Periodic Reporting for period 1 - HuSME (Human Skin Microbiome Engineering)
Reporting period: 2020-12-01 to 2022-11-30
Skin diseases affect millions of people worldwide but are often poorly addressed, due to lack of resources, expensive or inefficient treatments, or their non-urgent, non-life-threatening nature. Atopic dermatitis (AD) alone affects 7-10% of adults (and up to 25% of children), making it the most common chronic inflammatory skin disease. While non-fatal (and sometimes perceived as relatively benign), AD severely affects patients’ quality of life. The characteristic itch/pruritus can lead to sleep deprivation and adverse psychosocial effects due to the high visibility of the lesions, including attention deficit, anxiety and depression. Besides, AD patients show a propensity towards skin infections, mainly by Staphylococcus aureus and herpes. The economic burden of AD amounts to $5.3 billion only in the US, and current treatments are either highly expensive ($37,000/patient/year for Dupilumab) or linked to important adverse effects, such as skin thinning linked to topical corticosteroids or immunodeficiency linked to systemic immunosuppressants.
This project aimed at producing the basic tools and knowledge for the future use of engineered human skin microbiome for diagnostics and treatment of skin diseases in general and of AD in particular. While the modification of the gut microbiome for medical uses is exponentially growing and starting to unfold its potential, the modification of the skin microbiome is clearly lagging behind. Cutibacterium acnes (formerly Propionibacterium acnes) an inhabitant of the pilosebaceous unit and the most abundant skin bacterium; a Gram-positive anaerobe contributing to skin homeostasis. C. acnes constitutes an ideal framework for the development of living biotherapeutics for several reasons, including its high abundance and low turnover, its relevance in both health and disease, its natural biocontainment within the pilosebaceous unit, and its engraftment capabilities when transferred to a recipient individual.
Despite its huge potential, there existed important limitations to the modification of C. acnes that prevented further advancement. For example, almost no tool was available for C. acnes engineering, and successful delivery of DNA into this bacterium had been scarcely reported. The first goal of the project was therefore to improve the transformability of C. acnes and to develop tools for its programming as a living medical device. Next, I aimed to use this toolset to develop C. acnes strains able to sense inflammation and respond to it by releasing anti-inflammatory molecules. Finally, I sought to validate the engraftment and activity of the engineered strains in a mouse model of atopic dermatitis.
The research performed has accomplished most of the initial objectives, and notably those that serve as basis for future development of the technology. We are now able to routinely deliver exogenous DNA into C. acnes, and have developed the molecular biology tools to engineer these bacteria for medical (or other) purposes. Some of our engineer strains secrete active anti-inflammatory proteins, and we have demonstrated successful engraftment in model mice. We continue developing inflammation sensors, and the in vivo validation of a strain that senses inflammation and responds to it will follow.
Other than research, during the course of the fellowship I have continued to develop transversal skills of high relevance for my career development, such as leadership and mentoring (courses, Master student or iGEM team (>10 student) supervision), teaching (undergraduates), or communication (talks, outreach).
This project aimed at producing the basic tools and knowledge for the future use of engineered human skin microbiome for diagnostics and treatment of skin diseases in general and of AD in particular. While the modification of the gut microbiome for medical uses is exponentially growing and starting to unfold its potential, the modification of the skin microbiome is clearly lagging behind. Cutibacterium acnes (formerly Propionibacterium acnes) an inhabitant of the pilosebaceous unit and the most abundant skin bacterium; a Gram-positive anaerobe contributing to skin homeostasis. C. acnes constitutes an ideal framework for the development of living biotherapeutics for several reasons, including its high abundance and low turnover, its relevance in both health and disease, its natural biocontainment within the pilosebaceous unit, and its engraftment capabilities when transferred to a recipient individual.
Despite its huge potential, there existed important limitations to the modification of C. acnes that prevented further advancement. For example, almost no tool was available for C. acnes engineering, and successful delivery of DNA into this bacterium had been scarcely reported. The first goal of the project was therefore to improve the transformability of C. acnes and to develop tools for its programming as a living medical device. Next, I aimed to use this toolset to develop C. acnes strains able to sense inflammation and respond to it by releasing anti-inflammatory molecules. Finally, I sought to validate the engraftment and activity of the engineered strains in a mouse model of atopic dermatitis.
The research performed has accomplished most of the initial objectives, and notably those that serve as basis for future development of the technology. We are now able to routinely deliver exogenous DNA into C. acnes, and have developed the molecular biology tools to engineer these bacteria for medical (or other) purposes. Some of our engineer strains secrete active anti-inflammatory proteins, and we have demonstrated successful engraftment in model mice. We continue developing inflammation sensors, and the in vivo validation of a strain that senses inflammation and responds to it will follow.
Other than research, during the course of the fellowship I have continued to develop transversal skills of high relevance for my career development, such as leadership and mentoring (courses, Master student or iGEM team (>10 student) supervision), teaching (undergraduates), or communication (talks, outreach).
The most pressing and fundamental challenge to be addressed was the extremely low transformation efficiency of C. acnes. Only few studies existed reporting the successful delivery of DNA into this bacterium, and the efficiency was remarkably low, with most attempts resulting in no transformants. I approached the challenge from several complementary angles (such as protocol optimization, vector optimization, or DNA or strain modification), which eventually increased transformation efficiency several orders of magnitude. We are now able to routinely transform C. acnes and obtain several hundreds of colonies, which enables the transformation of small/medium libraries.
The next critical step was to develop basic tools for the use of C. acnes as a living (medical) device; this included tools to produce and secrete custom proteins and to monitor the cell’s gene expression activity. My research has produced small libraries of such basic tools, such as promoters, ribosome binding sites (RBSs), or secretion signals, as well as validated fluorescent reporters, both aerobic (sfGFP, mCherry, mKO2, mCitrine) and anaerobic (FAST), more suitable for C. acnes. I have optimized the transformation vector and made it fully modular; combined with a fast and modular cloning strategy that I have developed, it renders the construction of designs quicker and more efficient. Together, these allow us (and the whole scientific community) to start modifying C. acnes for relevant applications.
I have also contributed to develop a C. acnes cell-free system (CFS), which facilitates the characterization of new components by eliminating the need to transform DNA and to grow transformants for testing, a process that takes 10-14 days on average. Results related to the C. acnes CFS have already been published (https://pubs.acs.org/doi/full/10.1021/
acsbiomaterials.1c00894).
I have managed to produce several anti-inflammatory proteins in C. acnes, some of which are efficiently secreted and keep their efficiency in the extracellular milieu. Detecting inflammatory signals has revealed more challenging, and we are currently optimizing the process. We have validated the engraftment of C. acnes in mice, and we will next assess the capacity of our engineered strains to detect and treat inflammation in vivo.
My results are of high industrial interest, and we are currently exploring their patentability.
The next critical step was to develop basic tools for the use of C. acnes as a living (medical) device; this included tools to produce and secrete custom proteins and to monitor the cell’s gene expression activity. My research has produced small libraries of such basic tools, such as promoters, ribosome binding sites (RBSs), or secretion signals, as well as validated fluorescent reporters, both aerobic (sfGFP, mCherry, mKO2, mCitrine) and anaerobic (FAST), more suitable for C. acnes. I have optimized the transformation vector and made it fully modular; combined with a fast and modular cloning strategy that I have developed, it renders the construction of designs quicker and more efficient. Together, these allow us (and the whole scientific community) to start modifying C. acnes for relevant applications.
I have also contributed to develop a C. acnes cell-free system (CFS), which facilitates the characterization of new components by eliminating the need to transform DNA and to grow transformants for testing, a process that takes 10-14 days on average. Results related to the C. acnes CFS have already been published (https://pubs.acs.org/doi/full/10.1021/
acsbiomaterials.1c00894).
I have managed to produce several anti-inflammatory proteins in C. acnes, some of which are efficiently secreted and keep their efficiency in the extracellular milieu. Detecting inflammatory signals has revealed more challenging, and we are currently optimizing the process. We have validated the engraftment of C. acnes in mice, and we will next assess the capacity of our engineered strains to detect and treat inflammation in vivo.
My results are of high industrial interest, and we are currently exploring their patentability.
Current attempts to engineer the skin microbiome focus on Staphylococcus epidermidis, a Gram-positive bacterium that populates the skin surface. C. acnes, in contrast, lives deeper in the skin, which on one hand facilitates the engraftment and reduces the turnover, and on the other hand provides a closer contact with relevant cell types such as sebocytes and immune system cells. Despite these notorious advantages, the engineering of C. acnes has been hampered by its virtual genetic intractability.
Through this project I have generated the knowledge and tools to make the engineering of C. acnes a reality. I anticipate that my results will boost the interest on and use of this major skin inhabitant, both by academics and companies. I have myself started to modify C. acnes to detect and treat inflammatory skin diseases, and other attempts will likely follow. My research can critically contribute to the future development of skin therapies based on living bacteria, which can potentially result in better alternatives for patients and lower economic impact on healthcare systems.
Through this project I have generated the knowledge and tools to make the engineering of C. acnes a reality. I anticipate that my results will boost the interest on and use of this major skin inhabitant, both by academics and companies. I have myself started to modify C. acnes to detect and treat inflammatory skin diseases, and other attempts will likely follow. My research can critically contribute to the future development of skin therapies based on living bacteria, which can potentially result in better alternatives for patients and lower economic impact on healthcare systems.