A single injection of double-stranded adeno-associated viral vector expressing GH normalizes growth in GH-deficient mice

Abstract

GH is secreted by the somatotropic cells of the pituitary gland, and its deficiency (GHD) impairs longitudinal growth. Due to its short half-life, GH therapy needs administration of GH injections daily. Adeno-associated viral vectors (AAV) can deliver gene therapy to animals with possible future applications in humans. The new generation of double-stranded AAV vectors (dsAAV) provides widespread, strong, and stable transgene expression without toxicity and immune response. To determine whether such a new system could be used to deliver GH to a mouse model of isolated GHD due to ablation of the GHRH knock-out gene (GHRHKO), we have created AAV viral particles containing mouse GH cDNA driven by a cytomegalovirus promoter (dsAAV8-CMV-GH), and tested them in male GHRHKO mice. GHRHKO animals received either a single (low dose) or two (high dose) i.p. injections of dsAAV8-CMV-GH (1!10 11 particles) at the 10th and 11th days of age, or a placebo injection, and were followed up to the 6th or 24th week of life. A single dsAAV8-GH injection caused body length and weight normalization. At week 6, serum GH was higher in mice receiving both virus doses compared with controls, while it was normal at week 24. Serum IGF-1 increased in both virus-treated groups, and it was normal at 24 weeks. GH mRNA expression was detected in liver, skeletal, and heart muscle of virus-injected animals. These data show that normalization of longitudinal growth can be reached in GHD mice using a single injection of a doublestranded adeno-associated virus expressing GH.

Effects of GH deficiency and GH replacement on inter-male aggressiveness in mice

“…Indeed, normalization of growth was attained by the same GH dosage and schedule we have used in this work (in younger mice), without serum IGF-I normalization. As further proof that GH was administered at a dosage that had biological consequences, we found a significant increase in spleen weight induced by GH treatment, a finding that we had reported before [29]. A series of studies have shown that GH influences behavior in several species: in salmonid fish GH is involved in aggressive behavior by improving swimming activity [15] and appetite [9]; and similar behavioral changes have been reported in GH-transgenic salmon [16].…”

“…As previously reported [29], GHRHKO mice injected with GH exhibited a significant increase in spleen weight to a size comparable to that of the HTZ group.…”

“…Serum IGF-I levels are mostly an expression of liver IGF-I production [4], and do not necessarily correlate with growth [32]. Accordingly, we have previously reported a similar discrepancy between serum IGF-I levels and the effects of either GH or GHRH on growth in GHRHKO mice [24,29]. Indeed, normalization of growth was attained by the same GH dosage and schedule we have used in this work (in younger mice), without serum IGF-I normalization.…”

Different ex Vivo and Direct in Vivo DNA Administration Strategies for Growth Hormone Gene Therapy in Dwarf Animals

“…More recently, in 2008, a new generation of double-stranded adeno-associated viral vectors (dsAAV) encoding mGH cDNA driven by a universal promoter (CMV) were used by a group coordinated by Johns Hopkins University School of Medicine to prepare viral particles that were injected into GHRHKO mice, a model of isolated GH deficiency due to generalized ablation (knock-out, KO) of the GHRH gene 17,18 . These genetically modified dsAAV can infect dividing and non-dividing cells in vitro and in vivo with a long-term transgenic expression (up to 1 year) and elicit a lower toxicity and cellular immune response, therefore being generally considered to be safer than adenoviral vectors.…”

“…These genetically modified dsAAV can infect dividing and non-dividing cells in vitro and in vivo with a long-term transgenic expression (up to 1 year) and elicit a lower toxicity and cellular immune response, therefore being generally considered to be safer than adenoviral vectors. In an initial study, GHRHKO mice were injected intraperitoneally with either a single dose (low dose) or two doses (high dose) of 1 x 10 11 viral particles at the 10 th and 11 th days of age and were followed up to the 6 th or 24 th week of life 17 . Body weight and length of both viraltreated groups became normal at 6 months of age and normal femoral and tibial lengths, body composition and weight of organs (liver, spleen, heart and kidney) were also obtained.…”