UC Davis

Extended space missions in a microgravity environment alter normal physiology in the human body. One critical issue in spaceflight is the loss of bone mineral density. In the process of bone homeostasis, specialized cells facilitate bone remodeling 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 the duration of a mission, and in a limited-resource environment (LRE), 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. A whole plant can be thought of as a single-use organic bioreactor especially suited for spaceflight applications.

We are producing transgenic Lactuca sativa (lettuce) which expresses a fusion protein consisting of the PTH peptide linked with a fragment crystallizable (Fc) domain of human IgG1 via a flexible linker. The size increase by the addition of the Fc component and sequestration of the PTH-Fc within the plant endoplasmic reticulum is intended to increase bioavailability via oral delivery.

Developing a plant-based production system for use in LREs necessitates designing models to predict biomass production, which in turn requires the empirical analysis of basic plant growth characteristics. In Chapter 3, we discuss the characterization of plant growth, including biomass over time, water mass fraction in the plant, and seed production. We also describe the growth of a model plant in a vertical aeroponic growth system.

In Chapter 4, we discuss screening and selection strategies to verify the integration and expression of the PTH-Fc transgene. We present experimental results for a study of PTH-Fc expression over time and per leaf in transgenic lettuce. The expression level varied across different leaves and time points. It was found that the average PTH-Fc expression at a harvest time of 25 days after emergence was 68 mg/kg (fresh weight basis). We also show proof-of-concept expression in transgenic Nicotiana benthamiana.

While we suggest that stable expression of PTH-Fc in transgenic plants is the optimal solution to produce therapeutics in LREs for chronic disease states, transient expression, in which growing wild type plants are made to produce recombinant protein, is a valuable method for rapidly meeting health needs. We quantify transient expression of PTH-Fc in wild type Nicotiana benthamiana grown in a vertical aeroponic system in Chapter 5. It was found that plants grown in this system have expression levels comparable to plants grown in solid substrate in an environmentally controlled growth chamber. We show that the method used to induce expression in N. benthamiana is compatible with our reference cultivar of lettuce.

Modeling is a critical aspect of mission design, and our proposal to use plant biotechnology in space requires simulation of the growth system as part of a life support system, which includes the production of food and medicine. Chapter 6 concerns modeling of plant biomass growth using the NASA-sponsored Modified Energy Cascade model. We show an adaptation of the model for use in systems engineering and use cases for its output. The model is unable to account for the nitrogen required for plant growth and therefore must be extended. We discuss an approach to a nitrogen-dependent biomass growth model.

In Chapter 7, we discuss the purification of PTH-Fc from plant protein extracts via liquid chromatography. We show that purification methods that work well with N. benthamiana need to be modified for use with lettuce. Progress made toward purification of recombinant PTH-Fc from transgenic lettuce is discussed.

In Chapter 8, we review plant-based production of pharmaceuticals from a broader perspective. Production platforms and strategies are compared in the context of building a space medical foundry. Chapter 9 takes an even broader look at the use of biotechnology for long-duration space exploration surface missions. We discuss biotech-driven examples of production and recycling of food, pharmaceuticals, and materials and how they integrate with one another. In Chapter 10, the emerging field of space bioprocess engineering is given perspective. Metrics, technology deployment, and education are examined.

Finally, in Chapter 11, we discuss the challenges which lie ahead for spaceflight applications of plant biotechnology. We identify knowledge gaps and consider the difficulty of simulating microgravity conditions.