Abstract

Hair loss affects roughly 61 million people in the United States. More than 95% of hair thinning in men is attributed to male pattern baldness, or androgenetic alopecia. The hair loss product market is a multi-billion dollar market: in the United States alone, this market’s annual revenue is 7 billion dollars, with 3.5% annual growth rate from 2007 to 2012, yet a sufficient solution has yet to be made available. The trigger for androgenetic alopecia is believed to be dihydrotestosterone (DHT), a derivative of testosterone. This summer, we engineered Bacillus subtilis, a bacterium found naturally on the scalp, to secrete 3α-hydroxysteroid dehydrogenase (3α-HSD). This enzyme reduces DHT to a non-steroidically active compound, using NADPH and NADH as cofactors.

Our project consists of 3 main parts: 3α-HSD secretion by the bacterium, engineered bacterium for NADPH overproduction, and a comb as a tool to create a user-friendly experience. By synchronizing these parts, we hope to be able to show convincing evidence of DHT breakdown, providing a promising platform for treatment of male pattern baldness!

Problem

Hair loss affects roughly 40 million men and 21 million women in the United States. Approximately 25% of men begin balding by the age 30, and two-thirds begin balding by the age 60. Male pattern baldness (Androgenic alopecia) is the main cause of thinning hair in men - in more than 90% of the cases. This condition is characterized by hair receding from different areas of the scalp, beginning with the lateral sides of the forehead and the vertex¹.

The male sex hormone, testosterone (which is produced largely in the testicles), is partially converted into a more potent derivative: dihydrotestosterone (DHT). The DHT is synthesized from the testosterone in several areas in the body: on the hair follicles, testes and adrenal glands². The DHT is not only synthesized on the scalp, but is also transported to the scalp through the bloodstream. It causes the deterioration of the hair follicle through a hormonal cascade³.

Available solutions

The hair loss product market is a multi-billion dollar market: in the United States alone, this market’s annual revenue is $7 billion, with a 3.5% annual growth rate from 2007 to 2012 ⁴.In China, the world’s second largest economy, sales of minoxidil (one of the two major products on the market), jumped 90% to $16.4 million from 2007 to 2012 ⁵.

Two major non-surgical treatments are available today:

1. Finasteride- Finasteride is one of the leading drugs treating male pattern baldness orally. It holds 27.5 % of the market in the United States. The pills are taken orally on a daily basis. The drug blocks the production of DHT, hence reducing its activity on the scalp^1,6. According to a study, after five years of treatment with Finasteride, 48% of men had increased hair growth, while 75% of men who did not take the drug experienced hair loss⁷.The main drawback of Finasteride is it’s potential side effects (partially because the treatment is not a topical one), such as impotence & cancer⁸.
2. Minoxidil- holds 28.2 % combined market share in the United States. This is an over-the-counter medication approved for male pattern baldness ¹. It is rubbed into the scalp twice a day. The mechanism by which minoxidil promotes hair growth is not fully understood. It is most effective for people under 40 years of age whose hair loss is recent. Minoxidil has no effect on receding hairlines. Its use is advised for central (vertex) hair loss only. Hair regrowth can take eight to twelve months. However, only 30–40% of patients experience hair growth⁹.

Our solution strategy

With the recent advances that established a strong relationship between the human microbiome and human health and disease, new frontiers in synthetic biology have begun to emerge concomitantly. Namely, rather than engineering a single bacterium chassis as is normally done in conventional synthetic biology applications, we have opted to devise an engineering strategy for a microbiome in order to address health-related problems.

A national research program of the human microbiome has been established in the United States - The Human Microbiome Project (HMP). The goal of the HMP is to demonstrate that there are opportunities to improve human health through monitoring and/or manipulation of the human microbiome ¹⁰. New and promising startups have been established based on the idea of the utilization and adaptation of the human microbiome to our needs. For example, AOBiome, LLC. created a dedicated line of products (named ‘Motherdirt’) - the first line of products for the skin made, which includes live bacteria incorporated into the product itself (Ammonia Oxidizing Bacteria) ¹¹. This product was publicized extensively in the media - In “The New York Times” and “Good Morning America”. In the past several years, several other big cosmetic companies have released products incorporating natural bacteria - “Clinique Redness Solutions Makeup SPF 15” by Clinique and “Cosmeceuticals Unstress Total Serenity Serums” by Christina are few examples of this promising market ¹².

Using this trend in research and cosmetics, we decided to develop a new synthetic biology approach for engineering a microbiome. In our approach, we opt to genetically alter one or more of the bacterial populations that are naturally present to secrete a particular enzyme. Thus, on the one hand we minimally perturb the natural microbiome, while on the other we co-opt it to produce a sustainable treatment of some sort.

We decided to apply this approach on the problem of male pattern baldness. We planned to alter the most abundant bacterium which is naturally found on our scalp (Bacillus subtilis) ¹⁵ and genetically engineer it for our needs: taking advantage of its natural protein secretion pathways in order to secrete a DHT-reducing enzyme (as will be explained in the following paragraph). This new application of the human microbiome usage is a path, in our opinion, to solve a variety of conditions and enhance overall wellness in the future.

Solution

The proposed solution is to incorporate the gene of a DHT-inactivating enzyme into a bacterium that is naturally found on our scalp (B.subtillis). These transgenic microorganisms will be applied on the patient's scalp, and will secrete the enzyme to their environment, providing a constantly renewed supply of the treatment. The proposed DHT-inactivating enzyme is the 3α-hydroxysteroid dehydrogenase (3α-HSD) enzyme. DHT is inactivated by the 3α-HSD to form 3α-androstanediol ¹³, as shown in figure 1 below.

3α-HSD exists naturally in human prostate, performing the mentioned reaction¹⁴. However, according to previous research, the isoform originated from rat liver has greater specificity to DHT than the human isoform ¹⁴. Therefore, we chose to use the sequence of AKR1C9 for our project. We altered the sequence in order to delete forbidden restriction sites according to the iGEM regulations for BioBricks.

An illustration of our proposed solution and projecy components can be seen in Figure 2.

The components of the project

In order to better focus on the many different components of our project, we split up into groups to tackle each component: Expression of the 3α-HSD enzyme, secretion of the enzyme, and cofactor production. Additionally, a group worked on developing a comb- a tool which could provide a user-friendly solution for the consumer in applying our product to the scalp. For more information about each sub-group, enter the page from the menu above, or visit the links below:

• Secretion
• Expression
• Cofactor
• Comb and Design
• Results

Modeling

Our novel treatment for male pattern baldness is separated into three modules for modeling analysis, which constitute our entire system:

1. The Bacillus – our enzyme producer
2. The E.Coli – our cofactor producer.
3. 3α-HSD – the "engine" of our process.

Since the goal of our system is to break down the DHT molecule, we chose to focus on the enzymatic activity for our model, which has the key role in the breakdown process.

Using principles from statistical mechanics, we developed a completely new enzyme reaction function and simulated the breakdown process in a way that correlated with our wet lab results.

Our model allows us to take a look at the mechanism of this enzyme and gain insight into the reactions which eventually break down DHT.

For more information about our models, click here.

What’s next?

Our plans in the future consist of examining our product on model mice for androgenic alopecia (male pattern baldness) as a proof of concept. We would like to utilize this model to prove that our combined solution for this condition can actually show results (in-vivo). If this succeeds, we will continue on to perform human trials.

We strive to open a standalone startup which will take our concepts forward.

We also believe that our new application of utilizing the human microbiome (genetically modify it for a specific cause ) is a promising new way to treat different conditions.