RESUMEN
The safe correction of an inherited bleeding disorder in utero prior to the onset of organ damage is highly desirable. Here, we report long-term transgene expression over more than 6 years without toxicity following a single intrauterine gene transfer (IUGT) at 0.9G using recombinant adeno-associated vector (AAV)-human factor IX (hFIX) in the non-human primate model we have previously described. Four of six treated animals monitored for around 74 months expressed hFIX at therapeutic levels (3.9%-120.0%). Long-term expression was 6-fold higher in males and with AAV8 compared to AAV5, mediated almost completely at this stage by random genome-wide hepatic proviral integrations, with no evidence of hotspots. Post-natal AAV challenge without immunosuppression was evaluated in two animals exhibiting chronic low transgene expression. The brief neutralizing immune reaction elicited had no adverse effect and, although expression was not improved at the dose administered, no clinical toxicity was observed. This long-term surveillance thus confirms the safety of late-gestation AAV-hFIX transfer and demonstrates that postnatal re-administration can be performed without immunosuppression, although it requires dose optimization for the desired expression. Nevertheless, eventual vector genotoxicity and the possibility of germline transmission will require lifelong monitoring and further evaluation of the reproductive function of treated animals.
Asunto(s)
Dependovirus/genética , Factor IX/genética , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Hemofilia B/sangre , Hemofilia B/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Dependovirus/inmunología , Modelos Animales de Enfermedad , Femenino , Terapia Genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/efectos adversos , Hemofilia B/terapia , Humanos , Tolerancia Inmunológica , Hígado/metabolismo , Macaca fascicularis , Masculino , Embarazo , Factores de Tiempo , Transducción Genética , TransgenesRESUMEN
Recombinant adeno-associated viral vectors (rAAV) currently constitute a real therapeutic strategy for the sustained correction of diverse genetic conditions. Though a wealth of preclinical and clinical studies have been conducted with rAAV, the oncogenic potential of these vectors is still controversial, particularly when considering liver-directed gene therapy. Few preclinical studies and the recent discovery of incomplete wild-type AAV2 genomes integrated in human hepatocellular carcinoma biopsies have raised concerns on rAAV safety. In the present study, we have characterized the integration of both complete and partial rAAV2/5 genomes in nonhuman primate tissues and clinical liver biopsies from a trial aimed to treat acute intermittent porphyria. We applied a new multiplex linear amplification-mediated polymerase chain reaction (PCR) assay capable of detecting integration events that are originated throughout the rAAV genome. The integration rate was low both in nonhuman primates and patient's samples. Importantly, no integration clusters or events were found in genes previously reported to link rAAV integration with hepatocellular carcinoma development, thus showing the absence of genotoxicity of a systemically administered rAAV2/5 in a large animal model and in the clinical context.
Asunto(s)
Dependovirus/fisiología , Vectores Genéticos/administración & dosificación , Hígado/efectos de los fármacos , Porfiria Intermitente Aguda/terapia , Animales , Dependovirus/genética , Terapia Genética , Vectores Genéticos/efectos adversos , Humanos , Macaca fascicularis , Recombinación Genética , Análisis de Secuencia de ADN/métodos , Transducción Genética , Integración ViralRESUMEN
High-throughput integration site (IS) analysis of wild-type adeno-associated virus type 2 (wtAAV2) in human dermal fibroblasts (HDFs) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site (trs)-like motif leads to a 4-fold-increased probability of wtAAV integration. Electrophoretic mobility shift assays (EMSAs) confirmed binding of Rep to off-target RBSs. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.
Asunto(s)
Secuencias de Aminoácidos , Proteínas de Unión al ADN/metabolismo , Dependovirus/fisiología , Proteínas Virales/metabolismo , Integración Viral , Línea Celular , Células Epiteliales/virología , Fibroblastos/virología , HumanosRESUMEN
The comprehensive characterization of recombinant adeno-associated viral (rAAV) integration frequency and persistence for assessing rAAV vector biosafety in gene therapy is severely limited due to the predominance of episomal rAAV vector genomes maintained in vivo. Introducing rAAV insertional standards (rAIS), we show that linear amplification-mediated (LAM)-PCR and deep sequencing can be used for validated measurement of rAAV integration frequencies. Integration of rAAV2/1 or rAAV2/8, following intramuscular (IM) or regional intravenous (RI) administration of therapeutically relevant vector doses in nine adult non-human primates (NHP), occurs at low frequency between 10(-4) and 10(-5) both in NHP liver and muscle, but with no preference for specific genomic loci. High resolution mapping of inverted terminal repeat (ITR) breakpoints in concatemeric and integrated vector genomes reveals distinct vector recombination hotspots, including large deletions of up to 3 kb. Moreover, retrieval of integrated rAAV genomes indicated approximately threefold increase in liver compared to muscle. This molecular analysis of rAAV persistence in NHP provides a promising basis for a reliable genotoxic risk assessment of rAAV in clinical trials.
Asunto(s)
Dependovirus/genética , Vectores Genéticos , Músculo Esquelético/metabolismo , Primates/metabolismo , Recombinación Genética , Integración Viral , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Dosificación de Gen , Técnicas de Transferencia de Gen , Humanos , Hígado , Músculo Esquelético/virología , Primates/virología , Provirus/genéticaRESUMEN
Intrauterine gene transfer (IUGT) offers ontological advantages including immune naiveté mediating tolerance to the vector and transgenic products, and effecting a cure before development of irreversible pathology. Despite proof-of-principle in rodent models, expression efficacy with a therapeutic transgene has yet to be demonstrated in a preclinical nonhuman primate (NHP) model. We aimed to determine the efficacy of human Factor IX (hFIX) expression after adeno-associated-viral (AAV)-mediated IUGT in NHP. We injected 1.0-1.95 × 10(13) vector genomes (vg)/kg of self-complementary (sc) AAV5 and 8 with a LP1-driven hFIX transgene intravenously in 0.9G late gestation NHP fetuses, leading to widespread transduction with liver tropism. Liver-specific hFIX expression was stably maintained between 8 and 112% of normal activity in injected offspring followed up for 2-22 months. AAV8 induced higher hFIX expression (P = 0.005) and milder immune response than AAV5. Random hepatocellular integration was found with no hotspots. Transplacental spread led to low-level maternal tissue transduction, without evidence of immunotoxicity or germline transduction in maternal oocytes. A single intravenous injection of scAAV-LP1-hFIXco to NHP fetuses in late-gestation produced sustained clinically-relevant levels of hFIX with liver-specific expression and a non-neutralizing immune response. These data are encouraging for conditions where gene transfer has the potential to avert perinatal death and long-term irreversible sequelae.
Asunto(s)
Dependovirus/genética , Factor IX/genética , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/genética , Hemofilia B/terapia , Animales , Línea Celular , Dependovirus/inmunología , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/farmacocinética , Células HEK293 , Hemofilia B/genética , Humanos , Inyecciones , Macaca fascicularis , Placenta/metabolismo , Embarazo , Transducción Genética , Integración ViralRESUMEN
The clinical application of adeno-associated virus vectors (AAVs) is limited because of concerns about AAV integration-mediated tumorigenicity. We performed integration-site analysis after AAV1-LPL(S447X) intramuscular injection in five lipoprotein lipase-deficient subjects, revealing random nuclear integration and hotspots in mitochondria. We conclude that AAV integration is potentially safe and that vector breakage and integration may occur from each position of the vector genome. Future viral integration-site analyses should include the mitochondrial genome.
Asunto(s)
Dependovirus/genética , Terapia Genética , Hiperlipoproteinemia Tipo I/genética , Integración Viral/fisiología , Animales , Sitios de Unión/genética , Dependovirus/fisiología , Terapia Genética/efectos adversos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/efectos adversos , Vectores Genéticos/genética , Vectores Genéticos/fisiología , Humanos , Inyecciones Intramusculares , Lipoproteína Lipasa/administración & dosificación , Lipoproteína Lipasa/deficiencia , Lipoproteína Lipasa/genética , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Mutagénesis Insercional/genética , Mutagénesis Insercional/fisiología , Transcriptoma , Integración Viral/genéticaRESUMEN
Acute intermittent porphyria (AIP) results from haplo-insufficient activity of porphobilinogen deaminase (PBGD) and is characterized clinically by life-threatening, acute neurovisceral attacks. To date, liver transplantation is the only curative option for AIP. The aim of the present preclinical nonhuman primate study was to determine the safety and transduction efficacy of an adeno-associated viral vector encoding PBGD (recombinant AAV serotype 5-codon-optimized human porphobilinogen deaminase, rAAV5-cohPBGD) administered intravenously as part of a safety program to start a clinical study in patients with AIP. Macaques injected with either 1 × 10(13) or 5 × 10(13) vector genomes/kg of clinical-grade rAAV5-cohPBGD were monitored by standardized clinical parameters, and vector shedding was analyzed. Liver transduction efficacy, biodistribution, vector integration, and histopathology at day 30 postvector administration were determined. There was no evidence of acute toxicity, and no adverse effects were observed. The vector achieved efficient and homogenous hepatocellular transduction, reaching transgenic PBGD expression levels equivalent to 50% of the naturally expressed PBGD mRNA. No cellular immune response was detected against the human PBGD or AAV capsid proteins. Integration site analysis in transduced liver cells revealed an almost random integration pattern supporting the good safety profile of rAAV5-cohPBGD. Together, data obtained in nonhuman primates indicate that rAAV5-cohPBGD represents a safe therapy to correct the metabolic defect present in AIP patients.
Asunto(s)
Terapia Genética , Haploinsuficiencia/genética , Hidroximetilbilano Sintasa/genética , Porfiria Intermitente Aguda/terapia , Animales , Dependovirus , Vectores Genéticos , Hepatocitos/metabolismo , Humanos , Hidroximetilbilano Sintasa/uso terapéutico , Macaca , Porfiria Intermitente Aguda/genética , Porfiria Intermitente Aguda/patología , Distribución Tisular/genética , Transducción GenéticaRESUMEN
Zinc-finger nucleases (ZFNs) allow gene editing in live cells by inducing a targeted DNA double-strand break (DSB) at a specific genomic locus. However, strategies for characterizing the genome-wide specificity of ZFNs remain limited. We show that nonhomologous end-joining captures integrase-defective lentiviral vectors at DSBs, tagging these transient events. Genome-wide integration site analysis mapped the actual in vivo cleavage activity of four ZFN pairs targeting CCR5 or IL2RG. Ranking loci with repeatedly detectable nuclease activity by deep-sequencing allowed us to monitor the degree of ZFN specificity in vivo at these positions. Cleavage required binding of ZFNs in specific spatial arrangements on DNA bearing high homology to the intended target site and only tolerated mismatches at individual positions of the ZFN binding sites. Whereas the consensus binding sequence derived in vivo closely matched that obtained in biochemical experiments, the ranking of in vivo cleavage sites could not be predicted in silico. Comprehensive mapping of ZFN activity in vivo will facilitate the broad application of these reagents in translational research.