Bioactive, full-length parathyroid hormone delivered using an adeno-associated viral vector

Abstract

Delivering the parathyroid hormone (PTH) gene has been attempted preclinically in a handful of studies, but delivering full-length PTH (1-84) using adeno-associated viral (AAV) vectors has not. Given the difficulty in achieving therapeutic levels of secreted proteins using gene therapy, this study seeks to determine the feasibility of doing so with PTH. An AAV vector was used to deliver human PTH driven by a strong promoter. We demonstrate the ability to secrete full-length PTH from various cell types in vitro. PTH secretion from hepatocytes was measured over time and a fluorescent marker was used to compare the secretion rate of PTH in various cell types. Potency was measured by the ability of PTH to act on the PTH receptors of osteosarcoma cells and induced proliferation. PTH showed potency in vitro by inducing proliferation in two osteosarcoma cell lines. In vivo, AAV was administered systemically in immunocompromised mice which received xenografts of osteosarcoma cells. Animals that received the highest dose of AAV-PTH had higher liver and plasma concentrations of PTH. All dosing groups achieved measurable plasma concentrations of human PTH that were above the normal range. The high-dose group also had significantly larger tumors compared to control groups on the final day of the study. The tumors also showed dose-dependent differences in morphology. When looking at endocrine signaling and endogenous bone turnover, we observed a significant difference in tibial growth plate width in animals that received the high-dose AAV as well as dose-dependent changes in blood biomarkers related to PTH. This proof-of-concept study shows promise for further exploration of an AAV gene therapy to deliver full-length PTH for hypoparathyroidism. Additional investigation will determine efficacy in a disease model, but data shown establish bioactivity in well-established models of osteosarcoma.

Keywords: AAV vector; AAV-PTH; Hypoparathyroidism; parathyroid hormone.

Figure 1.

Exogenous cell types can be engineered to secrete PTH in vitro. (a) The AAV vector used for all experiments contains the human cDNA sequence for the full 115 residue human prepro-PTH peptide. Through endogenous post-translational modification pathways, secreted PTH is 84 amino acids in full form. (b) Kinetic secretion of PTH by HepG2 cells after transduction with AAV2-EF1α-PTH. Cells were perfused with media at 0.2 mL/h over 110 h to track secretion and expression over time. At maximal rates, 10⁵ cells secreted 5 pg/h of human PTH. N = 4 per group. (c) Three cell lines were transduced with AAV-EF1α-PTH-IRES-GFP at an MOI of 5000. Per cell secretion rate was obtained by sampling the PTH concentration in the well at 72 h post-transduction/the number of transduced cells per well. Mean values shown are three well replicates in two experiments for total of n = 6 per group. Data are shown as mean values ± SD. (A color version of this figure is available in the online journal.)

Figure 2.

Secreted PTH is potent and capable of inducing osteoblast proliferation. (a) Osteosarcoma (U2OS and SaOS-2) cells were transduced with AAV-EF1α-PTH-IRES-GFP at varying multiplicity of infection (MOI). (b) The concentrations of PTH across different groups from U2OS wells correlated positively with GFP+ cell populations. (c) Transduction efficiency for each MOI and cell type as determined by GFP+ cell number compared to total cell population. Normalized proliferation of U2OS (d) and SaOS-2 (e) human osteosarcoma cell lines over time. The cells transduced at the highest MOI (5000) exhibited the highest levels of proliferation at all timepoints for both groups. Mean values shown are from four well replicates in two experiments for total of n = 8 wells per group. Data are shown as mean values ± SD. (A color version of this figure is available in the online journal.)

Figure 3.

PTH expression in mice after AAV2 injection. (A) Plasma PTH concentrations (pg/mL) 20 days after AAV injection for each dose group. (b) Protein was extracted from liver samples after euthanasia (day 42) and measured via ELISA for PTH concentration shown as pg/mg liver tissue for each group. N = 4 mice per group. Data are shown as mean values ± SD. (c) AAV viral genome copies were measured in DNA extracted from liver samples using qPCR and are represented as copy number/µg genomic DNA. (A color version of this figure is available in the online journal.) NS: non-significant. ****P < 0.0001.

Figure 4.

PTH induces tumor growth in osteosarcoma model mice. (a) Osteosarcoma model development timeline showing AAV and SaOS-2 cell injection. Arrows indicate the tumor tracking timepoints. Endpoint was day 42. (b) Tumor growth rates over time as measured by caliper showed the high-dose group grew faster than all other groups. (c) The final tumor weights for each group. Bars shown are mean values ± SD. N = 4 mice per group. (d) and (e) Representative images of tumors from low-dose (d) and high-dose groups (e). Scale bar shown is 1 cm in length. (A color version of this figure is available in the online journal.)

Figure 5.

High-dose AAV influences growth plate width of the tibia. (a) Average growth plate width for each dosing group taken from five measurements from one H&E section from each mouse tibia. Bars represent the mean values ± SD. (b) Representative H&E images of the tibial heads from each group. (A color version of this figure is available in the online journal.) NS: non-significant. **P < 0.01.

Figure 6.

PTH induces dose–dependent changes in blood biomarkers. Title of each figure shows the protein analyte. Plasma samples at the time of euthanasia were used for measurement. Bars represent the mean value ± SD of animals in each group. Significance denotes a dose–dependent relationship. (A color version of this figure is available in the online journal.) NS: non-significant; ACTH: adrenocorticotropic hormone; FGF23: fibroblast growth factor 23; DKK1: Dickkopf WNT signaling pathway inhibitor 1; IL-6: interleukin-6; TNFA: tumor necrosis factor alpha. *P < 0.05; **P < 0.01.

Figure 7.

High-dose AAV8 osteosarcoma study. Comparative results for dose-matched groups in the AAV2 (black) and AAV8 (pink) studies for (a) PTH concentration at 21 days postinjection of AAV (pg/mL), (b) endpoint tumor diameter at day 42 postinjection of SaOS-2 cells (mm), and (c) liver expression as measured by qPCR (vg copies/µg DNA). Blood analytes for the AAV8 study for (d) calcium (mmol/L), (e) phosphorus (mmol/L), (f) P1NP, and (g) CTX. Bars represent the mean value ± SD for n = 4 animals/group. (A color version of this figure is available in the online journal.) NS: non-significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.