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1.
Hum Gene Ther ; 22(8): 999-1009, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21320035

ABSTRACT

Recombinant adeno-associated virus 5 (rAAV5) represents a candidate vector with unique advantages for the treatment of hepatic disorders because of its narrow hepatic tropism. Noninvasive in vivo imaging of transgene expression provides an important tool with which to quantify the transduction efficiency, and duration and location, of transgene expression. In this study, we used positron emission tomography (PET) and positron emission tomography-computed tomography (PET-CT) imaging to monitor liver transduction efficacy in rodents and nonhuman primates that received rAAV5 vector encoding herpes simplex virus thymidine kinase (HSV-TK). HSV-TK expression in liver was also measured by immunohistochemistry. Notable differences in liver transduction efficiency were found, dependent on the animal species and sex. Male rodents were better transduced than females, as previously described. Moreover, male nonhuman primates also displayed increased hepatic expression of the rAAV5-delivered transgene, indicating that differences in rAAV-mediated liver transduction can be anticipated in humans. Our results demonstrate the high sensitivity and reproducibility of PET, using HSV-TK and [(18)F]FHBG, to detect gene expression after rAAV vector administration into living animals, confirming the utility of this technology in the quantification of transgene expression, even at low expression levels. However, we also describe how an immune response against HSV-TK hampered analysis of long-term expression in nonhuman primates.


Subject(s)
Dependovirus/genetics , Liver/metabolism , Animals , Female , Fluorine Radioisotopes , Genes, Reporter , Genetic Vectors , Guanine/analogs & derivatives , Liver/diagnostic imaging , Macaca fascicularis , Male , Mice , Mice, Inbred C57BL , Positron-Emission Tomography , Radiopharmaceuticals , Rats , Rats, Sprague-Dawley , Sex Factors , Simplexvirus/enzymology , Simplexvirus/immunology , Thymidine Kinase/genetics , Thymidine Kinase/metabolism , Transduction, Genetic , Transgenes
2.
Mol Ther ; 18(4): 715-24, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20179682

ABSTRACT

For many experiments in the study of the peripheral nervous system, it would be useful to genetically manipulate primary sensory neurons. We have compared vectors based on adeno-associated virus (AAV) serotypes 1, 2, 3, 4, 5, 6, and 8, and lentivirus (LV), all expressing green fluorescent protein (GFP), for efficiency of transduction of sensory neurons, expression level, cellular tropism, and persistence of transgene expression following direct injection into the dorsal root ganglia (DRG), using histological quantification and qPCR. Two weeks after injection, AAV1, AAV5, and AAV6 had transduced the most neurons. The time course of GFP expression from these three vectors was studied from 1 to 12 weeks after injection. AAV5 was the most effective serotype overall, followed by AAV1. Both these serotypes showed increasing neuronal transduction rates at later time points, with some injections of AAV5 yielding over 90% of DRG neurons GFP(+) at 12 weeks. AAV6 performed well initially, but transduction rates declined dramatically between 4 and 12 weeks. AAV1 and AAV5 both transduced large-diameter neurons, IB4(+) neurons, and CGRP(+) neurons. In conclusion, AAV5 is a highly effective gene therapy vector for primary sensory neurons following direct injection into the DRG.


Subject(s)
Dependovirus/classification , Ganglia, Spinal , Genetic Therapy , Genetic Vectors , Animals , Dependovirus/genetics , Female , Plasmids , Rats , Rats, Wistar , Serotyping , Transduction, Genetic
3.
Hum Gene Ther ; 20(8): 908-17, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19419275

ABSTRACT

Recombinant adeno-associated viral (AAV) vectors have unique properties, which make them suitable vectors for gene transfer. Here we assess the liver transduction efficiency and biodistribution of AAV-pseudotyped capsids (serotypes) 1, 5, 6, and 8, combined with single-stranded and double-stranded genomic AAV2 structures carrying the luciferase reporter gene after systemic administration. The analysis was performed in vivo and ex vivo, in male and female mice. Gender-related differences in AAV-mediated transduction and biodistribution were shown for the four serotypes. Our data confirm the superiority of AAV8 over the rest of the serotypes, as well as a significant advantage of double-stranded genomes in terms of liver transduction efficiency, particularly in females. Regarding biodistribution, AAV5 displayed a narrower tropism than the other serotypes tested, transducing, almost exclusively, the liver. Interestingly, AAV1 and AAV8, in particular those having single-stranded genomes, showed high transduction efficiency of female gonads. However, no inadvertent germ line transmission of AAV genomes was observed after breeding single-stranded AAV8-injected female mice with untreated males. In conclusion, double-stranded AAV8 vectors led to the highest levels of liver transduction in mice, as demonstrated by luciferase expression. Nevertheless, the transduction of other organs with AAV8 vectors could favor the use of less efficient serotypes, such as AAV5, which display a narrow tropism.


Subject(s)
Dependovirus/classification , Dependovirus/genetics , Genome, Viral/genetics , Liver/metabolism , Liver/virology , Sex Characteristics , Transduction, Genetic , Animals , Female , Germ Cells/virology , Injections, Intravenous , Luciferases/metabolism , Luminescent Measurements , Male , Mice , Mice, Inbred BALB C , Ovary/cytology , Ovary/virology , Serotyping , Time Factors , Tissue Distribution
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