Patent Application
20240000863
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled B21-024-2US.xml, was created on Aug. 30, 2023, and is 15,682 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
Existing topical treatments of the skin suffer from short half-lives and repeated numerous applications is not feasible. Patches that deliver continuous treatment are uncomfortable and impractical for large skin areas. Continuous delivery of peptides, antimicrobial peptides and proteins to treat skin disease and skin aging through skin probiotics could be a transformational improvement in skin diseases, skin care, and health-care more broadly.
WO2015184134 (U.S. Pat. No. 10,702,558) relates to treating skin diseases with engineered, skin commensal microorganisms, like Staphylococcus epidermidia. U.S. Pat. No. 9,234,204 relates to protecting human skin by recombinantly expressing mycosporine-like amino acids in plasmids in skin commensal organisms. U.S. Ser. No. 10/293,007 relates to the the treatment of skin diseases with engineered human skin microorganisms, like Propionibacterium acnes.
While providing sustainable infection, skin flora commensal microbes can also be opportunistic pathogens, and impose engineering constraints for producing and secreting compounds at a therapeutic level.
The invention provides engineering Corynebacterium glutamicum to produce beneficial topical agents like cosmetics, neutraceuticals, sunscreens, and therapies on the skin surface. The invention provides use of genetically engineered Corynebacterium glutamicum as a host for the production of beneficial compounds to the skin surface. An auxotrophic C. glutamicum (the auxotrophy provides a guarantee preventing unwanted spread) is applied to the skin surface as part of a fermentation medium that can also provide a moisturizer. The formulation includes sugars and other media components that the engineered C. glutamicum use as a nutrient source. C. glutamicum converts the nutrient source to the production of beneficial compounds such as amino acids, oligopeptides, antimicrobial peptides, and proteins.
C. glutamicum has been engineered for over 40 years with a diverse genetic toolbox already developed. Moreover, the C. glutamicum is non-commensal, and will not be engineered to grow on the lipids and proteins of the skin; rather, our media formulations provide growth nutrient(s).
In an aspect the invention provides a topical, skin probiotic formulation comprising a living population of Corynebacterium glutamicum bacteria genetically-engineered to produce a skin-bioactive agent, and a nutrient source for the bacteria.
In an aspect the invention provides use of an engineered strain of Corynebacterium glutamicum that is specially engineered through adaptive laboratory evolution for better growth and bioproduction in the acidic and lipidic environment of the skin.
In embodiments:
In an aspect the invention provides a method of using a subject formulation comprising applying the formulation to the skin of a person in need thereof under conditions wherein the bacteria produce an effective amount of the agent on the skin over a predetermined time range.
In embodiments:
The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited, such as wherein
Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or. The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.
We disclose genetically engineering innocuous bacterium Corynebacterium glutamicum to temporarily populate the skin microbiome and deliver molecules and proteins that treat disease as well as improve cosmetic appearance. While Corynebacterium is one of the three most abundant bacterial genera on human skin, the Generally Recognized as Safe (GRAS) organism C. glutamicum is not native to the skin microbiome. Temporary colonization of C. glutamicum, a well-studied organism for protein production, provides an added safety control feature. This platform for continuous production of enzymes and small molecules to the skin surface is a transformative advance in skin therapies.
While manipulation of the skin microbiome through modulation of native bacteria has been conceived in both academia and industry (MatriSys, Azitra, Xycrobe), we take a radically different approach to skin therapy. 9 While the abundance of Corynebacteria in the skin microbiome portends the compatibility of C. glutamicum, our approach is based on engineering a non-native microbe to temporarily colonize the skin. This is achieved through formulation engineering to manipulate the residence time on the skin, akin to media optimization in traditional fermentations. This approach provides increased safety as C. glutamicum cannot colonize the skin environment long-term, and unlike S. epidermidis or C. acnes, is not associated with any skin diseases. The benefits of this approach include slow, stable, consistent and effective release of therapies, at the level of the target cell in the skin. With adaptive laboratory evolution (ALE), we also use an engineered strain of C. glutamicum especially suited to grow and produce bioproducts in the acidic and lipidic environment of the skin.
While C. glutamicum is a GRAS (Generally Recognized as Safe), we tested its safety in a human equivalent tissue (EpiDerm). EpiDerm is a highly differentiated 3D tissue model consisting of human-derived epidermal keratinocytes. In a mixture of components common to moisturizers (glycerol, glucose, LB and biotin), we dosed C. glutamicum (˜107 CFUs/cm2) and media alone on the apical surface of EpiDerm. Tissue cell viability assays showed a similar tissue viability, compared to the cell death caused by soap (
As a proof-of-concept, we dosed the EpiDerm system with a C. glutamicum strain that overproduces lysine, a common moisturizer component. We showed the production of approximately 20 mM more of lysine, thereby illustrating our overall goal of longer effective peptide concentrations through continuous microbial production (
We have also shown that C. glutamicum is active when mixed in with common moisturizer ingredients (water, glycerol, stearic acid, Span60, xanthan gum, dimethicone) in an emulsion for over 28 days (
Additional examples below demonstrate, inter alia, stable temporary colonization of the human skin by providing nutrients in a moisturizer, the continuous production of glutamate on the skin surface, and the heterologous production of bioactive LEKTI to treat skin diseases such as Netherton Syndrome, atopic dermatitis, psoriasis, and acne rosacea
Corynebacterium glutamicum is a common soil bacterium and cannot survive on the skin surface. As C. glutamicum consumes sugars and fatty acids, it cannot use the lipids and proteins on the skin surface to produce bioproducts. We have shown that by adding the components for growth to common moisturizer ingredients, we can adjust the environment of the skin where C. glutamicum cannot only stably colonize the skin, but produce bioproducts.
We used common moisturizer ingredients (paraffin oil, water, Span60, dimethicone, and stearic acid) with the needed components for C. glutamicum to grow (glucose, glycerol, yeast extract and biotin) so that C. glutamicum could produce nutrients on the skin surface. The moisturizer is combined in a 1:1 mixture with Corynebacterium glutamicum resuspended at a concentration of 1010 CFU/mL. Human testing was performed to determine both skin colonization of the soil bacterium and production of glutamate, an amino acid secreted and produced C. glutamicum. We used a control of moisturizer alone to determine local microbiome conditions. After 0, 2, 4, 8 and 24 hours we used a skin swab and determine the colony forming units in a 16 cm2 portion on the skin. We determined that C. glutamicum stably colonizes the surface of the skin for 8 hours before dropping back to control conditions after 24 hours. We also found increasing and continuous production of C. glutamicum over 8 hours before dropping back to control conditions after 24 hours. Highlighting the power of C. glutamicum continuous production, a topical glutamate formula of 0.1% glutamate was added to the skin, and was undetectable after 15 minutes likely because of absorption and degradation on the skin surface, demonstrating the utility of bacterial produced compounds continuously on the surface. Our safety data from human trials also demonstrate that applying this organism on human skin is non-irritating and non-allergenic, confirming both safety and efficacy; see, e.g.
This example uses glutamate, a common moisturizer ingredient, as a proof-of-concept for the production of other therapeutic and aesthetic proteins. Notably, the nutrients in the moisturizer enable the bacterium to colonize the skin, and adjusting the concentration of the nutrient source adjusts the bioproduction and colonization time. We can vary the concentration of glucose and glycerol from 20% of the moisturizer for the longest level of colonization to lacking it completely where the bacterium would die off very quickly.
Netherton Syndrome is a disease driven by mutations in the SPINK5 gene that encodes lymphoepithelial Kazal-Type-related protease inhibitor (LEKTI) serine protease inhibitors. These mutations cause insufficient inhibition of kallikreins on the surface of the skin, particularly KLK5. Kallikreins are proteases that break down the structural proteins of the epidermis, and insufficient inhibition results in uncontrolled protease activity leading to loss of skin barrier function. Other diseases such as atopic dermatitis, psoriasis, and acne rosacea also have been implicated to have insufficient inhibition of kallikriens by LEKTI.
We engineered C. glutamicum to continuously produce LEKTI proteins, and apply them topically to the lesional skin surface of NS patients, thereby inhibiting KLK5 and restoring the skin surface. The active drug is a modified version LEKTI protein that is known to inhibit KLK5. It has been modified with an N-terminal secretion amino acid signal so that C. glutamicum can secrete the protein out of the cell.
We genetically inserted the LEKTI-D6 and LEKTI-D5 protein into the genome of Corynebacterium glutamicum. The coding sequence is driven from the pH36 promoter and includes an N-terminus secretion tag (porB). The secretion tag improves secretion of the protein into the extracellular environment of C. glutamicum and the secretion tag cleaves the amino acid sequence, resulting in the mature protein secreted to the extracellular environment. The strain was then fermented, and the protein was purified. An SDS page gel of the purified protein shows a clear band at the molecular weights of the encoded proteins (
MKLSHRIAAMAATAGITVAAFAAPASA
MESGKATSYAELCNEYRKLVRNG
KLACTRENDPIQGPDGKVHGNTCSMCEVFFQAEEEEKKKKEGESRNKR
MKLSHRIAAMAATAGITVAAFAAPASA
MEIVKLCSQYQNQAKNGILFCTR
ENDPIRGPDGKMHGNLCSMCQAYFQAENEEKKKAEARARN
*Bold font is secretion tag' **Underlined font is active moiety
We showed a correctly folded and bioactive protein by the microbial secretion of LEKTI by C. glutamicum using a KLK5 bioassay. The KLK5 inhibition assay is the foremost method to determine inhibition of KLK5. In the assay, KLK5 is incubated with the substrate Acetyl-YASR-paranitroanilide Uninhibited, KLK5 cleaves the substrate Acetyl-YASR at the arginine position (P4), releasing para-nitroanilide, a chromophore with absorbance 405 nm, resulting in increased absorption at that wavelength. When inhibited, the substrate Acetyl-YASR-paranitroanilide remains intact, and there is no increase in absorption at 405 nm. As a positive control, we used the absence of any inhibitor and the addition of purified Red Fluorescent Protein (RFP). As negative controls, we used a substrate only addition (no KLK5 is added) as well as a 100 uM addition of purified leupeptin, which is known to inhibit KLK5 at high concentrations. Our results showed that RFP and no inhibitor resulted in steadily increasing absorbance over 30 minutes. On the other hand, substrate only or the addition of leupeptin resulted in very little increasing absorbance in that time period. Increasing concentration of LEKTI-D6 showed a dose-dependent quenching of KLK5 with an IC50 of 89 nM.
Continuous production of antimicrobial peptides can treat skin infections and ameliorate bacterial dysbiosis. Current topical approaches often result in recalcitrant re-emergence of the harmful bacteria. By applying C. glutamicum topically, we use competitive inhibition and the continuous production of antimicrobials to permanently eliminate harmful bacteria such as Staphylococcus aureus. Pediocin is a narrow-range antibiotic, and we show that continuous on-site production can be used to treat and prevent S. aureus infections and colonization. In one embodiment, using the native sequence and transporter, we express pediocin as a three gene member family where pedC is the transporter, pedA is the precursor, and pedD cleaves pedA to provide the active pediocin in the supernatant.
Pediocin (Expressed Through 3 Proteins)
In another embodiment, we use C. glutamicum secretion tags to secrete the active compound directly.
Pediocin (Expressed Through a Single Protein)
MKLSHRIAAMAATAGITVAAFAAPASAKYYGNGVTCGKHSCSVDWGKATT
We use the same protocol to deliver alternative antimicrobial peptides, including nisin, a polycyclic antibacterial peptide produced by the bacterium Lactococcus lactis.
We constructed cassettes for on-site production of anti-inflammatory antibodies, antibody fragments, and nanobodies to treat a immunological disorders such as Netherton Syndrome, atopic dermatitis, psoriasis and acne rosacea; the protocols are readily extended to other antibodies targeting IgG, CD20 and other immune targets.
Single Chain Variable Fragment Anti-Tumor Necrosis Factor Alpha
MKLSHRIAAMAATAGITVAAFAAPASAEVKLEESGGGLVQPGGSMKLSCV
We constructed cassettes for on-site production of interleukins, including IL-10, to treat immune hypersensitivity responses on the skin.
IL-10
MKLSHRIAAMAATAGITVAAFAAPASASPGQGTQSENSCTHFPGNLPNML
In addition to LEKTI, other protease inhibitors can be used to inhibit skin diseases by continuous production by C. glutamicum, including Secretory leukocyte protease inhibitor and elafin.
MKLSHRIAAMAATAGITVAAFAAPASASGKSFKAGVCPPKKSAQCLRYKK
MKLSHRIAAMAATAGITVAAFAAPASAMGSSAVTGVPVKGQDTVKGRVPF
We also validated the invention with representative short peptide sequences that are beneficial to aesthetics by upregulating the production of collagen or relaxing facial muscles to reduce wrinkling. Continuous production by bacteria greatly increases their residence time, as normally they are absorbed and broken down by the skin. Examples include (encoded aesthetic peptide sequences are underscored):
This application is a continuation of PCT/US22/19051, filed Mar. 5, 2022, which claims priority to U.S. Provisional Application No. 63/157,564, filed Mar. 5, 2021, the disclosures of which is hereby incorporated by reference in its entirety for all purposes.
This invention was made with government support under Grant Number GM125179 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63157564 | Mar 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US22/19051 | Mar 2022 | US |
| Child | 18458139 | US |
