Extended space missions in a microgravity environment alter normal physiology in the human body. One critical issue in spaceflight is loss of bone mineral density. In the process of bone homeostasis, specialized cells facilitate bone remodelling with coupled bone resorption and formation. The reduced mechanical loading in microgravity causes resorption to outpace formation, which results in loss of bone mass. Medications such as parathyroid hormone (PTH) [amino acids 1-34], a peptide fragment of naturally occurring human PTH, stimulate bone formation and may be able to restore bone mass in microgravity. However, this requires a daily subcutaneous injection for a number of months, and in a limited resource environment, this is impractical. An alternative to transporting an injectable medication is to instead build capacity to produce it during the mission, which we propose to do in transgenic plants. We are producing transgenic lettuce which expresses a fusion protein consisting of PTH [amino acids 1-34] linked with a fragment crystallizable (Fc) domain of human IgG1 via a flexible linker. The Fc component and sequestration of the PTH-Fc within the plant endoplasmic reticulum is intended to increase bioavailability via oral delivery. We constructed a binary vector for expression of this PTH-Fc protein and used it to constitutively produce PTH-Fc in two varieties of transgenic lettuce via Agrobacterium tumefaciens mediated transformation of wild type lettuce. We have successfully generated transgenic (T) plants and screened T1 and T2 generations using a selectable marker and PCR. An extraction protocol coupled with Western blotting has enabled the detection of PTH-Fc in plant extracts by targeting either the PTH or the Fc regions of the fusion protein. An optimized ELISA protocol is being developed to quantify expression levels of PTH-Fc. It is estimated from preliminary results that the expression level is on the order of 10-12 mg PTH-Fc per kilogram of fresh weight biomass.

Development of a plant generated therapeutic made in-flight to treat microgravity osteopenia

ACS Spring 2022

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