOP), a transcription factor induced by ER stress, was found among the most up-regulated genes in livers of PiZ mice that express ATZ and in human livers of patients homozygous for the Z allele. Compared with controls, juvenile PiZ/Chop-/- mice showed reduced hepatic ATZ and a transcriptional response indicative of decreased ER stress by RNA-Seq analysis. Livers of PiZ/Chop-/- mice also showed reduced SERPINA1 mRNA levels. By chromatin immunoprecipitations and luciferase reporter-based transfection assays, CHOP was found to up-regulate SERPINA1 cooperating with c-JUN, which was previously shown to up-regulate SERPINA1, thus aggravating hepatic accumulation of ATZ. Increased CHOP levels were detected in diseased livers of children homozygous for the Z allele. In summary, CHOP and c-JUN up-regulate SERPINA1 transcription and play an important role in hepatic disease by increasing the burden of proteotoxic ATZ, particularly in the pediatric population.


Asunto(s)
Hepatopatías/metabolismo , Hígado/metabolismo , Mutación , Agregación Patológica de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción CHOP/metabolismo , alfa 1-Antitripsina/biosíntesis , Alelos , Animales , Estrés del Retículo Endoplásmico/genética , Humanos , Hígado/patología , Hepatopatías/genética , Hepatopatías/patología , Ratones , Ratones Noqueados , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Pliegue de Proteína , Proteínas Proto-Oncogénicas c-jun/genética , Factor de Transcripción CHOP/genética , Transcripción Genética , Regulación hacia Arriba , alfa 1-Antitripsina/genética
4.
Mol Ther ; 28(3): 747-757, 2020 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-31982038

RESUMEN

With the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) approvals for Zolgensma, Luxturna, and Glybera, recombinant adeno-associated viruses (rAAVs) are considered efficient tools for gene transfer. However, studies in animals and humans demonstrate that intramuscular (IM) AAV delivery can trigger immune responses to AAV capsids and/or transgenes. IM delivery of rAAV1 in humans has also been described to induce tolerance to rAAV characterized by the presence of capsid-specific regulatory T cells (Tregs) in periphery. To understand mechanisms responsible for tolerance and parameters involved, we tested 3 muscle-directed administration routes in rhesus monkeys: IM delivery, venous limb perfusion, and the intra-arterial push and dwell method. These 3 methods were well tolerated and led to transgene expression. Interestingly, gene transfer in muscle led to Tregs and exhausted T cell infiltrates in situ at both day 21 and day 60 post-injection. In human samples, an in-depth analysis of the functionality of these cells demonstrates that capsid-specific exhausted T cells are detected after at least 5 years post-vector delivery and that the exhaustion can be reversed by blocking the checkpoint pathway. Overall, our study shows that persisting transgene expression after gene transfer in muscle is mediated by Tregs and exhausted T cells.


Asunto(s)
Proteínas de la Cápside/inmunología , Dependovirus/genética , Dependovirus/inmunología , Técnicas de Transferencia de Gen , Vectores Genéticos/efectos adversos , Vectores Genéticos/genética , Músculos , Transducción Genética , Animales , Expresión Génica , Vectores Genéticos/administración & dosificación , Humanos , Inmunidad Celular , Inmunidad Humoral , Inyecciones Intramusculares , Recuento de Linfocitos , Macaca mulatta , Músculos/metabolismo , Especificidad de Órganos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Transgenes
5.
Mol Ther ; 25(11): 2477-2489, 2017 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-29032169

RESUMEN

Hepatocytes represent an important target for gene therapy and editing of single-gene disorders. In α-1 antitrypsin (AAT) deficiency, one missense mutation results in impaired secretion of AAT. In most patients, lung damage occurs due to a lack of AAT-mediated protection of lung elastin from neutrophil elastase. In some patients, accumulation of misfolded PiZ mutant AAT protein triggers hepatocyte injury, leading to inflammation and cirrhosis. We hypothesized that correcting the Z mutant defect in hepatocytes would confer a selective advantage for repopulation of hepatocytes within an intact liver. A human PiZ allele was crossed onto an immune-deficient (NSG) strain to create a recipient strain (NSG-PiZ) for human hepatocyte xenotransplantation. Results indicate that NSG-PiZ recipients support heightened engraftment of normal human primary hepatocytes as compared with NSG recipients. This model can therefore be used to test hepatocyte cell therapies for AATD, but more broadly it serves as a simple, highly reproducible liver xenograft model. Finally, a promoterless adeno-associated virus (AAV) vector, expressing a wild-type AAT and a synthetic miRNA to silence the endogenous allele, was integrated into the albumin locus. This gene-editing approach leads to a selective advantage of edited hepatocytes, by silencing the mutant protein and augmenting normal AAT production, and improvement of the liver pathology.


Asunto(s)
Dependovirus/genética , Terapia Genética/métodos , Hepatocitos/trasplante , Transgenes , Deficiencia de alfa 1-Antitripsina/terapia , alfa 1-Antitripsina/genética , Animales , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Edición Génica , Expresión Génica , Silenciador del Gen , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Supervivencia de Injerto , Hepatocitos/enzimología , Hepatocitos/patología , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , MicroARNs/genética , MicroARNs/metabolismo , Mutación , Trasplante Heterólogo , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/enzimología , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/patología
6.
Mol Ther ; 25(6): 1387-1394, 2017 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-28408179

RESUMEN

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%-3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%-2.5% of the target level from years 1-5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated.


Asunto(s)
Expresión Génica , Neutrófilos/metabolismo , Deficiencia de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Biomarcadores , Biopsia , Cápside/inmunología , Dependovirus/genética , Dependovirus/inmunología , Epítopos de Linfocito T/inmunología , Terapia Genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Inmunohistoquímica , Inmunofenotipificación , Músculos/metabolismo , Músculos/patología , Neutrófilos/enzimología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factores de Tiempo , Transgenes , Deficiencia de alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/terapia
7.
Mol Ther ; 22(9): 1605-13, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24954473

RESUMEN

The robustness and safety of liver-directed gene therapy can be substantially improved by enhancing expression of the therapeutic transgene in the liver. To achieve this, we developed a new approach of rational in silico vector design. This approach relies on a genome-wide bio-informatics strategy to identify cis-acting regulatory modules (CRMs) containing evolutionary conserved clusters of transcription factor binding site motifs that determine high tissue-specific gene expression. Incorporation of these CRMs into adeno-associated viral (AAV) and non-viral vectors enhanced gene expression in mice liver 10 to 100-fold, depending on the promoter used. Furthermore, these CRMs resulted in robust and sustained liver-specific expression of coagulation factor IX (FIX), validating their immediate therapeutic and translational relevance. Subsequent translational studies indicated that therapeutic FIX expression levels could be attained reaching 20-35% of normal levels after AAV-based liver-directed gene therapy in cynomolgus macaques. This study underscores the potential of rational vector design using computational approaches to improve their robustness and therefore allows for the use of lower and thus safer vector doses for gene therapy, while maximizing therapeutic efficacy.


Asunto(s)
Sitios de Unión , Biología Computacional/métodos , Dependovirus/genética , Hígado/metabolismo , Macaca/virología , Factores de Transcripción/genética , Animales , Secuencia de Bases , Secuencia Conservada , Factor IX/genética , Factor IX/metabolismo , Vectores Genéticos/administración & dosificación , Genoma , Humanos , Hígado/virología , Macaca/genética , Ratones , Especificidad de Órganos , Elementos Reguladores de la Transcripción , Factores de Transcripción/metabolismo
8.
Hum Gene Ther ; 35(9-10): 355-364, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38581431

RESUMEN

Recombinant adeno-associated virus (rAAV) vectors appear, more than ever, to be efficient viral vectors for in vivo gene transfer as illustrated by the approvals of 7 drugs across Europe and the United States. Nevertheless, preexisting immunity to AAV capsid in humans remains one of the major limits for a successful clinical translation. Whereas a preexisting humoral response to AAV capsid is well documented, the prevalence of preexisting capsid-specific T cell responses still needs to be studied and characterized. In this study, we investigated the prevalence of AAV-specific circulating T cells toward AAV2, 4, 5, 8, 9, and rh10 in a large cohort of healthy donors using the standard IFNγ ELISpot assay. We observed the highest prevalence of preexisting cellular immunity to AAV9 serotype followed by AAV8, AAV4, AAV2, AAVrh10, and AAV5 independently of the donors' serological status. An in-depth analysis of T cell responses toward the 2 most prevalent serotypes 8 and 9 shows that IFNγ secretion is mainly mediated by CD8 T cells for both serotypes. A polyfunctional analysis reveals different cytokine profiles between AAV8 and AAV9. Surprisingly, no IL-2 secretion was mediated by anti-AAV9 immune cells suggesting that these cells may rather be exhausted or terminally differentiated than cytotoxic T cells. Altogether, these results suggest that preexisting immunity to AAV may vary depending on the serotype and support the necessity of using multiparametric monitoring methods to better characterize anticapsid cellular immunity and foresee its impact in rAAV-mediated clinical trials.


Asunto(s)
Proteínas de la Cápside , Dependovirus , Vectores Genéticos , Inmunidad Celular , Humanos , Dependovirus/genética , Dependovirus/inmunología , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/genética , Vectores Genéticos/genética , Voluntarios Sanos , Cápside/inmunología , Linfocitos T CD8-positivos/inmunología , Interferón gamma/metabolismo , Adulto , Serogrupo , Masculino , Femenino , Citocinas/metabolismo , Linfocitos T/inmunología
9.
Mol Ther Methods Clin Dev ; 32(1): 101200, 2024 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-38445045

RESUMEN

Alpha-1 antitrypsin deficiency (AATD) is characterized by both chronic lung disease due to loss of wild-type AAT (M-AAT) antiprotease function and liver disease due to toxicity from delayed secretion, polymerization, and aggregation of misfolded mutant AAT (Z-AAT). The ideal gene therapy for AATD should therefore comprise both endogenous Z-AAT suppression and M-AAT overexpression. We designed a dual-function rAAV3B (df-rAAV3B) construct, which was effective at transducing hepatocytes, resulting in a considerable decrease of Z-AAT levels and safe M-AAT augmentation in mice. We optimized df-rAAV3B and created two variants, AAV3B-E12 and AAV3B-G3, to simultaneously enhance the concentration of M-AAT in the bloodstream to therapeutic levels and silence endogenous AAT liver expression in cynomolgus monkeys. Our results demonstrate that AAV3b-WT, AAV3B-E12, and AAV3B-G3 were able to transduce the monkey livers and achieve high M-AAT serum levels efficiently and safely. In this nondeficient model, we did not find downregulation of endogenous AAT. However, the dual-function vector did serve as a potentially "liver-sparing" alternative for high-dose liver-mediated AAT gene replacement in the context of underlying liver disease.

10.
Mol Ther Methods Clin Dev ; 25: 425-438, 2022 Jun 09.
Artículo en Inglés | MEDLINE | ID: mdl-35592360

RESUMEN

α1-antitrypsin deficiency is a rare genetic condition that can cause liver and/or lung disease. There is currently no cure for this disorder, although repeated infusions of plasma-purified protein may slow down emphysema progression. Gene therapy in which a single recombinant adeno-associated viral vector (rAAV) administration would lead to sustained protein expression could therefore similarly affect disease progression, and provide the added benefits of reducing treatment burden and thereby improving the patient's quality of life. The study presented here tests whether treating the Serpina1a-e knockout mouse model of α1-antitrypsin-deficiency lung disease with gene therapy would have an impact on the disease course, either on spontaneous disease caused by aging or on accelerated disease caused by exposure to cigarette smoke. Liver-directed gene therapy led to dose-dependent levels of biologically active human α1-antitrypsin protein. Furthermore, decreased lung compliance and increased elastic recoil indicate that treated mice had largely preserved lung tissue elasticity and alveolar wall integrity compared with untreated mice. rAAV-mediated gene augmentation is therefore able to compensate for the loss of function and restore a beneficial lung protease-antiprotease balance. This work constitutes a preclinical study report of a disease-modifying treatment in the Serpina1a-e knockout mouse model using a liver-specific rAAV serotype 8 capsid.

11.
Nat Med ; 28(2): 251-259, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-35145305

RESUMEN

Tay-Sachs disease (TSD) is an inherited neurological disorder caused by deficiency of hexosaminidase A (HexA). Here, we describe an adeno-associated virus (AAV) gene therapy expanded-access trial in two patients with infantile TSD (IND 18225) with safety as the primary endpoint and no secondary endpoints. Patient TSD-001 was treated at 30 months with an equimolar mix of AAVrh8-HEXA and AAVrh8-HEXB administered intrathecally (i.t.), with 75% of the total dose (1 × 1014 vector genomes (vg)) in the cisterna magna and 25% at the thoracolumbar junction. Patient TSD-002 was treated at 7 months by combined bilateral thalamic (1.5 × 1012 vg per thalamus) and i.t. infusion (3.9 × 1013 vg). Both patients were immunosuppressed. Injection procedures were well tolerated, with no vector-related adverse events (AEs) to date. Cerebrospinal fluid (CSF) HexA activity increased from baseline and remained stable in both patients. TSD-002 showed disease stabilization by 3 months after injection with ongoing myelination, a temporary deviation from the natural history of infantile TSD, but disease progression was evident at 6 months after treatment. TSD-001 remains seizure-free at 5 years of age on the same anticonvulsant therapy as before therapy. TSD-002 developed anticonvulsant-responsive seizures at 2 years of age. This study provides early safety and proof-of-concept data in humans for treatment of patients with TSD by AAV gene therapy.


Asunto(s)
Enfermedad de Tay-Sachs , Anticonvulsivantes , Dependovirus/genética , Terapia Genética , Humanos , Enfermedad de Tay-Sachs/genética , Enfermedad de Tay-Sachs/terapia
12.
Mol Ther Methods Clin Dev ; 20: 660-674, 2021 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-33718516

RESUMEN

Adeno-associated virus (AAV) vectors are considered efficient vectors for gene transfer, as illustrated by recent successful clinical trials targeting retinal or neurodegenerative disorders. However, limitations as host immune responses to AAV capsid or transduction of limited regions must still be overcome. Here, we focused on locoregional (LR) intravenous perfusion vector delivery that allows transduction of large muscular areas and is considered to be less immunogenic than intramuscular (IM) injection. To confirm this hypothesis, we injected 6 cynomolgus monkeys with an AAV serotype 8 (AAV8) vector encoding for the highly immunogenic GFP driven by either a muscle-specific promoter (n = 3) or a cytomegalovirus (CMV) promoter (n = 3). We report that LR delivery allows long-term GFP expression in the perfused limb (up to 1 year) despite the initiation of a peripheral transgene-specific immune response. The analysis of the immune status of the perfused limb shows that LR delivery induces persisting inflammation. However, this inflammation is not sufficient to result in transgene clearance and is balanced by resident regulatory T cells. Overall, our results suggest that LR delivery promotes persisting transgene expression by induction of Treg cells in situ and might be a safe alternative to IM route to target large muscle territories for the expression of secreted therapeutic factors.

13.
Methods Mol Biol ; 2164: 75-85, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32607885

RESUMEN

We recently described the generation of a novel mouse strain that efficiently and readily engrafts human primary hepatocytes to create liver xenografts (Borel et al., Mol Ther, 25: 2477-89, 2017). A transgenic mouse strain expressing a human PiZ allele for the SerpinA1 gene was crossed with the NOD-SCID-gamma chain knockout (NSG) strain to create a recipient strain (PiZ-NSG) for human hepatocyte xenotransplantation. In this chapter we provide a description of the methods to achieve these liver xenografts in the PiZ-NSG mouse.


Asunto(s)
Hepatocitos/trasplante , Alelos , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Trasplante Heterólogo/métodos , alfa 1-Antitripsina/genética
14.
Hum Gene Ther ; 30(7): 802-813, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30808235

RESUMEN

Anti-transgene immune responses elicited after intramuscular (i.m.) delivery of recombinant adeno-associated virus (rAAV) have been shown to hamper long-term transgene expression in large-animal models of rAAV-mediated gene transfer. To overcome this hurdle, an alternative mode of delivery of rAAV vectors in nonhuman primate muscles has been described: the locoregional (LR) intravenous route of administration. Using this injection mode, persistent inducible transgene expression for at least 1 year under the control of the tetracycline-inducible Tet-On system was previously reported in cynomolgus monkeys, with no immunity against the rtTA transgene product. The present study shows the long-term follow-up of these animals. It is reported that LR delivery of a rAAV2/1 vector allows long-term inducible expression up to at least 5 years post gene transfer, with no any detectable host immune response against the transactivator rtTA, despite its immunogenicity following i.m. gene transfer. This study shows for the first time a long-term regulation of muscle gene expression using a Tet-On-inducible system in a large-animal model. Moreover, these findings further confirm that the rAAV LR delivery route is efficient and immunologically safe, allowing long-term skeletal muscle gene transfer.


Asunto(s)
Dependovirus/genética , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Transgenes , Animales , Anticuerpos Antivirales/inmunología , Citocinas/metabolismo , Dependovirus/inmunología , Estudios de Seguimiento , Vectores Genéticos/administración & dosificación , Vectores Genéticos/efectos adversos , Genoma Viral , Inmunidad , Macaca fascicularis , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Factores de Tiempo
15.
Mol Ther Methods Clin Dev ; 13: 233-242, 2019 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-30828586

RESUMEN

Phase 1 and phase 2 gene therapy trials using intramuscular (IM) administration of a recombinant adeno-associated virus serotype 1 (rAAV1) for replacement of serum alpha-1 antitrypsin (AAT) deficiency have shown long-term (5-year) stable transgene expression at approximately 2% to 3% of therapeutic levels, arguing for the long-term viability of this approach to gene replacement of secreted serum protein deficiencies. However, achieving these levels required 100 IM injections to deliver 135 mL of vector, and further dose escalation is limited by the scalability of direct IM injection. To further advance the dose escalation, we sought to bridge the rAAV-AAT clinical development program to regional limb perfusion, comparing two methods previously established for gene therapy, peripheral venous limb perfusion (VLP) and an intra-arterial push and dwell (IAPD) using rAAV1 and rAAV8 in a non-human primate (rhesus macaque) study. The rhesus AAT transgene was used with a c-myc tag to enable quantification of transgene expression. 5 cohorts of animals were treated with rAAV1-IM, rAAV1-VLP, rAAV1-IAPD, rAAV8-VLP, and rAAV8-IAPD (n = 2-3), with a dose of 6 × 1012 vg/kg. All methods were well tolerated clinically. Potency, as determined by serum levels of AAT, of rAAV1 by the VLP method was twice that observed with direct IM injection; 90 µg/mL with VLP versus 38 µg/mL with direct IM injection. There was an approximately 25-fold advantage in estimated vector genomes retained within the muscle tissue with VLP and a 5-fold improvement in the ratio of total vector genomes retained within muscle as compared with liver. The other methods were intermediate in the potency and retention of vector genomes. Examination of muscle enzyme (CK) levels indicated rAAV1-VLP to be equally safe as compared with IM injection, while the IAPD method showed significant CK elevation. Overall, rAAV1-VLP demonstrates higher potency per vector genome injected and a greater total vector retention within the muscle, as compared to IM injection, while enabling a much greater total dose to be delivered, with equivalent safety. These data provide the basis for continuation of the dose escalation of the rAAV1-AAT program in patients and bode well for rAAV-VLP as a platform for replacement of secreted proteins.

16.
Nat Biotechnol ; 37(8): 884-894, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31375812

RESUMEN

Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospinal fluid. Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker. In mice, di-siRNAs induced the potent silencing of huntingtin, the causative gene in Huntington's disease, reducing messenger RNA and protein throughout the brain. Silencing persisted for at least 6 months, with the degree of gene silencing correlating to levels of guide strand tissue accumulation. In cynomolgus macaques, a bolus injection of di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. This siRNA design may enable RNA interference-based gene silencing in the CNS for the treatment of neurological disorders.


Asunto(s)
Sistema Nervioso Central/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Huntingtina/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , Animales , Proteína Huntingtina/genética , Ratones , Mutación , ARN Mensajero , ARN Interferente Pequeño/metabolismo
17.
Sci Transl Med ; 10(465)2018 10 31.
Artículo en Inglés | MEDLINE | ID: mdl-30381409

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease caused by degeneration of motor neurons leading to rapidly progressive paralysis. About 10% of cases are caused by gain-of-function mutations that are transmitted as dominant traits. A potential therapy for these cases is to suppress the expression of the mutant gene. Here, we investigated silencing of SOD1, a gene commonly mutated in familial ALS, using an adeno-associated virus (AAV) encoding an artificial microRNA (miRNA) that targeted SOD1 In a superoxide dismutase 1 (SOD1)-mediated mouse model of ALS, we have previously demonstrated that SOD1 silencing delayed disease onset, increased survival time, and reduced muscle loss and motor and respiratory impairments. Here, we describe the preclinical characterization of this approach in cynomolgus macaques (Macaca fascicularis) using an AAV serotype for delivery that has been shown to be safe in clinical trials. We optimized AAV delivery to the spinal cord by preimplantation of a catheter and placement of the subject with head down at 30° during intrathecal infusion. We compared different promoters for the expression of artificial miRNAs directed against mutant SOD1 Results demonstrated efficient delivery and effective silencing of the SOD1 gene in motor neurons. These results support the notion that gene therapy with an artificial miRNA targeting SOD1 is safe and merits further development for the treatment of mutant SOD1-linked ALS.


Asunto(s)
Silenciador del Gen , MicroARNs/metabolismo , Superóxido Dismutasa-1/genética , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Encéfalo/metabolismo , Dependovirus/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Inmunidad , Inyecciones Espinales , Hígado/enzimología , Vértebras Lumbares/patología , Macaca , MicroARNs/genética , Neuronas Motoras/metabolismo
18.
Hum Gene Ther ; 29(6): 663-673, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29207890

RESUMEN

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a genetic expansion of the CAG repeat region in the huntingtin (HTT) gene. Studies in HD mouse models have shown that artificial miRNAs can reduce mutant HTT, but evidence for their effectiveness and safety in larger animals is lacking. HD transgenic sheep express the full-length human HTT with 73 CAG repeats. AAV9 was used to deliver unilaterally to HD sheep striatum an artificial miRNA targeting exon 48 of the human HTT mRNA under control of two alternative promoters: U6 or CßA. The treatment reduced human mutant (m) HTT mRNA and protein 50-80% in the striatum at 1 and 6 months post injection. Silencing was detectable in both the caudate and putamen. Levels of endogenous sheep HTT protein were not affected. There was no significant loss of neurons labeled by DARPP32 or NeuN at 6 months after treatment, and Iba1-positive microglia were detected at control levels. It is concluded that safe and effective silencing of human mHTT protein can be achieved and sustained in a large-animal brain by direct delivery of an AAV carrying an artificial miRNA.


Asunto(s)
Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , MicroARNs/metabolismo , Proteínas Mutantes/metabolismo , Neostriado/metabolismo , Animales , Animales Modificados Genéticamente , Dependovirus/genética , Modelos Animales de Enfermedad , Electrólitos/metabolismo , Vectores Genéticos/metabolismo , Genoma Viral , Humanos , Inmunoensayo , Inyecciones , Riñón/fisiopatología , Hígado/fisiopatología , MicroARNs/genética , Microglía/metabolismo , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ovinos
19.
Hum Gene Ther ; 28(4): 338-349, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28323492

RESUMEN

Recombinant adeno-associated viruses (AAVs) are quickly becoming the preferred viral vector for viral gene delivery for the treatment of a wide variety of genetic disorders. However, since their use in a clinical trial targeting hemophilia B patients 10 years ago, immune responses to the AAV capsid appear to have hampered some of the early clinical gene transfer efficacy. Indeed, AAV-based gene transfer has been shown to reactivate capsid-specific memory T cells, which have correlated with a decline in AAV-transduced tissue in some patients. Importantly, clinical trials have also shown that this reactivation can be quelled by administering time-course taper of glucocorticoid steroids before or after dosing. More recently, two clinical studies have shown that AAV gene transfer is not only able to induce a deleterious immune response, but also can result in the initiation of a tolerance to the AAV capsid mediated by regulatory T cells and exhausted T cells. This article reviews clinical trials describing immune responses to AAV, as well as the mechanisms responsible for immune tolerance in chronic infections and how it could apply to AAV-based gene transfer. A better understanding of both cytotoxic and tolerogenic immune responses to recombinant AAV will lead to safer gene transfer protocols in patients.


Asunto(s)
Proteínas de la Cápside/uso terapéutico , Dependovirus/genética , Terapia Genética/efectos adversos , Inmunidad Celular , Proteínas de la Cápside/inmunología , Dependovirus/inmunología , Técnicas de Transferencia de Gen , Vectores Genéticos/efectos adversos , Vectores Genéticos/inmunología , Vectores Genéticos/uso terapéutico , Humanos , Linfocitos T/inmunología
20.
Hum Gene Ther ; 27(1): 19-31, 2016 01.
Artículo en Inglés | MEDLINE | ID: mdl-26710998

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease; survival in ALS is typically 3-5 years. No treatment extends patient survival by more than three months. Approximately 20% of familial ALS and 1-3% of sporadic ALS patients carry a mutation in the gene encoding superoxide dismutase 1 (SOD1). In a transgenic ALS mouse model expressing the mutant SOD1(G93A) protein, silencing the SOD1 gene prolongs survival. One study reports a therapeutic effect of silencing the SOD1 gene in systemically treated adult ALS mice; this was achieved with a short hairpin RNA, a silencing molecule that has raised multiple safety concerns, and recombinant adeno-associated virus (rAAV) 9. We report here a silencing method based on an artificial microRNA termed miR-SOD1 systemically delivered using adeno-associated virus rAAVrh10, a serotype with a demonstrated safety profile in CNS clinical trials. Silencing of SOD1 in adult SOD1(G93A) transgenic mice with this construct profoundly delayed both disease onset and death in the SOD1(G93A) mice, and significantly preserved muscle strength and motor and respiratory functions. We also document that intrathecal delivery of the same rAAVrh10-miR-SOD1 in nonhuman primates significantly and safely silences SOD1 in lower motor neurons. This study supports the view that rAAVrh10-miR-SOD1 merits further development for the treatment of SOD1-linked ALS in humans.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/terapia , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Humanos , Ratones , Mutación , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/uso terapéutico , Superóxido Dismutasa-1
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