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Trastuzumab improves overall survival for HER2+ breast cancer, but its short half-life in the cerebrospinal fluid (~2-4 days) and delivery limitations restrict the ability to target HER2+ central nervous system (CNS) disease. We developed an adeno-associated virus (AAV) vector expressing a codon-optimized, ubiquitin C (UbC)-promoter-driven trastuzumab sequence (AAV9.UbC.trastuzumab) for intrathecal administration. Transgene expression was evaluated in adult Rag1 knockout mice and rhesus nonhuman primates (NHPs) after a single intracerebroventricular (ICV) or intra-cisterna magna (ICM) AAV9.UbC.trastuzumab injection, respectively, using real-time PCR, ELISA, Western blot, in situ hybridization, single-nucleus RNA sequencing, and liquid chromatography-mass spectrometry; antitumor efficacy was evaluated in brain xenografts using HER2+ breast cancer cell lines (BT-474, MDA-MB-453). Transgene expression was detected in brain homogenates of Rag1 knockout mice following a single ICV injection of AAV9.UbC.trastuzumab (1 × 1011 vector genome copies [GC]/mouse) and tumor progression was inhibited in xenograft models of breast-to-brain metastasis. In NHPs, ICM delivery of AAV9.UbC.trastuzumab (3 × 1013 GC/animal) was well tolerated (36-37 days in-life) and resulted in transgene expression in CNS tissues and cerebrospinal fluid at levels sufficient to induce complete tumor remission in MDA-MB-453 brain xenografts. With AAV9's proven clinical safety record, this gene therapy may represent a viable approach for targeting HER2 + CNS malignancies.
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Neoplasias Encefálicas , Dependovirus , Receptor ErbB-2 , Trastuzumab , Trastuzumab/administración & dosificación , Trastuzumab/farmacología , Trastuzumab/uso terapéutico , Dependovirus/genética , Animales , Humanos , Ratones , Receptor ErbB-2/metabolismo , Receptor ErbB-2/genética , Femenino , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundario , Neoplasias Encefálicas/patología , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Neoplasias de la Mama/genética , Neoplasias de la Mama/tratamiento farmacológico , Ratones Noqueados , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Macaca mulatta , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Antineoplásicos Inmunológicos/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto , Sistema Nervioso Central/metabolismo , Línea Celular TumoralRESUMEN
We analyzed retrospective data from toxicology studies involving administration of high doses of adeno-associated virus expressing different therapeutic transgenes to 21 cynomolgus and 15 rhesus macaques. We also conducted prospective studies to investigate acute toxicity following high-dose systemic administration of enhanced green fluorescent protein-expressing adeno-associated virus to 10 rhesus macaques. Toxicity was characterized by transaminitis, thrombocytopenia, and alternative complement pathway activation that peaked on post-administration day 3. Although most animals recovered, some developed ascites, generalized edema, hyperbilirubinemia, and/or coagulopathy that prompted unscheduled euthanasia. Study endpoint livers from animals that recovered and from unscheduled necropsies of those that succumbed to toxicity were analyzed via hypothesis-driven histopathology and unbiased single-nucleus RNA sequencing. All liver cell types expressed high transgene transcript levels at early unscheduled timepoints that subsequently decreased. Thrombocytopenia coincided with sinusoidal platelet microthrombi and sinusoidal endothelial injury identified via immunohistology and single-nucleus RNA sequencing. Acute toxicity, sinusoidal injury, and liver platelet sequestration were similarly observed with therapeutic transgenes and enhanced green fluorescent protein at doses ≥1 × 1014 GC/kg, suggesting it was the consequence of high-dose systemic adeno-associated virus administration, not green fluorescent protein toxicity. These findings highlight a potential toxic effect of high-dose intravenous adeno-associated virus on nonhuman primate liver microvasculature.
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Dependovirus , Trombocitopenia , Animales , Dependovirus/genética , Macaca mulatta/genética , Estudios Prospectivos , Estudios Retrospectivos , Hígado/metabolismo , Transgenes , Trombocitopenia/metabolismo , Células Endoteliales , Vectores Genéticos/genéticaRESUMEN
Adeno-associated virus (AAV) vector gene therapy is a promising approach to treat rare genetic diseases; however, an ongoing challenge is how to best modulate host immunity to improve transduction efficiency and therapeutic outcomes. This report presents two studies characterizing multiple prophylactic immunosuppression regimens in male cynomolgus macaques receiving an AAVrh10 gene therapy vector expressing human coagulation factor VIII (hFVIII). In study 1, no immunosuppression was compared with prednisolone, rapamycin (or sirolimus), rapamycin and cyclosporin A in combination, and cyclosporin A and azathioprine in combination. Prednisolone alone demonstrated higher mean peripheral blood hFVIII expression; however, this was not sustained upon taper. Anti-capsid and anti-hFVIII antibody responses were robust, and vector genomes and transgene mRNA levels were similar to no immunosuppression at necropsy. Study 2 compared no immunosuppression with prednisolone alone or in combination with rapamycin or methotrexate. The prednisolone/rapamycin group demonstrated an increase in mean hFVIII expression and a mean delay in anti-capsid IgG development until after rapamycin taper. Additionally, a significant reduction in the plasma cell gene signature was observed with prednisolone/rapamycin, suggesting that rapamycin's tolerogenic effects may include plasma cell differentiation blockade. Immunosuppression with prednisolone and rapamycin in combination could improve therapeutic outcomes in AAV vector gene therapy.
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Ciclosporina , Sirolimus , Masculino , Humanos , Animales , Sirolimus/farmacología , Sirolimus/uso terapéutico , Sirolimus/metabolismo , Ciclosporina/metabolismo , Células Plasmáticas , Prednisolona/farmacología , Prednisolona/uso terapéutico , Prednisolona/metabolismo , Terapia Genética , Vectores Genéticos/genética , Macaca/genética , DependovirusRESUMEN
Integration of naturally occurring adeno-associated viruses (AAV; wild-type AAV [wtAAV]) and those used in gene therapy (recombinant AAV [rAAV]) into host genomic DNA has been documented for over two decades. Results from mouse and dog studies have raised concerns of insertional mutagenesis and clonal expansion following AAV exposure, particularly in the context of gene therapy. This study aimed to characterize the genomic location, abundance, and expansion of wtAAV and rAAV integrations in macaque and human genomes. Using an unbiased, next-generation sequencing-based approach, we identified the genome-wide integration loci in tissue samples (primarily liver) in 168 nonhuman primates (NHPs) and 85 humans naïve to rAAV exposure and 86 NHPs treated with rAAV in preclinical studies. Our results suggest that rAAV and wtAAV integrations exhibit similar, broad distribution patterns across species, with a higher frequency in genomic regions highly vulnerable to DNA damage or close to highly transcribed genes. rAAV exhibited a higher abundance of unique integration loci, whereas wtAAV integration loci were associated with greater clonal expansion. This expansive and detailed characterization of AAV integration in NHPs and humans provides key translational insights, with important implications for the safety of rAAV as a gene therapy vector.
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Dependovirus , Macaca , Animales , Humanos , Dependovirus/genética , Terapia Genética , Vectores Genéticos/genética , Hígado , Macaca/genética , Integración Viral/genéticaRESUMEN
The development of liver-based adeno-associated virus (AAV) gene therapies is facing concerns about limited efficiency and durability of transgene expression. We evaluated nonhuman primates following intravenous dosing of AAV8 and AAVrh10 vectors for over 2 years to better define the mechanism(s) of transduction that affect performance. High transduction of non-immunogenic transgenes was achieved, although expression declined over the first 90 days to reach a lower but stable steady state. More than 10% of hepatocytes contained single nuclear domains of vector DNA that persisted despite the loss of transgene expression. Greater reductions in vector DNA and RNA were observed with immunogenic transgenes. Genomic integration of vector sequences, including complex concatemeric structures, were detected in 1 out of 100 cells at broadly distributed loci that were not in proximity to genes associated with hepatocellular carcinoma. Our studies suggest that AAV-mediated transgene expression in primate hepatocytes occurs in two phases: high but short-lived expression from episomal genomes, followed by much lower but stable expression, likely from integrated vectors.
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Crigler-Najjar syndrome is a rare disorder of bilirubin metabolism caused by uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) mutations characterized by hyperbilirubinemia and jaundice. No cure currently exists; treatment options are limited to phototherapy, whose effectiveness diminishes over time, and liver transplantation. Here, we evaluated the therapeutic potential of systemically administered, lipid nanoparticle-encapsulated human UGT1A1 (hUGT1A1) mRNA therapy in a Crigler-Najjar mouse model. Ugt1 knockout mice were rescued from lethal post-natal hyperbilirubinemia by phototherapy. These adult Ugt1 knockout mice were then administered a single lipid nanoparticle-encapsulated hUGT1A1 mRNA dose. Within 24 h, serum total bilirubin levels decreased from 15 mg/dL (256 µmol/L) to <0.5 mg/dL (9 µmol/L), i.e., slightly above wild-type levels. This reduction was sustained for 2 weeks before bilirubin levels rose and returned to pre-treatment levels by day 42 post-administration. Sustained reductions in total bilirubin levels were achieved by repeated administration of the mRNA product in a frequency-dependent manner. We were also able to rescue the neonatal lethality phenotype seen in Ugt1 knockout mice with a single lipid nanoparticle dose, which suggests that this may be a treatment modality appropriate for metabolic crisis situations. Therefore, lipid nanoparticle-encapsulated hUGT1A1 mRNA may represent a potential treatment for Crigler-Najjar syndrome.
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Transthyretin amyloidosis (ATTR) is a progressive and fatal disease caused by transthyretin (TTR) amyloid fibril accumulation in tissues, which disrupts organ function. As the TTR protein is primarily synthesized by the liver, liver transplantation can cure familial ATTR but is not an option for the predominant age-related wild-type ATTR. Approved treatment approaches include TTR stabilizers and an RNA-interference therapeutic, but these require regular re-administration. Gene editing could represent an effective one-time treatment. We evaluated adeno-associated virus (AAV) vector-delivered, gene-editing meganucleases to reduce TTR levels. We used engineered meganucleases targeting two different sites within the TTR gene. AAV vectors expressing TTR meganuclease transgenes were first tested in immunodeficient mice expressing the human TTR sequence delivered using an AAV vector and then against the endogenous TTR gene in rhesus macaques. Following a dose of 3 × 1013 genome copies per kilogram, we detected on-target editing efficiency of up to 45% insertions and deletions (indels) in the TTR genomic DNA locus and >80% indels in TTR RNA, with a concomitant decrease in serum TTR levels of >95% in macaques. The significant reduction in serum TTR levels following TTR gene editing indicates that this approach could be an effective treatment for ATTR.
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Neuropatías Amiloides Familiares , Dependovirus , Humanos , Ratones , Animales , Dependovirus/genética , Dependovirus/metabolismo , Macaca mulatta/genética , Macaca mulatta/metabolismo , Neuropatías Amiloides Familiares/terapia , Neuropatías Amiloides Familiares/tratamiento farmacológico , Prealbúmina/genética , Prealbúmina/metabolismo , Prealbúmina/uso terapéutico , ARN/uso terapéuticoRESUMEN
Gene therapy using neurotropic adeno-associated virus vectors represents an emerging solution for genetic disorders affecting the central nervous system. The first approved central nervous system-targeting adeno-associated virus gene therapy, Zolgensma®, for treating spinal muscular atrophy is administered intravenously at high doses that cause liver-associated adverse events in 20%-30% of patients. Intrathecal routes of vector administration, such as the intra-cisterna magna route, provide efficient gene transduction to central nervous system cells while reducing off-target liver transduction. However, significant levels of liver transduction often occur upon intra-cisterna magna vector delivery in preclinical studies. Using vectors expressing monoclonal antibody transgenes, we examined whether passive transfer of adeno-associated virus-neutralizing antibodies as intravenous immunoglobulin before intrathecal adeno-associated virus delivery improved the safety of viral gene therapy targeting the central nervous system in mice and nonhuman primates. We used intracerebroventricular and intra-cisterna magna routes for vector administration to mice and nonhuman primates, respectively, and evaluated transgene expression and vector genome distribution. Our data indicate that pretreatment with intravenous immunoglobulin significantly reduced gene transduction to the liver and other peripheral organs but not to the central nervous system in both species. With further refinement, this method may improve the safety of adeno-associated virus-based, central nervous system-targeting gene therapies in clinical settings.
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[This corrects the article DOI: 10.1016/j.omtm.2022.09.017.].
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Hemophilia A, a bleeding disorder, affects 1:5,000 males and is caused by a deficiency of human blood coagulation factor VIII (hFVIII). Studies in mice and macaques identified AAVhu37.E03.TTR.hFVIIIco-SQ.PA75 as a clinical candidate gene therapy vector to treat hemophilia A. In this study, we sought to determine the minimally effective dose (MED) of this vector in a hemophilia A mouse model. Mice received one of four vector doses (3 × 1011-1 × 1013 genome copies [GCs]/kg) via intravenous tail vein injection; one cohort received vehicle as a control. Animals were monitored daily after vector/vehicle administration. Blood samples were collected to evaluate hFVIII activity levels and anti-hFVIII antibodies. Animals were sacrificed and necropsied on days 28 and 56; tissues were harvested for histopathological examination and blood was collected for serum chemistry panel analysis. We found no significant differences in liver transaminase levels in mice administered any vector dose compared to those administered vehicle (except for one group administered 3 × 1011 GC/kg). Total bilirubin levels were significantly elevated compared to the vehicle group following two vector doses at day 56 (1 × 1012 and 1 × 1013 GC/kg). We observed no vector-related gross or histological findings. Most microscopic findings were in the vehicle group and considered secondary to blood loss, an expected phenotype of this mouse model. Since we observed no dose-limiting safety markers, we determined that the maximally tolerated dose was greater than or equal to the highest dose tested (1 × 1013 GC/kg). Since we detected hFVIII activity in all cohorts administered vector, we conclude that the MED is 3 × 1011 GC/kg-the lowest dose evaluated in this study.
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Dependovirus , Hemofilia A , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Factor VIII/genética , Factor VIII/uso terapéutico , Femenino , Terapia Genética , Vectores Genéticos/genética , Hemofilia A/genética , Hemofilia A/terapia , Humanos , Masculino , RatonesRESUMEN
Maple syrup urine disease (MSUD) is a rare, inherited metabolic disorder characterized by a dysfunctional mitochondrial enzyme complex, branched-chain alpha-keto acid dehydrogenase (BCKDH), which catabolizes branched-chain amino acids (BCAAs). Without functional BCKDH, BCAAs and their neurotoxic alpha-keto intermediates can accumulate in the blood and tissues. MSUD is currently incurable and treatment is limited to dietary restriction or liver transplantation, meaning there is a great need to develop new treatments for MSUD. We evaluated potential gene therapy applications for MSUD in the intermediate MSUD (iMSUD) mouse model, which harbors a mutation in the dihydrolipoamide branched-chain transacylase E2 (DBT) subunit of BCKDH. Systemic delivery of an adeno-associated virus (AAV) vector expressing DBT under control of the liver-specific TBG promoter to the liver did not sufficiently ameliorate all aspects of the disease phenotype. These findings necessitated an alternative therapeutic strategy. Muscle makes a larger contribution to BCAA metabolism than liver in humans, but a muscle-specific approach involving a muscle-specific promoter for DBT expression delivered via intramuscular (IM) administration only partially rescued the MSUD phenotype in mice. Combining the muscle-tropic AAV9 capsid with the ubiquitous CB7 promoter via IM or IV injection, however, substantially increased survival across all assessed doses. Additionally, near-normal serum BCAA levels were achieved and maintained in the mid- and high-dose cohorts throughout the study; this approach also protected these mice from a lethal high-protein diet challenge. Therefore, administration of a gene therapy vector that expresses in both muscle and liver may represent a viable approach to treating patients with MSUD.
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Dependovirus/genética , Terapia Genética/métodos , Enfermedad de la Orina de Jarabe de Arce/genética , Enfermedad de la Orina de Jarabe de Arce/terapia , Fenotipo , Administración Intravenosa , Aminoácidos de Cadena Ramificada/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/administración & dosificación , Masculino , Ratones , MutaciónRESUMEN
SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis.
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Enzima Convertidora de Angiotensina 2 , Tratamiento Farmacológico de COVID-19 , Dependovirus , Terapia Genética , Vectores Genéticos , SARS-CoV-2 , Administración Intranasal , Enzima Convertidora de Angiotensina 2/biosíntesis , Enzima Convertidora de Angiotensina 2/genética , Animales , COVID-19/genética , COVID-19/metabolismo , Humanos , Ratones , Ratones Transgénicos , SARS-CoV-2/genética , SARS-CoV-2/metabolismoRESUMEN
Our group previously used adeno-associated viral vectors (AAVs) to express an engineered meganuclease specific for a sequence in the PCSK9 gene (M2PCSK9), a clinical target for treating coronary heart disease. Upon testing this nuclease in non-human primates, we observed specific editing characterized by several insertions and deletions (indels) in the target sequence as well as indels in similar genomic sequences. We hypothesized that high nuclease expression increases off-target editing. Here, we reduced nuclease expression using two strategies. The first was a self-targeting strategy that involved inserting the M2PCSK9 target sequence into the AAV genome that expresses the nuclease and/or fusing the nuclease to a specific peptide to promote its degradation. The second strategy used a shortened version of the parental promoter to reduce nuclease expression. Mice administered with these second-generation AAV vectors showed reduced PCSK9 expression due to the nuclease on-target activity and reduced off-target activity. All vectors induced a stable reduction of PCSK9 in primates treated with self-targeting and short-promoter AAVs. Compared to the meganuclease-expressing parental AAV vector, we observed a significant reduction in off-target activity. In conclusion, we increased the in vivo nuclease specificity using a clinically relevant strategy that can be applied to other genome-editing nucleases.
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Dependovirus/genética , Endonucleasas/genética , Edición Génica , Vectores Genéticos/genética , Lipoproteínas LDL/sangre , Inhibidores de PCSK9 , Regiones Promotoras Genéticas , Animales , Humanos , Ratones , Primates , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via the original article.
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BACKGROUND: Identifying nuclease-induced double-stranded breaks in DNA on a genome-wide scale is critical for assessing the safety and efficacy of genome editing therapies. We previously demonstrated that after administering adeno-associated viral (AAV) vector-mediated genome-editing strategies in vivo, vector sequences integrated into the host organism's genomic DNA at double-stranded breaks. Thus, identifying the genomic location of inserted AAV sequences would enable us to identify DSB events, mainly derived from the nuclease on- and off-target activity. RESULTS: Here, we developed a next-generation sequencing assay that detects insertions of specific AAV vector sequences called inverted terminal repeats (ITRs). This assay, ITR-Seq, enables us to identify off-target nuclease activity in vivo. Using ITR-Seq, we analyzed liver DNA samples of rhesus macaques treated with AAV vectors expressing a meganuclease. We found dose-dependent off-target activity and reductions in off-target events induced by further meganuclease development. In mice, we identified the genomic locations of ITR integration after treatment with Cas9 nucleases and their corresponding single-guide RNAs. CONCLUSIONS: In sum, ITR-Seq is a powerful method for identifying off-target sequences induced by AAV vector-delivered genome-editing nucleases. ITR-Seq will help us understand the specificity and efficacy of different genome-editing nucleases in animal models and clinical studies. This information can help enhance the safety profile of gene-editing therapies.
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Roturas del ADN de Doble Cadena , Edición Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Animales , Proteína 9 Asociada a CRISPR , Dependovirus , Vectores Genéticos/genética , Macaca mulatta , Ratones , ARN Guía de Kinetoplastida , Secuencias Repetidas Terminales/genéticaRESUMEN
Wilson disease (WD), an autosomal recessive disease caused by mutations in a copper-transporting P-type ATPase (Atp7b), causes severe liver damage. This disease is currently treated with the lifelong use of copper chelation therapy, which has side effects and does not fix copper metabolism. Here, we thoroughly characterized a mouse model of WD, the toxic milk mouse, and used the model to test a gene therapy approach for treating WD. WD mice accumulated copper in the liver from birth; severe copper accumulation and concurrent liver disease were evident by 2 months of age. Intravenously administering an adeno-associated viral (AAV) 8 vector expressing a codon-optimized version of the human ATP7B transgene into 2-month-old WD mice significantly decreased liver copper levels compared with age-matched, uninjected, WD mice. We also observed a significant dose-dependent decrease in liver disease. Male mice injected with 1011 genome copies of AAV8 vector showed only mild histopathological findings with a complete lack of liver fibrosis. Therefore, we conclude that administering gene therapy at the early stages of disease onset is a promising approach for reducing liver damage and correcting copper metabolism in WD.
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ATPasas Transportadoras de Cobre/genética , Cobre/metabolismo , Terapia Genética , Degeneración Hepatolenticular/terapia , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Degeneración Hepatolenticular/genética , Degeneración Hepatolenticular/metabolismo , Humanos , Hígado/lesiones , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Transgénicos , MutaciónRESUMEN
Systemic delivery of adeno-associated viral (AAV) vectors has been evaluated for the treatment of several liver diseases, including homozygous familial hypercholesterolemia, ornithine transcarbamylase deficiency, and hemophilia. Here, we evaluated this approach for the treatment of Crigler-Najjar syndrome. We administered wild-type rhesus macaques with 1.0 × 1013 or 2.5 × 1013 genome copies/kg of an AAV serotype 8 vector expressing a codon-optimized version of human uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) from a liver-specific promoter. We extensively studied vector biodistribution, transgene expression, and immune responses following vector administration. All rhesus macaques survived until their scheduled necropsy at day 56 and showed no clinical abnormalities during the course of the study. Macaques administered with either vector dose developed a T cell response to the AAV capsid and/or transgene. We mapped the immunodominant epitope in the human UGT1A1 sequence, and we found no correlation between peripheral and tissue-resident lymphocyte responses. Upon further investigation, we characterized CD107a+, granzyme B+, CD4+, and CD8+ transgene-specific cellular responses that were restricted to tissue-resident T cells. This study highlights the importance of studying immune responses at the vector transduction site and the limited usefulness of blood as a surrogate to evaluate tissue-restricted T cell responses.
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Argininosuccinic aciduria (ASA) is the second most common genetic disorder affecting the urea cycle. The disease is caused by deleterious mutations in the gene encoding argininosuccinate lyase (ASL); total loss of ASL activity results in severe neonatal onset of the disease, which is characterized by hyperammonemia within a few days of birth that can rapidly progress to coma and death. The long-term complications of ASA, such as hypertension and neurocognitive deficits, appear to be resistant to the current treatment options of dietary restriction, arginine supplementation, and nitrogen scavenging drugs. Treatment-resistant disease is currently being managed by orthotopic liver transplant, which shows variable improvement and requires lifetime immunosuppression. Here, we developed a gene therapy strategy for ASA aimed at alleviating the symptoms associated with urea cycle disruption by providing stable expression of ASL protein in the liver. We designed a codon-optimized human ASL gene packaged within adeno-associated virus serotype 8 (AAV8) as a vector for targeted delivery to the liver. To evaluate the therapeutic efficacy of this approach, we utilized a murine hypomorphic model of ASA. Neonatal administration of AAV8 via the temporal facial vein extended survival in ASA hypomorphic mice, although not to wild-type levels. Intravenous injection into adolescent hypomorphic mice led to increased survival and body weight and correction of metabolites associated with the disease. Our results demonstrate that AAV8 gene therapy is a viable approach for the treatment of ASA.
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Argininosuccinatoliasa/genética , Aciduria Argininosuccínica/terapia , Terapia Genética , Hiperamonemia/terapia , Animales , Argininosuccinatoliasa/administración & dosificación , Aciduria Argininosuccínica/genética , Dependovirus/genética , Modelos Animales de Enfermedad , Humanos , Hiperamonemia/genética , Hiperamonemia/patología , Ratones , Urea/metabolismoRESUMEN
Liver metabolism disorders are attractive targets for gene therapy, because low vector doses can reverse the buildup of toxic metabolites in the blood. Crigler-Najjar syndrome is an inherited disorder of bilirubin metabolism that is caused by the absence of uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) activity. This syndrome is characterized by hyperbilirubinemia and jaundice. Unfortunately, current phototherapy treatment is not effective long term. We intravenously injected phototherapy-rescued adult UGT1 knockout mice with 2.5 × 1010-2.5 × 1013 genome copies (GC)/kg of a clinical candidate vector, AAV8.TBG.hUGT1A1co, to study the treatment of disease compared to vehicle-only control mice. There were no apparent vector-related laboratory or clinical sequelae; the only abnormalities in clinical pathology were elevations in liver transaminases, primarily in male mice at the highest vector dose. Minimal to mild histopathological findings were present in control and vector-administered male mice. At vector doses greater than 2.5 × 1011 GC/kg, we observed a reversal of total bilirubin levels to wild-type levels. Based on a significant reduction in serum total bilirubin levels, we determined the minimally effective dose in this mouse model of Crigler-Najjar syndrome to be 2.5 × 1011 GC/kg.