SBIR-STTR Award

Cannabinoids are a general class of natural compounds that have a wide range of proven therapeutic effects. Studies of cannabinoids led to the discovery of the endocannabinoid system (ECS), regarded as one of the most important modulators of nervous system processes. Cannabinoids have been found to have efficacy in treating pain, neuroinflammation, and bone loss, among many other conditions. Cannabinoids can be found in many plants, the most well-known is Cannabis sativa, but, like many other natural products that have been developed into pharmaceuticals, the production of most of these compounds by the plant are at low levels. The difficulties in sourcing these cannabinoids inhibits research and makes their eventual development and production into pharmaceuticals problematic. To increase the accessibility as well as our basic knowledge of cannabinoids and enable the pursuit of natural cannabinoids as therapeutic agents, Librede has developed a biosynthetic cannabinoid production/drug discovery platform based on genetically engineered yeast with selected portions of Cannabis sativa metabolic pathways. The production of cannabinoids in yeast is an ideal platform because fermentation and genetic engineering are well-established, low cost, and scalable. Since we can choose to engineer yeast strains to produce only a single cannabinoid, the effort and cost of separation/purification are minimized. Furthermore it is a modular platform technology-by adding or removing expression of different enzymes, different cannabinoids can be produced as desired, even cannabinoids produced in low abundance naturally. Using this approach, in preliminary work we have produced Cannabidiolic acid (CBDA) in yeast, the world's first biosynthetically- produced cannabinoids outside of plants. This preliminary work converted hexanoic acid, added to the growth media, to CBDA demonstrating that the production of cannabinoids was possible. In order to increase the yield to provide compounds for early testing and to prepare for large scale production Librede has developed a method to produce cannabinoids from glucose. In the work proposed here, we will add the published glucose?hexanoic acid pathways to our yeast allowing for the production of CBDA from glucose. The platform is modular and can be used to create a wide range of natural cannabinoids. By swapping out the last enzyme in the biosynthetic pathway (e.g change CBDA synthase to CBCA synthase) different cannabinoids can be produced. As a result, we will have a modular, high yield platform for synthesis of specific natural cannabinoids from glucose which is scalable for production of compounds at levels suitable for basic research, clinical trials, and for therapeutic use.

Public Health Relevance Statement:
PUBLIC HEALTH RELEVANCE Cannabinoids are natural products with the potential to treat a wide range of diseases, but most are expressed in plants in very low levels, making their production and extraction highly uneconomical. Librede has developed the world's first in vivo biosynthetic platform for cannabinoid production from genetically engineered yeast. We propose to enable practical production with our platform by engineering the yeast to use glucose as a feedstock.

NIH Spending Category:
Cannabinoid Research; Complementary and Alternative Medicine; Substance Abuse

Project Terms:
Acids; Address; Anabolism; base; Basic Science; Biology; Bone Growth; bone loss; cannabigerolic acid; Cannabinoids; Cannabis; Cannabis sativa plant; Chemicals; Chemistry; Clinical Trials; Cloning; cost; cost efficient; Culture Media; Development; Disease; drug discovery; endogenous cannabinoid system; Engineering; Environmental Impact; Enzymes; Fermentation; Gene Expression; Genes; Genetic Engineering; Glucose; glucose production; Growth; Guidelines; hexanoic acid; Human; improved functioning; in vivo; Inflammation; interest; Knowledge; large scale production; Marinol; Medical; Metabolic Pathway; Metabolism; Methods; Nabilone; National Institute of Drug Abuse; Natural Products; Nervous system structure; neuroinflammation; novel therapeutics; Pain; Pathway interactions; Patients; Peripheral Nervous System; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Physiologic Thermoregulation; Physiological Processes; phytocannabinoid; plant growth/development; Plants; Positioning Attribute; Process; Production; public health relevance; Publishing; Reporting; Reproduction; Research; research and development; research clinical testing; Research Personnel; Resources; Saccharomyces cerevisiae; screening; Sleep; Source; Technology; Testing; Tetrahydrocannabinol; Therapeutic; Therapeutic Agents; Therapeutic Effect; Therapeutic Uses; Work; Yeasts

Cannabinoids are a class of natural products shown to treat a wide range of diseases, including epilepsy, one of the most common neurological disorders. Epilepsy affects people of all ages, with over 50 million epileptics worldwide and over 150,000 new cases of epilepsy each year in the United States alone. Even with numerous anti-epileptic drugs (AED) available, 1/3 of patients have uncontrollable seizures that do not respond to treatment. Without proper seizure control the effects of epilepsy are more pronounced, and in children lack of seizure control not only interrupts early stage brain development but can also lead to social isolation. Recently, the endocannabinoid system (ECS) has been shown to play a powerful role in a number of neurological diseases, including epilepsy, creating a new therapeutic approach to control seizures where other AEDs have failed. One of the most effective and promising ECS-targeting therapeutics for seizure control is cannabidiol (CBD), a non- psychotropic naturally occurring cannabinoid. CBD is available over the counter in the United States and pharmaceuticals containing CBD are approved for use in many countries (currently in Phase III clinical trials in the United States). CBD is especially attractive since it has been shown to control seizures in patients with pharmaco-resistant seizures, especially children, with no significant side effects. Although CBD is effective with few side effects, pharmaceutical CBD has limited availability and high cost, primarily as a result of production shortcomings. The only source of pharmaceutical or consumer CBD is through agricultural production, purified from the cannabis or hemp plant. Further, pharmaceutical CBD is slated to cost $80-$160/day once approved in the United States. These high costs create a treatment gap between poor and wealthy patients and preclude access for the overwhelming majority of epileptics. To address this issue, Librede has developed a scalable yeast-based cannabinoid production technology capable of producing CBD in bulk for <10% of the cost of current production methods, allowing a broader population access to life-changing medicine. In Phase I work, we successfully engineered yeast to produce CBD using a low cost glucose feedstock. In the Phase II work proposed here, we will optimize CBD production through genetic, metabolic, and fermentation engineering to yields > 100 mg per liter of culture. Librede will combine targeted genetic optimization with a screened library approach to efficiently select yeast strains having improved CBD yield. Librede will also optimize growth and extraction conditions using commercial bioreactors (up to 300 L) to improve yield and develop methods compatible with high volume commercial production. At the conclusion of this work Librede will have a fully developed process for producing and purifying CBD at large scale and low cost. Widely available low cost CBD has the potential to make a significant difference in the lives of millions of epileptic sufferers worldwide.

Public Health Relevance Statement:
Narrative Cannabidiol (CBD) is a naturally occurring cannabinoid shown to be highly effective in treating pharmaco- resistant epilepsy with no side effects, particularly significant for children. Current plant-based production of CBD leads to high therapeutic costs and low availability, precluding access to treatment by the majority of the population. To address this problem, Librede has developed a yeast-based biosynthetic CBD production platform capable of large scale, low cost production. In this work we will optimize and scale CBD production yields.

Project Terms:
Address; Adverse effects; Affect; Age; Agriculture; Antiepileptic Agents; Artemisinins; base; Bioreactors; Brain; Cannabidiol; Cannabinoids; Cannabis; Cannabis sativa plant; Child; Climacteric; Clustered Regularly Interspaced Short Palindromic Repeats; cost; Cost Control; Country; Development; Disease; Dose; Drug Costs; endogenous cannabinoid system; Engineering; Epilepsy; Fermentation; Fertilizers; fighting; flasks; Future; Genes; Genetic; Glucose; Goals; Growth; Harvest; Health Services Accessibility; Healthcare; Hemp; Hybrids; improved; Income; Interruption; Knock-out; laboratory equipment; Lead; Libraries; Malaria; Medicine; Metabolic; Metabolic Pathway; Methods; Multiple Sclerosis; mutant; Natural Products; nervous system disorder; novel therapeutic intervention; Pathway interactions; Patients; Pesticides; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phase III Clinical Trials; phytocannabinoid; Plants; Play; Population; Preparation; Press Releases; Price; Process; Production; Property; Research; Resistance; Role; Sativex; scale up; Seizures; Social isolation; Source; System; targeted treatment; Technology; technology development; Therapeutic; Time; United States; United States National Institutes of Health; Water; Weather; Work; Yeasts