RESUMO
Organic acidemias (OA) are a group of rare autosomal recessive disorders of intermediary metabolism that result in a systemic elevation of organic acid. Despite optimal dietary and cofactor therapy, OA patients still suffer from potentially lethal metabolic instability and experience long-term multisystemic complications. Severely affected patients can benefit from elective liver transplantation, which restores hepatic enzymatic activity, improves metabolic stability, and provides the theoretical basis for the pursuit of gene therapy as a new treatment for patients. Because of the poor outcomes reported in those with OA, especially methylmalonic and propionic acidemia, multiple gene therapy approaches have been explored in relevant animal models. Here, we review the results of gene therapy experiments performed using MMA and PA mouse models to illustrate experimental paradigms that could be applicable for all forms of OA.
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Erros Inatos do Metabolismo dos Aminoácidos , Transplante de Fígado , Acidemia Propiônica , Animais , Camundongos , Humanos , Acidemia Propiônica/genética , Acidemia Propiônica/terapia , Acidemia Propiônica/complicações , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Transplante de Fígado/efeitos adversos , Terapia Genética , Modelos Animais de Doenças , Ácido MetilmalônicoRESUMO
On August 18, 2021, the American Society of Gene and Cell Therapy (ASGCT) hosted a virtual roundtable on adeno-associated virus (AAV) integration, featuring leading experts in preclinical and clinical AAV gene therapy, to further contextualize and understand this phenomenon. Recombinant AAV (rAAV) vectors are used to develop therapies for many conditions given their ability to transduce multiple cell types, resulting in long-term expression of transgenes. Although most rAAV DNA typically remains episomal, some rAAV DNA becomes integrated into genomic DNA at a low frequency, and rAAV insertional mutagenesis has been shown to lead to tumorigenesis in neonatal mice. Currently, the risk of rAAV-mediated oncogenesis in humans is theoretical because no confirmed genotoxic events have been reported to date. However, because insertional mutagenesis has been reported in a small number of murine studies, there is a need to characterize this genotoxicity to inform research, regulatory needs, and patient care. The purpose of this white paper is to review the evidence of rAAV-related host genome integration in animal models and possible risks of insertional mutagenesis in patients. In addition, technical considerations, regulatory guidance, and bioethics are discussed.
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Dependovirus , Vetores Genéticos , Animais , Dependovirus/genética , Vetores Genéticos/genética , Humanos , Camundongos , Mutagênese Insercional , Plasmídeos , Transgenes , Integração ViralRESUMO
Methylmalonic acidemia (MMA) is a rare and severe inherited metabolic disease typically caused by mutations of the methylmalonyl-CoA mutase (MMUT) gene. Despite medical management, patients with MMA experience frequent episodes of metabolic instability, severe morbidity, and early mortality. In several preclinical studies, systemic gene therapy has demonstrated impressive improvement in biochemical and clinical phenotypes of MMA murine models. One approach uses a promoterless adeno-associated viral (AAV) vector that relies upon homologous recombination to achieve site-specific in vivo gene addition of MMUT into the last coding exon of albumin (Alb), generating a fused Alb-MMUT transcript after successful editing. We have previously demonstrated that nuclease-free AAV mediated Alb editing could effectively treat MMA mice in the neonatal period and noted that hepatocytes had a growth advantage after correction. Here, we use a transgenic knock-out mouse model of MMA that recapitulates severe clinical and biochemical symptoms to assess the benefits of Alb editing in juvenile animals. As was first noted in the neonatal gene therapy studies, we observe that gene edited hepatocytes in the MMA mice treated as juveniles exhibit a growth advantage, which allows them to repopulate the liver slowly but dramatically by 8-10 months post treatment, and subsequently manifest a biochemical and enzymatic response. In conclusion, our results suggest that the benefit of AAV mediated nuclease-free gene editing of the Alb locus to treat MMA could potentially be therapeutic for older patients.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Metilmalonil-CoA Mutase , Camundongos , Animais , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Edição de Genes , Dependovirus/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Camundongos Knockout , Fígado/metabolismo , Hepatócitos/metabolismo , Albuminas/genética , Albuminas/metabolismo , Ácido Metilmalônico/metabolismoRESUMO
BACKGROUND AND AIMS: Adeno-associated viral (AAV) gene therapy has shown great promise as an alternative treatment for metabolic disorders managed using liver transplantation, but remains limited by transgene loss and genotoxicity. Our study aims to test an AAV vector with a promoterless integrating cassette, designed to provide sustained hepatic transgene expression and reduced toxicity in comparison to canonical AAV therapy. APPROACH AND RESULTS: Our AAV vector was designed to insert a methylmalonyl-CoA mutase (MMUT) transgene into the 3' end of the albumin locus and tested in mouse models of methylmalonic acidemia (MMA). After neonatal delivery, we longitudinally evaluated hepatic transgene expression, plasma levels of methylmalonate, and the MMA biomarker, fibroblast growth factor 21 (Fgf21), as well as integration of MMUT in the albumin locus. At necropsy, we surveyed for AAV-related hepatocellular carcinoma (HCC) in all treated MMA mice and control littermates. AAV-mediated genome editing of MMUT into the albumin locus resulted in permanent hepatic correction in MMA mouse models, which was accompanied by decreased levels of methylmalonate and Fgf21, and improved survival without HCC. With time, levels of transgene expression increased and methylmalonate progressively decreased, whereas the number of albumin-MMUT integrations and corrected hepatocytes in MMA mice increased, but not in similarly treated wild-type animals. Additionally, expression of MMUT in the setting of MMA conferred a selective growth advantage upon edited cells, which potentiates the therapeutic response. CONCLUSIONS: In conclusion, our findings demonstrate that AAV-mediated, promoterless, nuclease-free genome editing at the albumin locus provides safe and durable therapeutic benefit in neonatally treated MMA mice.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/terapia , Dependovirus/genética , Edição de Genes/métodos , Terapia Genética/métodos , Metilmalonil-CoA Mutase/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/sangue , Carcinoma Hepatocelular/patologia , Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/sangue , Hepatócitos , Neoplasias Hepáticas/patologia , Transplante de Fígado , Malonatos/sangue , Metilmalonil-CoA Mutase/genética , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC) and the progressive accumulation of the toxic metabolite psychosine. We showed previously that central nervous system (CNS)-directed, adeno-associated virus (AAV)2/5-mediated gene therapy synergized with bone marrow transplantation and substrate reduction therapy (SRT) to greatly increase therapeutic efficacy in the murine model of Krabbe disease (Twitcher). However, motor deficits remained largely refractory to treatment. In the current study, we replaced AAV2/5 with an AAV2/9 vector. This single change significantly improved several endpoints primarily associated with motor function. However, nearly all (14/16) of the combination-treated Twitcher mice and all (19/19) of the combination-treated wild-type mice developed hepatocellular carcinoma (HCC). 10 out of 10 tumors analyzed had AAV integrations within the Rian locus. Several animals had additional integrations within or near genes that regulate cell growth or death, are known or potential tumor suppressors, or are associated with poor prognosis in human HCC. Finally, the substrate reduction drug L-cycloserine significantly decreased the level of the pro-apoptotic ceramide 18:0. These data demonstrate the value of AAV-based combination therapy for Krabbe disease. However, they also suggest that other therapies or co-morbidities must be taken into account before AAV-mediated gene therapy is considered for human therapeutic trials.
Assuntos
Dependovirus/genética , Terapia Genética/efeitos adversos , Vetores Genéticos/genética , Leucodistrofia de Células Globoides/complicações , Leucodistrofia de Células Globoides/terapia , Animais , Transplante de Medula Óssea/métodos , Carcinoma Hepatocelular/etiologia , Terapia Combinada , Modelos Animais de Doenças , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Neoplasias Hepáticas/etiologia , CamundongosRESUMO
Niemann-Pick disease, type C1 (NPC1) is a heritable lysosomal storage disease characterized by a progressive neurological degeneration that causes disability and premature death. A murine model of NPC1 disease (Npc1-/-) displays a rapidly progressing form of NPC1 disease which is characterized by weight loss, ataxia, increased cholesterol storage, loss of cerebellar Purkinje neurons and early lethality. To test the potential efficacy of gene therapy for NPC1, we constructed adeno-associated virus serotype 9 (AAV9) vectors to deliver the NPC1 gene under the transcriptional control of the neuronal-specific (CamKII) or a ubiquitous (EF1a) promoter. The Npc1-/- mice that received a single dose of AAV9-CamKII-NPC1 as neonates (2.6 × 1011GC) or at weaning (1.3 × 1012GC), and the mice that received a single dose of AAV9-EF1a-NPC1 at weaning (1.2 × 1012GC), exhibited an increased life span, characterized by delayed weight loss and diminished motor decline. Cholesterol storage and Purkinje neuron loss were also reduced in the central nervous system of AAV9 treated Npc1-/- mice. Treatment with AAV9-EF1a-NPC1, as compared to AAV9-CamKII-NPC1, resulted in significantly increased survival (mean survival increased from 69 days to 166 and 97 days, respectively) and growth, and reduced hepatic-cholesterol accumulation. Our results provide the first demonstration that gene therapy may represent a therapeutic option for NPC1 patients and suggest that extraneuronal NPC1 expression can further augment the lifespan of the Npc1-/- mice after systemic AAV gene delivery.
Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos/administração & dosagem , Longevidade/genética , Doença de Niemann-Pick Tipo C/terapia , Proteínas/genética , Animais , Cerebelo/metabolismo , Cerebelo/patologia , Colesterol/metabolismo , Modelos Animais de Doenças , Feminino , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/genética , Células de Purkinje/metabolismo , Células de Purkinje/patologiaAssuntos
Hiperargininemia , Doenças Metabólicas , Nanopartículas , Arginase , Humanos , Lipídeos , RNA MensageiroRESUMO
Isolated methylmalonic acidemia (MMA), caused by deficiency of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT), is often complicated by end stage renal disease that is resistant to conventional therapies, including liver transplantation. To establish a viable model of MMA renal disease, Mut was expressed in the liver of Mut(-/-) mice as a stable transgene under the control of an albumin (INS-Alb-Mut) promoter. Mut(-/-);Tg(INS-Alb-Mut) mice, although completely rescued from neonatal lethality that was displayed by Mut(-/-) mice, manifested a decreased glomerular filtration rate (GFR), chronic tubulointerstitial nephritis and ultrastructural changes in the proximal tubule mitochondria associated with aberrant tubular function, as demonstrated by single-nephron GFR studies. Microarray analysis of Mut(-/-);Tg(INS-Alb-Mut) kidneys identified numerous biomarkers, including lipocalin-2, which was then used to monitor the response of the GFR to antioxidant therapy in the mouse model. Renal biopsies and biomarker analysis from a large and diverse patient cohort (ClinicalTrials.gov identifier: NCT00078078) precisely replicated the findings in the animals, establishing Mut(-/-);Tg(INS-Alb-Mut) mice as a unique model of MMA renal disease. Our studies suggest proximal tubular mitochondrial dysfunction is a key pathogenic mechanism of MMA-associated kidney disease, identify lipocalin-2 as a biomarker of increased oxidative stress in the renal tubule, and demonstrate that antioxidants can attenuate the renal disease of MMA.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Antioxidantes/farmacologia , Modelos Animais de Doenças , Túbulos Renais Proximais/fisiopatologia , Metilmalonil-CoA Mutase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Animais , Antioxidantes/uso terapêutico , Biomarcadores/metabolismo , Western Blotting , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Fluoresceína-5-Isotiocianato , Genótipo , Taxa de Filtração Glomerular/genética , Humanos , Imuno-Histoquímica , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Camundongos , Camundongos Knockout , Análise em Microsséries , Microscopia Eletrônica de Transmissão , Nefrite Intersticial/genética , Reação em Cadeia da Polimerase em Tempo Real , Transgenes/genética , Ubiquinona/farmacologiaRESUMO
While tail vein injections are frequently used as a systemic route of delivery in adult mice, retrobulbar injections are an alternative method for systemic delivery with fewer limitations. First, tail vein injections (TVIs) are limited to adult mice where the size of the tail vein is suitable for access. Being restricted to treating adult mice can be problematic when dealing with mouse models that do not survive to adulthood. Second, TVIs are not feasible for mouse models with growth retardation phenotypes where the mice never achieve the size of adult wildtype mice. Therefore, retrobulbar injections can be successfully used to treat both young and small adult mice. Lastly, retrobulbar injections are performed under anesthesia, which is less stressful on the mice than TVIs that are commonly performed without anesthesia. This article presents a protocol and detailed instructions for retrobulbar injections that can be used for systemic delivery to small and young mice.
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Injeções , Animais , Camundongos , Injeções/métodosRESUMO
Orphan drug designation (ODD) is an important program intended to facilitate the development of orphan drugs in the United States. An orphan drug benefiting pediatric patients can qualify as a drug for a Rare Pediatric Disease Designation (RPDD) as well. The ODD and RPDD programs provide financial incentives for development of diagnostic drugs, preventive measures, and treatment of diseases affecting small patient populations (adult and pediatric) for which commercial development would otherwise be very challenging. In 2019, a multidisciplinary group of collaborators at National Institutes of Health (NIH) embarked upon a gene therapy platform program called Platform Vector Gene Therapy (PaVe-GT) intended to develop gene therapies for four such rare disorders. An important part of PaVe-GT is to publicly share scientific and regulatory experience gained at different stages during the implementation of the PaVe-GT platform utilizing illustrative examples. The PaVe-GT team recently obtained ODD and RPDD for an adeno-associated virus gene therapy to treat propionic acidemia. Given an increasing interest in obtaining ODD for gene therapy, especially by small companies, research investigators, and patient groups, we overview the submission process and subsequently provide examples of our ODD and RPDD applications. Our ODD and RPDD applications and templates can also be found on the PaVe-GT website. Shared reference documents will have great utility to assist parties who may have limited experience with the preparation of similar applications for their orphan product.
Assuntos
Produção de Droga sem Interesse Comercial , Acidemia Propiônica , Humanos , Estados Unidos , Criança , United States Food and Drug Administration , Aprovação de Drogas , Doenças Raras/genética , Doenças Raras/terapia , Terapia Genética , National Institutes of Health (U.S.)RESUMO
Propionic acidemia (PA) is rare autosomal recessive metabolic disorder caused by defects in the mitochondrially localized enzyme propionyl-coenzyme A (CoA) carboxylase. Patients with PA can suffer from lethal metabolic decompensation and cardiomyopathy despite current medical management, which has led to the pursuit of gene therapy as a new treatment option for patients. Here we assess the therapeutic efficacy of a recently described adeno-associated virus (AAV) capsid, AAV44.9, to deliver a therapeutic PCCA transgene in a new mouse model of propionyl-CoA carboxylase α (PCCA) deficiency generated by genome editing. Pcca-/- mice recapitulate the severe neonatal presentation of PA and manifest uniform neonatal lethality, absent PCCA expression, and increased 2-methylcitrate. A single injection of the AAV44.9 PCCA vector in the immediate newborn period, systemically delivered at a dose of 1e11 vector genome (vg)/pup but not 1e10 vg/pup, increased survival, reduced plasma methylcitrate, and resulted in high levels of transgene expression in the liver and heart in treated Pcca-/- mice. Our studies not only establish a versatile and accurate new mouse model of PA but further demonstrate that the AAV44.9 vectors may be suitable for treatment of many metabolic disorders where hepato-cardiac transduction following systemic delivery is desired, such as PA, and, by extension, fatty acid oxidation defects and glycogen storage disorders.
RESUMO
We demonstrate that human methylmalonyl-CoA mutase (MUT), delivered using an AAV serotype 8 vector, rescues the lethal phenotype displayed by mice with MMA and provides long-term phenotypic correction. In addition to defining a lower limit of effective dosing, our studies establish that neither a species barrier to mitochondrial processing nor an apparent immune response to MUT limits the murine model as an experimental platform to test the efficacy of human gene therapy vectors for MMA.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/terapia , Dependovirus/genética , Terapia Genética , Metilmalonil-CoA Mutase/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/mortalidade , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Metilmalonil-CoA Mutase/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fenótipo , Alinhamento de Sequência , Taxa de SobrevidaRESUMO
Combined methylmalonic acidemia and homocystinuria, cblC type, is an inborn error of intracellular cobalamin metabolism with a wide spectrum of clinical manifestations that is stated to be the most common inherited disorder of cobalamin metabolism. This metabolic disease is caused by mutations in the MMACHC gene and results in impaired intracellular synthesis of adenosylcobalamin and methylcobalamin, cofactors for the methylmalonyl-CoA mutase and methionine synthase enzymes. Elevated methylmalonic acid and homocysteine with decreased methionine production are the biochemical hallmarks of this disorder. Awareness of the diverse clinical presentations associated with cblC disease is necessary to provide a timely diagnosis, to guide management of affected individuals and to establish a framework for the future treatment of individuals detected through expanded newborn screening. This article reviews the biochemistry, clinical presentations, genotype-phenotype correlations, diagnosis and management of cblC disease.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/genética , Proteínas de Transporte/genética , Homocistinúria/diagnóstico , Homocistinúria/genética , Mutação , Vitamina B 12/metabolismo , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Metionina/metabolismo , Ácido Metilmalônico/metabolismo , Modelos Biológicos , Modelos Genéticos , Triagem Neonatal/métodos , Oxirredutases , Gravidez , Diagnóstico Pré-Natal , Deficiência de Vitamina B 12/congênitoRESUMO
Methylmalonic acidemia (MMA) is a severe and potentially lethal autosomal recessive inborn error of metabolism most frequently caused by mutations in the methylmalonyl-CoA mutase (MMUT) gene. Proof-of-concept adeno-associated virus (AAV) gene therapy studies using mouse models of MMA have demonstrated promise for this therapeutic approach but translation to the clinic could be limited by preexisting capsid immunity and vector potency. Here we explore the efficacy of a novel clade E capsid, 44.9, as a serotype for systemic AAV gene therapy for MMA. An anti-AAV44.9 neutralizing antibody (NAb) survey in adult volunteers (n = 19) and a large cohort of MMA patients (n = 48) revealed a seroprevalence rate of â¼26% and 13%, respectively. The efficacy of AAV44.9 gene delivery was examined in two murine models of MMA, representing neonatal lethal and juvenile phenotypes of MMA. Systemic delivery of the AAV44.9-Mmut vector prevented lethality and lowered disease-related metabolites in MMA mice. Tissue biodistribution and transgene expression studies in treated MMA mice showed that AAV44.9 was efficient at transducing the liver and heart. In summary, we establish that AAV44.9 exhibits a low prevalence of preexisting NAb in humans, is highly efficacious in the treatment of clinically severe MMA mouse models and is therefore a promising vector for clinical translation.
RESUMO
Methylmalonic acidemia (MMA) is an organic acidemia caused by deficient activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). This disorder is associated with lethal metabolic instability and carries a poor prognosis for long-term survival. A murine model of MMA that replicates a severe clinical phenotype was used to examine the efficacy of recombinant adeno-associated virus (rAAV) serotype 8 gene therapy as a treatment for MMA. Lifespan extension, body weight, circulating metabolites, transgene expression, and whole animal propionate oxidation were examined as outcome parameters after gene therapy. One-hundred percent of the untreated Mut(-/-) mice (n = 58) died by day of life (DOL) 72, whereas >95% of the adeno-associated virus-treated Mut(-/-) mice (n = 27) have survived for > or = 1 year. Despite a gradual loss of transgene expression and elevated circulating metabolites in the treated Mut(-/-) mice, the animals are indistinguishable from unaffected control littermates in size and activity levels. These experiments provide the first definitive evidence that gene therapy will have clinical utility in the treatment of MMA and support the development of gene therapy for other organic acidemias.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/terapia , Terapia Genética/métodos , Metilmalonil-CoA Mutase/fisiologia , Erros Inatos do Metabolismo dos Aminoácidos/sangue , Erros Inatos do Metabolismo dos Aminoácidos/genética , Animais , Western Blotting , Dependovirus/genética , Vetores Genéticos/genética , Humanos , Ácido Metilmalônico/sangue , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Camundongos , Camundongos Mutantes , Reação em Cadeia da PolimeraseRESUMO
Methylmalonic acidemia (MMA) is a severe metabolic disorder most commonly caused by a mutation in the methylmalonyl-CoA mutase (MMUT) gene. Patients with MMA experience multisystemic disease manifestations and remain at risk for neurological disease progression, even after liver transplantation. Therefore, delivery of MMUT to the central nervous system (CNS) may provide patients with neuroprotection and, perhaps, therapeutic benefits. To specifically target the brain, we developed a neurotropic PHP.eB vector that used a CaMKII neuro-specific promoter to restrict the expression of the MMUT transgene in the neuraxis and delivered the adeno-associated virus (AAV) to mice with MMA. The PHP.eB vector transduced cells in multiple brain regions, including the striatum, and enabled high levels of expression of MMUT in the basal ganglia. Following the CNS-specific correction of MMUT expression, disease-related metabolites methylmalonic acid and 2-methylcitrate were significantly (p < 0.02) decreased in serum of treated MMA mice. Our results show that targeting MMUT expression to the CNS using a neurotropic capsid can decrease the circulating metabolite load in MMA and further highlight the benefit of extrahepatic correction for disorders of organic acid metabolism.
RESUMO
Methylmalonic acidemia (MMA) is a metabolic disorder most commonly caused by mutations in the methylmalonyl-CoA mutase (MMUT) gene. Although adeno-associated viral (AAV) gene therapy has been effective at correcting the disease phenotype in MMA mouse models, clinical translation may be impaired by loss of episomal transgene expression and magnified by the need to treat patients early in life. To achieve permanent correction, we developed a dual AAV strategy to express a codon-optimized MMUT transgene from Alb and tested various CRISPR-Cas9 genome-editing vectors in newly developed knockin mouse models of MMA. For one target site in intron 1 of Alb, we designed rescue cassettes expressing MMUT behind a 2A-peptide or an internal ribosomal entry site sequence. A second guide RNA targeted the initiator codon, and the donor cassette encompassed the proximal albumin promoter in the 5' homology arm. Although all editing approaches were therapeutic, targeting the start codon of albumin allowed the use of a donor cassette that also functioned as an episome and after homologous recombination, even without the expression of Cas9, as an integrant. Targeting the albumin locus using these strategies would be effective for other metabolic disorders where early treatment and permanent long-term correction are needed.
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A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model.
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Niemann-Pick C1 disease (NPC1) is a rare, fatal neurodegenerative disease caused by mutations in NPC1, which encodes the lysosomal cholesterol transport protein NPC1. Disease pathology involves lysosomal accumulation of cholesterol and lipids, leading to neurological and visceral complications. Targeting the central nervous system (CNS) from systemic circulation complicates treatment of neurological diseases with gene transfer techniques. Selected and engineered capsids, for example, adeno-associated virus (AAV)-PHP.B facilitate peripheral-to-CNS transfer and hence greater CNS transduction than parental predecessors. We report that systemic delivery to Npc1 m1N/m1N mice using an AAV-PHP.B vector ubiquitously expressing NPC1 led to greater disease amelioration than an otherwise identical AAV9 vector. In addition, viral copy number and biodistribution of GFP-expressing reporters showed that AAV-PHP.B achieved more efficient, albeit variable, CNS transduction than AAV9 in Npc1 m1N/m1N mice. This variability was associated with segregation of two alleles of the putative AAV-PHP.B receptor Ly6a in Npc1 m1N/m1N mice. Our data suggest that robust improvements in NPC1 disease phenotypes occur even with modest CNS transduction and that improved neurotrophic capsids have the potential for superior NPC1 AAV gene therapy vectors.