Isolated GH Deficiency Type II: Knockdown of the Harmful Δ3GH Recovers wt-GH Secretion in Rat Tumor Pituitary Cells

Isolated GH deficiency type II (IGHD II) is the autosomal dominant form of GHD. In the majority of the cases, this disorder is due to specific GH-1 gene mutations that lead to mRNA missplicing and subsequent loss of exon 3 sequences. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH (Δ3GH) isoform that reduces the accumulation and secretion of wild-type-GH. At present, patients suffering from this type of disease are treated with daily injections of recombinant human GH in order to maintain normal growth. However, this type of replacement therapy does not prevent toxic effects of the Δ3GH mutant on the pituitary gland, which can eventually lead to other hormonal deficiencies. We developed a strategy involving Δ3GH isoform knockdown mediated by expression of a microRNA-30-adapted short hairpin RNA (shRNA) specifically targeting the Δ3GH mRNA of human (shRNAmir-Δ3). Rat pituitary tumor GC cells expressing Δ3GH upon doxycycline induction were transduced with shRNAmir-Δ3 lentiviral vectors, which significantly reduced Δ3GH protein levels and improved human wild-type-GH secretion in comparison with a shRNAmir targeting a scrambled sequence. No toxicity due to shRNAmir expression could be observed in cell proliferation assays. Confocal microscopy strongly suggested that shRNAmir-Δ3 enabled the recovery of GH granule storage and secretory capacity. These viral vectors have shown their ability to stably integrate, express shRNAmir, and rescue IGHD II phenotype in rat pituitary tumor GC cells, a methodology that opens new perspectives for the development of gene therapy to treat IGHD patients. Impaired wt-GH secretion in rat pituitary tumor cell line is rescued after transduction with lentiviral vector-encoding shRNAmir targeting autosomal dominant Δ3GH mRNA.