An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene

Abstract

Adenoviral (Ad)-mediated in vivo gene transfer and expression are limited in part by cellular immune responses to viral-encoded proteins and͞or transgene immunogenicity. In an attempt to diminish the former responses, we have previously developed and described helper-dependent (HD) Ad vectors in which the viral protein coding sequences are completely eliminated. These HD vectors have up to 37 kb insert capacity, are easily propagated in a Cre recombinasebased system, and can be produced to high concentration and purity (>99.9% helper-free vector). In this study, we compared safety and efficacy of leptin gene delivery mediated by an HD vector (HD-leptin) and a first-generation E1-deleted Ad vector (Ad-leptin) in normal lean and ob͞ob (leptindeficient) mice. In contrast to evidence of liver toxicity, inf lammation, and cellular infiltration observed with Adleptin delivery in mice, HD-leptin delivery was associated with a significant improvement in associated safety͞toxicity and resulted in efficient gene delivery, prolonged elevation of serum leptin levels, and associated weight loss. The greater safety, efficient gene delivery, and increased insert capacity of HD vectors are significant improvements over current Ad vectors and represent favorable features especially for clinical gene therapy applications. Adenoviral (Ad) vectors are currently among the most efficient gene transfer vehicles for both in vitro and in vivo delivery, but the utilization of current Ad vectors for many gene therapy applications is limited by the transient nature of transgene expression obtained by these vectors (1-7). Several factors have been shown to contribute to and modulate the duration of Ad-mediated gene expression and the immunogenicity of these vectors, including ''leaky'' viral protein expression and the transgene that is delivered (8-15). The development of Ad vectors that are deleted in all viral protein-coding sequences offers the prospect of a potentially safer, less immunogenic vector with an insert capacity of up to 37 kb (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). This vector is supplied in trans with the structural proteins required for packaging and rescue and is thus helper-dependent (HD) (24).Leptin has been recently identified as a potent modulator of weight and food intake. Daily delivery of recombinant leptin protein was shown to induce weight reduction, suppress appetite, and decrease blood insulin and glucose levels in ob͞ob (leptindeficient) mice (27)(28)(29). It has been shown that delivery of the leptin cDNA by first-generation Ad vectors (Ad-leptin) may substitute for daily recombinant leptin protein treatment, although the effects were transient in both lean and ob͞ob treated mice (7,30). In the present study, we delivered the leptin cDNA using the HD virus (HD-leptin), testing the hypothesis that elimination of the viral protein coding sequences would diminish the vector's cellular immunogenicity and toxicity, and hence support its longevity in vivo. Because both the viral proteins and the trans...

Efficient repetitive gene delivery to skeletal muscle using recombinant adenovirus vector containing the Coxsackievirus and adenovirus receptor cDNA

“…Some researchers have reported that immune reaction to rAd has been suppressed by FK506 immunosuppression agent, 7 CTLA4Ig, 8 and a gutted adenovirus vector. [9][10][11] Thus, repetitive transduction to skeletal muscle could seem valid for consecutive expression of a therapeutic gene. The latter is caused by a maturation-dependent loss of the myofiber's susceptibility to rAd uptake which is due to structural and biochemical changes during muscle maturation.…”

Prolonged and inducible transgene expression in the liver using gutless adenovirus: A potential therapy for liver cancer

“…11,14 -16 In addition, the elimination of all the adenoviral genes leaves room to allocate large expression cassettes, and therefore gutless adenoviruses are also designed as high-capacity adenoviral vectors. 11,14 -16 For correction of metabolic and/or hereditary diseases, different authors have used gutless vectors with nonregulable promoters showing long-term expression of transgenes such as ␣1-antitrypsin, 17,18 leptin, 19 apolipoprotein E, 20 and factor VIII and IX [21][22][23][24] at therapeutic levels without apparent hepatic toxicity.…”

Gutless adenovirus: last-generation adenovirus for gene therapy

“…41,62,63 Last-generation helper-dependent or Gutless adenovirus R Alba et al gutless adenovirus vectors display reduced long-term toxicity and prolonged transgene expression compared to first-generation vectors after administration to peripheral organs of immunologically naïve animals. 56,[64][65][66][67][68] Lack of coding viral genes may account for reduced adaptive cellular immune response after systemic delivery of gutless vectors. Initially, gutless vectors are capable of transducing dendritic cells and stimulating Ad-specific T-cell responses, independent of viral gene transcription.…”

“…68 Gutless vectors have been shown to express non-immunogenic transgenes during the whole life of the mouse, while expression driven by first-generation adenovirus lasted at most for 3 months. [80][81][82] Therapeutic genes carried by gutless vectors have been administered to the liver of mouse models for different diseases, such as hemophilia A and B, 83,84 obesity, 66 familial hypercholesterolemia, 81,85-87 ornithine transcarbamylase deficiency, 88 diabetes 89 and chronic viral hepatitis. 90,91 Therapeutic levels of most proteins have been documented for a long-term duration.…”