215. Treating Hypoparathyroidism through Regulated Expression of Parathyroid Hormone in Skeletal Muscle
Authors: Maja Z. Salva, Stephen D. Hauschka
Journal: Molecular Therapy (2006)

Hypoparathyroidism is a disorder in which reduced serum calcium levels cause muscle irritability ranging from mild to severe. If left untreated, hypoparathyroidism can lead to seizures, tetany, and in extreme cases death. Currently, hypoparathyroid patients are treated with vitamin D and calcium supplements. While this treatment normalizes serum calcium levels, urine calcium levels remain elevated and over time this can lead to renal dysfunction. A recent study showed that one or two daily injections of recombinant human PTH1-34 normalized both serum and urine calcium levels (Winer et al, J Clin Endocrinol Metab. 2003, 88(9):4214). However, the spikes in parathyroid hormone levels associated with this delivery method can lead to undesirable bone turnover. Continuous delivery of low levels of PTH would provide a better solution, and we propose to achieve this using gene therapy. Our strategy involves rAAV6- mediated transduction of skeletal muscle with an externally regulated PTH expression cassette and fine-tuning of expression to achieve physiological levels by careful dosing of an orally available inducer molecule. We developed vectors for regulated expression of both full length hormone (PTH1-84) and PTH1-34 using ARIAD Pharmaceuticals' ARGENT system. This is a dual-vector system with the PTH gene placed downstream of the ARGENT inducible promoter on one vector and the ARGENT transcription factor subunits driven by the CMV promoter on the other, each containing ITRs for packaging into rAAV particles. Expression in cells containing both vectors is induced by addition of a rapamycin analog AP21967. We first evaluated the vectors by transient transfection of MM14 mouse muscle cells in culture. Addition of AP21967 to transfected cells increased PTH secretion into the media by about 100-fold. Expression of PTH1-84 was about 30-fold higher than PTH1-34, at 10 ng/100,000 cells/day. We then evaluated the vectors in mice following direct injection of rAAV6 vectors into the TA muscles. A single 10 mg/kg IP injection of AP21967 led to a transient increase in serum PTH to supraphysiological levels, with 5-fold more PTH1-84 than PTH1-34. However, maximum levels achieved with constitutive PTH1-84 expression (4000 pg/ml) were even higher than with regulated expression (500 pg/ml) and caused serious illness in mice that required euthanasia. This highlights the need for tight regulation of PTH levels. To further improve regulation of PTH expression, we plan to conduct studies involving continuous delivery of low levels of AP21967 from subcutaneously implanted minipumps. We also plan to evaluate the ability of this system to normalize serum calcium levels in parathyroidectomized mice. Finally, to limit PTH expression to muscle cells, we placed the ARIAD transcription factor subunits under control of MHCK7, our strongest muscle-specific regulatory cassette. Initial experiments in MM14 cells indicate that these vectors can achieve similar levels of regulated PTH expression as with CMV-driven transcription factors. We plan to further evaluate regulated muscle-specific expression of PTH in normal and parathyroidectomized mice.