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Biological Chemistry

Producing Protein Therapeutics: Could Water Buffalo Be The Next Big Thing?

Indian scientists isolate genetic information needed for the large mammals to produce biologics

by Biplab Das
May 11, 2015 | A version of this story appeared in Volume 93, Issue 19

Water buffalo (Bubalus bubalis) wading in a pond.
Credit: Shutterstock
Water buffalo are a common sight in rural India.

Water buffalo wallowing in muddy pools are a common sight in rural India and other Asian countries. When not relaxing, the large black mammals plow the rice fields, haul carts laden with produce, or drive sugarcane presses. Their horns, hides, meat, and milk have supported rural life for ages.

The beasts of burden are now primed to take on one more task: The animals may one day churn out therapeutic proteins—also called biologics—to treat human ailments.

A team of Indian biologists has demonstrated that a key component of the water buffalo genome can be manipulated to produce foreign proteins in the buffalo’s milk (J. Biotechnol. 2015, DOI:10.1016/j.jbiotec.2015.02.001). The findings suggest the possibility of modifying the genetic material of water buffalo—that is, creating transgenic buffalo—to produce protein drugs, says team member Subeer S. Majumdar, of the National Institute of Immunology, in New Delhi.

The key component, a DNA sequence called a promoter, initiates the process by which a neighboring gene gets translated into a protein. In the new study, the Indian researchers isolated the water buffalo promoter that helps translate the gene for β-casein, the most abundant protein in buffalo milk. Then they showed that if they attached a foreign gene next to the promoter in a DNA strand, they could produce the protein encoded by that foreign gene in milk-secreting cultured cells as well as in mice.

Certain human diseases can be treated with therapeutic proteins. Because of their complexity, however, current protein drugs tend to be produced in bacteria or mammalian cells. But these vehicles have limitations: Bacteria are unable to generate human proteins. And building a production-scale (10,000 L) bioreactor for mammalian cells costs $250 million to $500 million, according to a review by Yanli Wang of Xi’an Jiaotong University School of Medicine, in China, and colleagues (BioMed. Res. Int. 2013, DOI: 10.1155/2013/580463). Drugmakers have looked to plants and insects as alternatives, but these organisms produce proteins slowly, and purifying the proteins they churn out is also expensive, Wang and colleagues note.

Another way to make biologics is through milk-generating animals. Unlike bacteria, animals can synthesize complex human proteins, and unlike plants and insects, they can do it quickly. Plus, the technique is less expensive: A transgenic animal farm with a single purification facility would cost only about $80 million, according to Wang and colleagues.

The potential to produce biologics at low cost has encouraged some companies to use transgenic animals. Although several protein drugs made by transgenic animals are in clinical trials, commercialization has been slow. “Milk has produced only two recombinant therapeutic proteins that have moved from lab to market over the past 20 years,” says Louis-Marie Houdebine, a researcher who studies protein drug production in transgenic animals at the French National Institute for Agricultural Research, in Paris.

The two approved biologics are ATryn, an anti-blood-clotting protein, and Ruconest, a protein that treats rapid tissue swelling. Transgenic goats synthesize both therapeutics. Houdebine speculates that pharmaceutical companies do not yet feel enough financial pressure to adopt the technology widely.

Compared with cell cultures, milk can be produced in larger amounts and with higher protein content, according to Houdebine. Annually, a female goat can produce 800 L of milk containing 4 kg of protein.

Water buffalo could do better. According to Majumdar, a female buffalo can produce on average 2,800 L of milk annually and potentially yield 14 kg of protein per year. This capability would make water buffalo the second-largest production vehicle for therapeutic proteins, after the cow, which can annually yield 8,000 L of milk containing 40 kg of protein.

Encouraged by this potential, Majumdar and colleagues began exploring the possibility of producing therapeutic proteins in water buffalo milk. After they isolated the β-casein promoter, they placed it next to a foreign gene, one that encodes green fluorescent protein (GFP). Then they used fluorescence imaging to show that the promoter can initiate expression of the GFP gene in milk-secreting cultured cells. Next, they tested the promoter-GFP gene pair in a milk-producing animal. In transgenic mice, the researchers detected GFP only in the mammary glands.

The results indicate that the promoter-GFP gene pair didn’t leak into any other organs. “This is very encouraging,” Majumdar explains, “because had [it] leaked to other organs such as the brain or liver, it could affect the organ, causing harm to the transgenic animal.”

Moving to another foreign gene, for a human protein this time, Majumdar’s group attached the buffalo promoter next to a gene that encodes human interferon gamma, a key protein that stimulates the human immune system to strike at pathogens. After they delivered the promoter-gene pair into cultured human milk-secreting cells, they detected the interferon gamma inside the cells. “The buffalo promoter can work between species and drive the production of any tagged protein in milk of other mammals,” Majumdar contends. Producing human interferon gamma in human milk-secreting cells “is proof of that principle.”

“Undoubtedly, the group has in hand a promoter that’s active and driving the specific expression of foreign genes in milk,” Houdebine says. “However, the promoter from water buffalo will likely need several additional regulatory elements to drive the synthesis of pharmaceutical proteins repeatedly at a high level.”

So more work is needed to upgrade water buffalo from beasts of burden to biologics producers. Majumdar says his team will next use the buffalo-promoter-based technique to produce human proteins in the milk of transgenic rabbits and goats. Eventually, they hope to work their way up to replicating the feat in buffalo.

A graphic showing how biologics from transgenic animal milk promise treatments for many ailments.
Biologics from transgenic animal milk promise treatments for many ailments.
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