# Patent

## Title
Aspartic proteases

## Abstract

Methods and composition, thereof, relate to aspartic proteases, and particularly to aspartic proteases for plants. Disclosed are modified plant aspartic proteases, and methods for their manufacture, and uses thereof. Particularly contemplated are the uses of aspartic proteases in inducing skin desquamation.

## Background
RELATED APPLICATIONS
This application is a 35 U.S.C. § 371 national phase application of PCT/PT2014/000018, filed Mar. 19, 2014 (WO/2014/148932). PCT/PT2014/000018 claims priority to GB Application Serial No. 1305023.2, filed Mar. 19, 2013, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to aspartic proteases and particularly to aspartic proteases from plants.
Incorporated by reference herein in its entirety is the Sequence Listing entitled “Sequence-List.txt”, created Oct. 28, 2015, size of 27 kilobytes.
BACKGROUND TO THE INVENTION
The human epidermis is composed of five layers of stratified epithelial cells and is an organ in constant renewal. New cells are formed in the basal layer and after a differentiation process the cells reach the outermost layer of the skin. Renewal and maintenance requires the cell shedding of corneocytes from the stratum corneum (SC). Aging and certain skin diseases can disturb this process leading to a decrease in desquamation rate, resulting in an increase in thickness of the SC and in skin scales formation.
Skin desquamation is the shedding of corneocytes from the stratum corneum (SC), and is part of the self-renewal and maintenance of the skin. Desquamation or exfoliation of epidermal layers of human skin induces an increased rate of epidermal cell renewal. Desquamation of corneocytes from epidermis requires the enzymatic degradation of corneodesmosome structures, composed of corneodesmosin, desmoglein-1 and desmocolin-1 proteins. Several serine, cysteine and aspartic proteases participate in this exfoliation process including cathepsin D, cathepsin E and SASpase.
Compositions comprising the acid protease pepsin, and the stratum corneum trypsin-like serine proteases have been proposed for a number of uses including improving the texture or appearance of the skin, enhancing epidermal exfoliation, inducing skin desquamation and causing cell renewal (U.S. Pat. No. 6,656,701; WO95/07688). The acid proteases described in U.S. Pat. No. 6,656,701 exhibit peptidyl hydrolase (proteolytic) activity below the average pH of the surface of the skin, but which are significantly inactive at a pH greater than the average pH of the surface of the skin (about pH 5.5 for humans). The aspartic protease cathepsin D is also thought to facilitate desmosomal degradation, and has been exploited in some cosmetic/cosmeceutical preparations, for example purified oligosaccharides extracted from the fruit of prickly pear (Opuntia ficus indicia) have been used in facial acid peel treatments to enhance cathepsin D activity.
Aspartic proteases are peptidases present in animals, plants, fungi and viruses and exhibit a wide range of functions and activities, including: mammalian digestion e.g. chymosin and pepsin A, activation and degradation of polypeptide hormones and growth factors e.g. cathepsin D, regulation of blood pressure e.g. rennin, degradation of haemoglobin by parasites e.g. plasmepsins, proteolytic processing of the HIV polyprotein e.g. retropepsin, involvement in pollen-pistil interactions in plants e.g. Cardosin A.
Aspartic proteases are synthesised as preproenzymes and contain a signal peptide, which is cleaved resulting in a proenzyme which can be secreted and activated autocatalytically. Generally, aspartic proteases consist of a single peptide chain of approximately 320-360 amino acid residues, composed mainly of β-strand structures arranged into two lobes. The catalytic site of the enzyme is located between these two lobes, each containing an aspartate residue which are within hydrogen-bonding distance of each other and act together to activate a water molecule which results in cleavage of the substrate peptide bond (via nucleophilic attack).
Plant aspartic proteases differ from other aspartic proteases in that they comprise a Plant Specific Insert which is cleaved out during protein maturation, besides a signal peptide (responsible for translocation to the ER); a prosegment of 40-50 amino acid residues (involved in the correct folding, stabilisation and sorting of the enzyme); and a mature enzyme possessing two catalytic sequence motifs. The two catalytic aspartate residues in plant aspartic proteases are contained within Asp-Thr-Gly and Asp-Ser-Gly motifs.
Plant Specific Insert (PSI)
Many plant aspartic proteases differ from their mammalian and microbial counterparts by the presence of a plant-specific insert (PSI), typically having about 104 amino acids. In phytepsin, from barley, removal of the PSI led to secretion of the mutated phytepsin when expressed in Tobacco protoplasts, whilst retaining enzymatic activity4. The presence of PSI was shown to be at least necessary for vacuolar sorting4.
Vacuolar Sorting
The final destination of a protein after synthesis is a highly complex and regulated process and is usually dependent on the presence of specific targeting information (e.g. sorting signals, post-translational modifications) which is specifically recognized by receptors that target nascent proteins to their final localizations in the cell1.
One of the most complex biosynthetic routes is the secretory pathway. In a very simplified way, this system comprises several membrane-bound subcellular compartments and proteins are exchanged between these compartments by vesicle trafficking. Proteins resident in the endoplasmic reticulum (ER), Golgi apparatus, vacuoles or plasma membrane/extracellular matrix have to enter this endomembrane system and some of them undergo processing and post-translational modifications along their passage through the ER and Golgi network. Targeting to ER is determined cotranslationally by the presence of a signal peptide at the N-terminus of a nascent protein1. Although recent evidence indicates that the system may be more complex than first expected2, it is still generally accepted that proteins are actively sorted to vacuoles, meaning that they contain specific vacuolar sorting signals (VSS's).
Different types of vacuolar sorting signals (VSS's) have been identified1,3. Even though no consensus sequence has been yet defined for these signals they are currently divided into three categories: sequence-specific VSS (ssVSS's) which comprise N-terminal propeptides (e.g. sporamin) or internal sequences (e.g. ricin); C-terminal propeptides (CTPP's) (e.g. lectin and chitinase) and physical structure VSS (psVSS's) [e.g. plant specific insert (PSI) of phytepsin]3. Given the number of soluble vacuolar proteins that lack these types of VSS's, it is expected that novel motifs for vacuolar sorting are yet to be identified.
The ability to manipulate protein sorting is particularly important if considering high value-protein expression in heterologous or homologous systems. Specifically sorting a selected protein to storage vacuoles may be highly advantageous for accumulation of large quantities of recombinant proteins and, thereby, increase the food value of a plant. Conversely, redirecting a native vacuolar protein for secretion may be particularly useful considering, for example, expression in heterologous systems like yeasts where protein secretion into the media greatly facilitates recombinant protein handling and purification.
The relevance of these vacuolar sorting signals in various applications is confirmed by different issued patents: U.S. Pat. Nos. 6,9723,504, 7,368,6285, 5,360,7266 and 60,546,377, where the last two describe the VSS's of lectin and chitinase, respectively.
Typical aspartic proteases are widely distributed in plants and have been purified from a variety of tissues. In general, these enzymes share high levels of amino acid sequence identity (over 60%) and the majority of them accumulate inside plant vacuoles. However, there are exceptions to this intracellular localization and several plant aspartic proteases were shown to be extracellular.
SUMMARY OF THE INVENTION
The inventors have discovered that plant aspartic proteases, in particular Cardosins, and particularly Cardosin B, show skin desquamation activity, without affecting cellular viability. These plant aspartic proteases have enhanced ability to induce skin desquamation compared to mammalian aspartic proteases, including Cathepsin D.
The present invention therefore provides a method of medical treatment, the method comprising administering a plant aspartic protease to a subject in need of such treatment. The method may be for the treatment of a skin disorder. The method preferably involves inducing skin desquamation in the subject. The subject is preferably one suffering from at least one of xerosis, eczema, acne, psoriasis or ichthyosis, and the method involves the treatment of at least one of xerosis, eczema, acne, psoriasis or ichthyosis. The plant aspartic protease may be a mutant that lacks a functional plant specific insert (PSI) domain. The plant aspartic protease may be a Cardosin, such as one of Cardosin B or Cardosin A, or a mutant thereof. The plant aspartic protease may have at least 70% sequence identity to SEQ ID NO: 1, or SEQ ID NO: 2.
The present invention also provides the use of a plant aspartic protease in the manufacture of a medicament for treating a skin disorder. The treatment preferably involves inducing skin desquamation. The medicament may be provided to treat a skin disorder, such as xerosis, eczema, acne, psoriasis or ichthyosis. The plant aspartic protease may be a mutant that lacks a functional plant specific insert (PSI) domain. The plant aspartic protease may be a Cardosin, such as one of Cardosin B or Cardosin A, or a mutant thereof. The plant aspartic protease may have at least 70% sequence identity to SEQ ID NO: 1, or SEQ ID NO: 2.
In another aspect of the present invention a plant aspartic protease is provided for use in a method for treatment of the human or animal body by therapy. This may involve the treatment of a skin disorder and may preferably involve the induction of skin desquamation. As such, the plant aspartic protease may be provided for use in the treatment of at least one of xerosis, eczema, acne, psoriasis or ichthyosis. The plant aspartic protease may be a mutant that lacks a functional plant specific insert (PSI) domain. The plant aspartic protease may be a Cardosin, such as one of Cardosin B or Cardosin A, or a mutant thereof. The plant aspartic protease may have at least 70% sequence identity to SEQ ID NO: 1, or SEQ ID NO: 2.
In a further aspect of the present invention a cosmetic method for improving the appearance of the skin is provided, the method comprising administering a plant aspartic protease to the skin. The method may involve the induction of skin desquamation. The plant aspartic protease may be a mutant that lacks a functional plant specific insert (PSI) domain. The plant aspartic protease may be a Cardosin, such as one of Cardosin B or Cardosin A, or a mutant thereof. The plant aspartic protease may have at least 70% sequence identity to SEQ ID NO: 1, or SEQ ID NO: 2.
In another aspect of the present invention a composition is provided, the composition comprising an isolated plant aspartic protease. The plant aspartic protease may lack a functional plant specific insert (PSI) domain. The plant aspartic protease may be a Cardosin, such as one of Cardosin B or Cardosin A, or a mutant thereof. The plant aspartic protease may have at least 70% sequence identity to SEQ ID NO: 1, or SEQ ID NO: 2. The composition may be formulated for topical administration. The composition may be formulated as a pharmaceutical composition or medicament. Alternatively, the composition may be formulated as a cosmetic composition.
The inventors have also determined that the normal VSS function of the  ̃100 amino acid plant specific insert (PSI) may be inactivated by recombinant DNA manipulation to enhance secretion of plant aspartic proteases in either homologous or heterologous expression systems (preferably heterologous, non-plant, expression systems), whilst retaining the aspartic protease activity of the secreted protein. Thus, the inventors have provided a novel and advantageous means of producing high volumes of plant aspartic proteases in a form that is convenient to isolate and purify.
Accordingly, the present invention also provides methods for the expression in cells of mutant plant aspartic proteases modified such that the PSI domain is inactivated, the expression preferably being in non-plant eukaryotic cells. Preferably, the mutant plant aspartic proteases retain their protease activity.
In one aspect of the present invention an isolated mutant Cardosin plant aspartic protease is provided, wherein the Cardosin plant aspartic protease is mutated such that it lacks a functional plant specific insert (PSI) domain. The Cardosin plant aspartic protease may have an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, or SEQ ID NO: 2.
In another aspect of the present invention a method for producing a mutant plant aspartic protease is provided, the method comprising expressing, in a cell that is preferably not a plant cell of plant protoplast, a mutant plant aspartic protease, wherein the plant aspartic protease is mutated such that it lacks a functional plant specific insert (PSI) domain.
The method preferably involves secretion of the mutant plant aspartic protease from the cell, the method further comprising obtaining or collecting mutant plant aspartic protease secreted from the cell. Obtaining or collection of plant aspartic protease may involve partitioning of protein material from the culture media/fermentation broth and isolation of the plant aspartic protease fraction.
The nucleotide sequence encoding the mutant plant aspartic protease may be provided on a vector such that it is expressed from a vector contained in the cell. Alternatively, the nucleotide sequence encoding the mutant plant aspartic protease may be incorporated in the genome of the cell and expressed from the genome.
The cell is preferably a eukaryotic cell. In some embodiments it is a fungal cell, e.g. a yeast cell. The cell is optionally not a plant cell or plant protoplast.
The method may further comprise the step of mixing mutant plant aspartic protease obtained from said cell with a carrier, adjuvant or diluent to form a product comprising a composition containing said mutant plant aspartic protease.
In some aspects the vector comprises nucleic acid encoding a plant aspartic protease mutant lacking a functional plant specific insert (PSI) domain. Preferably, the plant aspartic protease mutant is either (i) the polypeptide of SEQ ID NO: 1, or SEQ ID NO: 2, or (ii) a polypeptide having at least 70% sequence identity to SEQ ID NO: 1, or SEQ ID NO: 2. The nucleic acid of (i) or (ii) is preferably operably linked to a regulatory sequence to control expression of the nucleic acid in a host cell, and may thereby form an expression cassette. A cell comprising the vector is also provided.
In other aspects a cell is provided having a genome modified to contain nucleic acid encoding a plant aspartic protease mutant lacking a functional plant specific insert (PSI) domain. Preferably, the plant aspartic protease mutant is either (i) the polypeptide of SEQ ID NO: 1, or SEQ ID NO: 2, or (ii) a polypeptide having at least 70% sequence identity to SEQ ID NO: 1, or SEQ ID NO: 2. The nucleic acid of (i) or (ii) is preferably operably linked to a regulatory sequence to control expression of the nucleic acid, and may thereby form an expression cassette
In a further aspect of the present invention a method for promoting accumulation of a polypeptide of interest in the vacuole of a cell, optionally a plant cell or plant protoplast, is provided, the method comprising expressing a polypeptide construct in the cell, the polypeptide construct comprising the amino acid sequence of the polypeptide of interest covalently linked to the amino acid sequence of a PSI domain. The PSI domain may have at least 70% sequence identity to SEQ ID NO: 3, or SEQ ID NO: 4.
In some aspects a vector is provided comprising nucleic acid encoding the polypeptide construct. The nucleic acid is preferably operably linked to a regulatory sequence to control expression of the nucleic acid in a host cell, and may thereby form an expression cassette. A cell comprising the vector is also provided.
In other aspects a cell having a genome modified to contain nucleic acid encoding the polypeptide construct is provided. The nucleic acid is preferably operably linked to a regulatory sequence to control expression of the nucleic acid in the cell, and may thereby form an expression cassette.
The present invention therefore also provides a plant aspartic protease that is modified so as to lack a functional plant specific insert (PSI) domain. The PSI domain may be entirely deleted, or partially deleted. For example, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 or more amino acids of the PSI domain may be deleted. The number of amino acids deleted may be calculated by comparing the plant aspartic protease sequence or PSI domain sequence to the sequence of an unmodified plant aspartic protease, such as a wild-type plant aspartic protease. The PSI domain may be wholly or partially replaced with a linker, such as a linker of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues. The amino acids of the linker may be all the same, for example, they may all be glycine residues. Alternatively, the linker may comprise a plurality of different amino acids. However, this linker does not comprise all, or substantially all, the amino acid residues of the functional PSI domain.
The modification to the PSI domain may confer altered trafficking on the plant aspartic protease. For example, trafficking of the plant aspartic protease within a cell may be modified as compared to the trafficking of a plant aspartic protease which does not have a modified PSI domain, such as a wild type plant aspartic protease. The plant aspartic proteases according to the invention have caseinolytic activity.
The plant aspartic protease according to the invention may have a pro segment. The N-terminal of the plant aspartic protease may not be modified with respect to, or different to, wild-type plant aspartic protease.
The plant aspartic protease according to the invention may be modified at the C-terminus. For example, the plant aspartic protease according to the invention may not have sequence AEAA or AEAV at the C-terminus.
The plant aspartic protease of the invention may be a modified cardosin, cyprosin, cenprosin, phytepsin, or cynarase. It may have a sequence of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to a known cardosin, cyprosin, cenprosin, phytepsin, or cynarase sequence. In some cases, the plant aspartic protease may be a cardosin, such as cardosin A or cardosin B. In some cases, the plant aspartic protease according to the invention is cardosin B.
In some cases, the plant aspartic protease according to the invention has at least 70% identity to SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, it has at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity to SEQ ID NO: 1 or SEQ ID NO: 2.
A plant aspartic protease according to the invention may have been expressed in a eukaryotic cell. For example, the plant aspartic protease may have been expressed in a yeast cell, for example a Kluyveromyces lactis cell. In some cases, the plant aspartic protease according to the invention has not been produced in a plant protoplast. In some cases the plant aspartic protease has not been produced in E. coli. 
The invention also provides nucleic acid encoding a plant aspartic protease according to the invention. For example, nucleic acid encoding a plant aspartic protease which lacks a functional PSI domain; a polypeptide having amino acid sequence SEQ ID NO: 1, or SEQ ID NO: 2, or a polypeptide having at least 70% sequence identity to SEQ ID NO: 1, or SEQ ID NO: 2, wherein the polypeptide lacks a functional plant specific insert (PSI) domain. A vector comprising the nucleic acid is also provided, for example a yeast expression vector.
Also provided is a cell which encodes a plant aspartic protease according to the invention. The cell may have a genome modified to encode the plant aspartic protease, or may include a vector that encodes the plant aspartic protease. The cell may have nucleic acid, for example, its genome may be modified to include nucleic acid, which encodes a plant aspartic protease which lacks a functional PSI domain; the polypeptide of SEQ ID NO: 1, or SEQ ID NO: 2, or a polypeptide having at least 70% sequence identity to SEQ ID NO: 1, or SEQ ID NO: 2, wherein the polypeptide encoded lacks a functional plant specific insert (PSI) domain. The cell may be a yeast cell, such as Kluyveromyces lactis. 
The invention also provides a method for producing a plant aspartic protease in which a cell, preferably a cell which is not a plant cell, or a plant protoplast, or an E. coli, which expresses a plant aspartic protease which lacks a functional plant specific insert (PSI) domain.
In some methods the plant aspartic protease is secreted from the cell, and the method may comprise collecting the plant aspartic protease that has been secreted from the cell, for example by partitioning the secreted plant aspartic protease from other components secreted from the cell or otherwise contained within the media in which the cells are growing. The method may comprise expressing the plant aspartic protease from a vector contained within the cell, or from the genome of the cell. The cell may be a eukaryotic cell. The cell may be a fungal cell such as a yeast cell, for example Kluyveromyces lactis. 
DETAILED DESCRIPTION
The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.



BRIEF DESCRIPTION OF THE FIGURES
Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:
FIG. 1. SDS-PAGE gel electrophoresis of enzyme purified from K. lactis (pCBΔPSI, hereby named pAP).
FIG. 2. Effect of pAP enzymatic activity on epidermal cell desquamation. The aspartic protease was applied to model surface at a final concentration of 1 mg/ml for 30 min, 3 h, 6 h and 12 h. The detached cells were counted on a hemocytometer.
FIG. 3. Study of enzymatic desquamation of skin models. The skin models were exposed to the pAP at the final concentrations 0.1% and 0.5%, at pH 4.5 and pH 5.5 for the incubations times: 30 min, 3 h and 6 h. The detached cells were counted by using a Neubaur chamber.
FIG. 4. Effect of pH on the enzymatic desquamation activity of pAP. The pAP was tested at a final concentration of 0.1%, at different pHs. After 1 h incubation time the samples were collected from the models surface and the detached cells were counted.
FIG. 5. Cell viability determination. The skin models exposed to pAP enzymatic activity were subjected to MTT assay in order determine the tissue cellular viability. After the detachment experiments, the models were incubated for 42 h at 37° C. atmosphere with 5% CO2. The models were incubated in MTT assay reagent for 3 h, the formed dye was solubilized in isopropanol, and the absorbance was measured at 570 nm. For cell viability calculation, the absorbance of the model incubated with PBS was taken as 100%.
FIG. 6. Effect of different aspartic proteases on epidermal cell desquamation. The aspartic proteases were applied to model surface and after 4 h incubation time the detached cells were counted on a hemocytometer. About 10 HUT of each enzyme were tested in citrate buffer pH5.0 excepting for Renin that was tested at pH6.0.
FIG. 7. Sequences of Cardosins B and A with and without PSI sequence, PSI sequences, and sequences used in the examples.



PREFERRED EMBODIMENTS OF THE INVENTION
The inventors have discovered that plant aspartic proteases, in particular Cardosins, and particularly Cardosin B, show skin desquamation activity, without affecting cellular viability.
Desquamation is the shedding of the skin, in particular the stratum corneum, the outermost layer of the epidermis. Desquamation occurs normally, under non-pathological conditions, and may also result from injury or disease of the akin, for example following the rash of measles or sunburn.
Desquamation of corneocytes from epidermis requires the enzymatic degradation of corneodesmosome structures, composed of corneodesmosin, desmoglein-1 and desmocolin-1 proteins. It is thought that the process of desquamation involves proteolytic degradation of desmosomes, causing the cohesive links between the cells to break down thereby allowing detachment of peripheral corneocytes from the surface of the stratum corneum. Several serine, cysteine and aspartic proteases participate in this exfoliation process, including cathepsin D, cathepsin E and SASpase. For example, WO95/07688 discloses a role for stratum corneum trypsin-like enzymes (serine proteases) from skin in the induction of skin desquamation.
The inventors have shown that plant aspartic proteases, particularly Cardosins, and particularly Cardosin B, have enhanced ability to induce skin desquamation compared to mammalian aspartic proteases, including Cathepsin D. The plant aspartic proteases of the invention induce greater skin desquamation over a given period of time than the level of skin desquamation induced by Cathepsin D. The amount of skin desquamation may be measured by any suitable method known in the art, such as by counting the number of detached cells on a hemocytometer. The number of detached cells may be measured after 1 hour, after 2 hours, after 3 hours, after 4 hours, after 5 hours or after 6 hours or more of exposure to the aspartic protease.
The plant aspartic proteases according to the invention preferably do not reduce cell viability, i.e. cell viability is not significantly different to the cell viability observed when the cells are not exposed to a plant aspartic protease according to the invention. Thus, the plant aspartic proteases according to the invention do not exhibit significant cytotoxic activity. Cell viability may be measured by any method known in the art, such as by MTT assay.
The plant aspartic proteases of the present invention exhibit skin desquamation activity over a broad range of pH values. Preferably, the plant aspartic proteases according to the invention are active at mildly acidic pH. The plant aspartic proteases according to the invention may be active between about pH 3 and about pH 7 between about pH 4 and about pH 7, between about pH 4 and about pH 5, between about pH 4 and about pH 6, between about pH 5 and about pH 7, between about pH 5 and about pH 6 or between about pH 6 and about pH 7.
Therapeutic Applications
The compounds and compositions of the present invention are useful in the treatment of a wide range of diseases and conditions. In particular, the compounds and compositions of the present invention are useful in the treatment of disorders, diseases and conditions of the skin and find application in the treatment of fibrosis (including post-surgical fibrosis), tissue adhesion formation, and scar formation. The compounds and compositions of the invention are useful in the treatment of acne, eczema, ichthyosis and psoriasis. Compounds, compositions and methods described herein may be used to enhance epidermal exfoliation and/or enhance epidermal cell renewal, and to improve the texture and/or appearance of the skin.
The plant aspartic proteases and compositions disclosed herein are useful for the treatment of any condition for which the induction or enhancement of skin desquamation would be beneficial. In particular for treating xerosis (dryness of the skin, such as dry skin resulting from Indinavir treatment of HIV), acne, psoriasis, eczema, and dermal or epidermal proliferations, certain benign or malignant tumor lesions, reactive hyperkeratoses, preventing epidermal and/or dermal atrophy, combating dysfunction of cell proliferation and/or differentiation, and avoiding the effects of HIV medication such as Indinavir.
Acne
Acne is a skin condition which involves plugged pores (blackheads and whiteheads), inflamed pimples (pustules), and deeper lumps (nodules). Acne occurs on the face, as well as the neck, chest, back, shoulders, and upper arms. Although most teenagers get some form of acne, adults in their 20's, 30's, 40's, or even older, can develop acne. Often, acne clears up after several years, even without treatment. Untreated acne can leave permanent scars. To avoid acne scarring, treating acne is important. There are three major factors that contribute to the formation and exacerbation of acne, overproduction of oil (sebum), irregular shedding of dead skin cells resulting in irritation of the hair follicles of skin and buildup of bacteria.
Acne is the most common skin disease of adolescence, affecting over 80% of teenagers (aged 13-18 years) at some point12. Estimates of prevalence vary depending on study populations and the method of assessment used. Prevalence of acne in a community Sample of 14- to 16-year-olds in the UK has been recorded as 50%13. In a sample of adolescents from schools in New Zealand, acne was present in 91% of males and 79% of females, and in a similar population in Portugal the prevalence was 82%14. The number of adults with acne, including people over 25 years, is increasing, although the reasons for this increase are uncertain15.
Eczema
Eczema (atopic dermatitis) is a particular type of inflammatory reaction of the skin in which there are typically vesicles (tiny blister-like raised areas) in the first stage followed by erythema (reddening), edema (swelling), papules (bumps), and crusting of the skin followed, finally, by lichenification (thickening) and scaling of the skin. Eczema characteristically causes itching and burning of the skin.
Eczema is very common and can first occur at any age. It is frequently chronic and difficult to treat, and it tends to disappear and recur. Itching can be extreme and severe. There are a number of types of eczema. The prevalence of ezcema is high with an estimated 33 million people in the USA alone affected.
Ichthyosis
Ichthyosis vulgaris is a genetic skin disease that is characterised by the presence of excessive amounts of dry surface scales on the skin caused by abnormal epidermal differentiation or metabolism. It is usually most severe over the legs but may also involve the arms, hands, and trunk in some cases. It may also be associated with atopic dermatitis, keratosis pilaris (small bumps on the back of the arms), or other skin disorders. It usually disappears during adulthood, but may recur when elderly.
Other types of ichthyosis include X-linked ichthyosis, ichthyosis lamellaris, epidermolytic hyperkeratosis, harlequin type ichthyosis, Netherton's syndrome and Sjogren-Larsson syndrome, although ichthyosis vulgaris, accounts for 95% of all ichthyosis cases. Hereditary (congenital) ichthyosis vulgaris accounts for more than 95% of cases of ichthyosis vulgaris. It first appears in early childhood. The gene responsible for ichthyosis vulgaris has been mapped to chromosome band 1q21. The product of this gene is a substance called filaggrin (FLG) which may act as the “keratin matrix protein” in cells of the stratum corneum. The inheritance pattern is autosomal dominant. Acquired ichthyosis vulgaris, typically develops in adulthood and results from an internal disease of the use of certain medications. Hereditary ichthyosis vulgaris is a common disease in United States, with a prevalence of approximately 1 case in 300 persons. Because symptoms improve with age, the true prevalence is probably higher. Acquired ichthyosis is extremely rare. Its prevalence in United States is unknown. In the United Kingdom, the incidence of ichthyosis vulgaris was reported to be 1 in 250. In China, ichthyosis vulgaris has a prevalence of 2.29%.
Psoriasis
Psoriasis is a chronic, genetic, non-contagious skin disorder characterised by itchy or sore patches of thick, red skin with silvery scales. The scaly patches caused by psoriasis, called psoriatic plaques, are areas of inflammation and excessive skin production. The disorder is a chronic recurring condition which varies in severity from minor localised patches to complete body coverage. Psoriasis can also cause inflammation of the joints, which is known as psoriatic arthritis. There are a number of forms of the disease. According to the National Institutes of Health (NIH), as many as 7.5 million Americans (approximately 2.2 percent of the population) have psoriasis, with an estimated 125 million being affected globally. Between 150,000 and 260,000 new cases of psoriasis occurring each year. Incidence of psoriasis tends to be affected by the climate and genetic heritage of the population. It is less common in the tropics and in dark-skinned persons, and most common in Caucasians. Total direct and indirect health care costs of psoriasis for patients are calculated at $11.25 billion annually, with work loss accounting for 40% of the cost burden16. Approximately 60% of psoriasis patients missed an average of 26 days of work a year due to their illness17.
Subjects
The subject to be treated (therapeutically or cosmetically) may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient. Therapeutic and cosmetic uses may be in humans or animals (veterinary use).
Cosmetic Applications
In some aspects the invention relates to a cosmetic treatment comprising the administration of a plant aspartic protease. “Cosmetic” as used herein is non-therapeutic. The cosmetic treatment may be used to improve the appearance and/or texture of the skin. The cosmetic treatment may be used to enhance epidermal exfoliation and/or enhance epidermal cell renewal.
In some aspects the invention relates to a method of cosmetic treatment comprising the administration of a plant aspartic protease. “Cosmetic” as used herein is non-therapeutic. Such methods do not involve the treatment of the human or animal body by therapy. As used herein the term “cosmetic method” does not include a method for treatment of the human or animal body by surgery or therapy, or a diagnostic method practised on the human or animal body according to Article 53(c) EPC.
The subject treated by the cosmetic methods disclosed herein may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject does not require inhibition of induction of skin desquamation for therapeutic benefit. In some cases the subject does not require therapeutic skin desquamation at the site at which the cosmetic treatment is to be applied.
The invention also provides a cosmetic composition comprising a plant aspartic protease. The composition may be used to improve the appearance of the skin. Cosmetic compositions may be formulated similarly to pharmaceutical compositions, as described below. A cosmetically effective amount of a plant aspartic protease may be administered to the subject. That is, an amount of plant aspartic protease effective to induce a cosmetic benefit. This is within the sound judgement of a relevant practitioner, who will appreciate that the appropriate dosages of the active compound or a composition containing the active compound can vary from subject to subject.
Administration
The compound of the invention may be formulated for pharmaceutical administration. Any convenient route of administration may be used, whether systemically, peripherally or topically (i.e. at the site of desired action). Preferably, the compound is formulated for topical administration. In particular embodiments of the invention, the compounds are administered to the skin.
The compound of the invention may be formulated for cosmetic administration. Any convenient route of administration may be used, whether systemically, peripherally or topically (i.e. at the site of desired action). Preferably, the compound is formulated for topical administration. In particular embodiments of the invention, the compounds are administered to the skin.
For therapeutic applications, administration is preferably in a “therapeutically effective amount”, this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.
While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical or cosmetic formulation (e.g., composition, preparation, medicament) comprising at least one active compound, as defined above, together with one or more other pharmaceutically or cosmetically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically or cosmetically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents. The formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents.
Thus, the present invention further provides pharmaceutical and cosmetic compositions, as defined above, and methods of making a pharmaceutical or cosmetic composition comprising admixing at least one active compound, as defined above, together with one or more other pharmaceutically or cosmetically acceptable ingredients well known to those skilled in the art, e.g., carriers, adjuvants, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the active compound.
The terms “pharmaceutically acceptable” and “cosmetically acceptable” as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound judgment of the relevant practitioner, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, adjuvant, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs.
The formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.
Ointments may be prepared using the plant aspartic protease and a paraffinic or a water-miscible ointment base.
Creams may be prepared from the active compound and an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
Emulsions may be prepared from the active compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
The inventors have demonstrated that plant aspartic proteases of the invention are active at across a range of pH. As such, compounds and compositions of the present invention may be formulated, and/or administered in, neutral, mildly alkaline, or acid buffers, diluents, carriers or adjuvants.
A buffer is a composition which, when topically administered to the skin, temporarily alters the pH of the surface of the skin. An acid buffer may lower the pH of the skin to pH 6.0 of lower, pH 5.5 or lower, pH 5.0 or lower, pH 4.5 or lower, pH 4.0 or lower, pH 3.5 or lower or pH 3.0 or lower. Preferably the pH of the surface of the skin is not lower than pH 2.5 or lower than pH 3.0. A neutral buffer may adjust the pH of the skin to between about pH 6 to 8. A mildly alkaline buffer may adjust the pH of the skin to between about pH 8 to 10. The buffer contains an acid, neural or alkali buffering component and a pharmaceutically acceptable carrier, vehicle or excipient. The buffer is susceptible to neutralisation to the average normal pH of the surface of the skin over time by normal skin processes such as perspiration. Acid buffers may be an organic acid, inorganic acid or mixture thereof. Suitable acidic buffers include lactic acid, citric acid, sorbic acid, glycolic acid, malic acid, gluconic acid, glucoronic acid, succinic acid, sodium citrate, sodium sulfate, phosphoric acid, sodium bisulfate, potassium bisulfate and mixtures thereof.
It will be appreciated by one of skill in the art that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient.
A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
Plant Aspartic Proteases
In this specification a “plant aspartic protease” refers to and includes aspartic proteases that can be obtained from plant cells, or tissue, including whole plants. Plant aspartic proteases include cardosins, cyprosins, cenprosins, phytepsins and cynarases. In some cases, the plant aspartic protease according to the invention is not a phytepsin. As used herein the term “plant aspartic protease” includes mutants of such proteases, particularly mutants in which the PSI domain has been made non-functional. It is preferred that the mutation does not inactivate the aspartic protease function of the protein. In preferred embodiments the mutation is such that the resulting polypeptide retains at least 50%, more preferably one of at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of the wild type aspartic protease.
The term “modified plant aspartic protease” as used herein describes a plant aspartic protease which contains one or more modifications as compared with a wild type plant aspartic protease. For example, it may contain one or more amino acid deletions, substitutions or additions as compared to the sequence of the plant aspartic protease as produced in a plant.
Cardosins are examples of plant aspartic proteases, obtained from cardoon (Cynara cardunculus). The amino acid sequence of Cardosin A and Cardosin B is known (see SEQ ID NOs: 5 and 6).
The inventors have developed a heterologous method of production for plant aspartic proteases in a GRAS yeast (K. lactis) that could be effectively transferred to scale-up production. They have used the K. lactis Protein Expression System from New England Biolabs and several optimization procedures were undertaken in order to enhance protein expression and secretion levels. Cardosin A and Cardosin B (a vacuolar and an extracellular aspartic protease from cardoon (Cynara cardunculus), respectively) were used as working models.
Although some trafficking mechanisms in plants appear to be similar to those in yeasts there are several variations, particularly regarding the presence in plants of multiple vacuole types, that could result in the non-recognition of aspartic protease VSS's by yeast vacuolar sorting receptors. In fact, other plant VSS's of the CTPP type were previously shown not to be recognized in yeasts. Conversely, the results described herein indicate that some VSS's identified in plant aspartic proteases are recognized by yeast trafficking mechanisms and can be used to redirect protein sorting. These results show that the PSI domain is functional in plants and yeasts.
Plant Specific Insert (PSI)
When the inventors generated a construct of Cardosin B (which is normally localised extracellularly), lacking the PSI domain and expressed this in the K. lactis yeast, higher levels of expression and secretion were observed in the absence of the PSI, when compared to the full-length wild type construct. These results demonstrate that removal of the PSI domain from all plant aspartic proteases (either vacuolar or secreted) may have a positive impact on their secretion, in yeasts or in plants.
The PSI is an insertion of approximately 100 amino acids located between the N-terminal domain and a C-terminal domain of the precursor “preproenzymes” of the majority of plant aspartic proteases so far identified. The PSI is only identified in plant aspartic proteases, and is highly similar to saposins and saposin-like proteins, whose biological function has not been completely established. Structurally, the PSI comprises five amphipathic α-helices folded into a compact globular domain and linked with each other by three disulphide bridges (discussed in Simoes and Faro 20043). The PSI sequence shown no homology with mammalian or microbial aspartic proteases but is highly similar to that of saposin-like proteins (SAPLIPs). A unique feature of the PSI is the swap of the N- and C-terminal portions of the saposin-like domain, where the C-terminal portion of one saposin is linked to the N-terminal portion of the other saposin. Hence the PSI is not a true saposin but a swaposin.
The plant aspartic proteases described herein may lack a functional PSI domain. The PSI domain may be entirely or partially deleted, or mutated such that it is rendered non functional. Mutation may involve modification of an oligonucleotide sequence encoding the aspartic protease. For example, the modification may be an addition, deletion, insertion or substitution in the coding sequence.
A PSI domain may have substantial identity to SEQ ID NO: 3, or SEQ ID NO: 4. A PSI domain may have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 3 or SEQ ID NO: 4.
The PSI domain may have a length of any one of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, amino acids. The PSI domain may have a length in the range 80, to 120 amino acids, or 90 to 110 amino acids, or 95 to 105 amino acids, or 98 to 108 amino acids. The PSI domain may have a minimum length of about 80 amino acids, more preferably one of 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106 amino acids. The PSI domain may have a maximum length of about 130 amino acids, more preferably one of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 129 amino acids.
The PSI domain of a plant aspartic protease functions to regulate trafficking of cardosins in the cell, for example targeting the protein to the vacuole. Thus, plant aspartic proteases according to the invention, which lack a functional PSI domain have altered trafficking, for example as compared to plant aspartic proteases that contain a complete PSI domain. For example, a modified aspartic protease that lacks a functional PSI domain may not be targeted to the vacuole, whereas the unmodified aspartic protease, such as the wild type protein, might be targeted to the vacuole.
The skilled person may readily determine whether an aspartic protease lacks a functional PSI domain by any suitable method known in the art. For example agents known to affect protein trafficking (e.g. glycosidases) may be applied to a cell to determine whether trafficking of the modified aspartic protease is affected by the agent in the same or a similar way to the complete, or wild type, plant aspartic protease. Alternatively, or additionally, subcellular fractionation may be used to determine whether the modified and complete, or wild-type, aspartic proteases are present in a similar distribution within a cell. Alternatively, or additionally, immunocytochemistry may be used to determine whether the protein is secreted from the cell, or present in a different cellular compartment, to a complete, or wild type, plant aspartic protease.
The lack of a functional PSI domain may be sufficient to stop the plant aspartic protease collecting in the vacuole and/or to increase secretion of the plant aspartic protease from the cell. In some cases, the lack of a functional PSI domain may entirely prevent the plant aspartic protease being localised to the vacuole such that substantially all of the plant aspartic protease produced by the cell is secreted from the cell.
The plant aspartic acid which lacks a functional PSI domain may be any plant aspartic acid. Preferably, the plant aspartic acid is from the Cardosin family of plant aspartic proteases, i.e. a mutant or modified Cardosin that lacks a functional PSI domain. In some cases the plant aspartic protease may be further mutated.
Modification of the PSI domain to render it non-functional may affect the kinetic properties of the plant aspartic protease. For example, the modified plant aspartic protease may be less caseinolytic as compared to the naturally occurring, or wild-type, plant aspartic protease. In some cases, modification of the PSI may increase the specificity of the plant aspartic protease for a substrate. For example, it may increase the specificity of the plant aspartic protease for α-casein.
Pro Segment
The prosegment is located in the N-terminal of plant aspartic proteases. It is present in the precursor protein and is normally removed by proteolysis during production of the mature, active, enzyme from the inactive zymogen. In some cases, the plant aspartic proteases according to the invention are expressed with a prosegment. The prosegment comprises approximately 40 amino acids.
C-Terminal Sequence
The C-terminal sequence is a putative enzyme sorting signal. Certain plant aspartic proteases may lack 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues, preferably 4 amino acids, from the C terminus, as compared to the naturally occurring form of the aspartic protease. For example, modified cardosin A according to the invention may lack AEAA from the C-terminus and cardosin B may lack AEAV.
Linkers
As used herein, the term “linker” denotes a series of amino acid resides which are introduced into a protein sequence to replace amino acid residues which have been removed. For example, the plant aspartic proteases of the present invention lack a functional PSI domain. Where the PSI domain is fully or partially deleted from the plant aspartic protease of the invention, the deleted amino acids may be replaced by a linker. The linker may allow two or more regions of the protein containing it to fold into the correct three dimensional configuration.
The linker may comprise one or more amino acids. The amino acids may all be the same, for example a plurality of glycine residues. Alternatively, the amino acids may be different. The linker may comprise a sequence corresponding to a scrambled sequence of the PSI domain.
The linker may comprise between 1 and 100, between 1 and 50, between 1 and 25 or between 1 and 10 amino acids. The linker may comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In some cases, the linker consists of 1 to 7 amino acid residues.
The presence of a linker may affect the kinetic properties of the plant aspartic protease. For example, the introduction of a linker may render the plant aspartic protease less caseinolytic as compared to the naturally occurring, or wild-type, plant aspartic protease. In some cases, the linker may increase the specificity of the plant aspartic protease for a substrate. For example, the introduction of a linker may increase the specificity of the plant aspartic protease for α-casein.
Cardosins
In this specification, a Cardosin nucleic acid may be any nucleic acid (DNA or RNA) having a nucleotide sequence which encodes a polypeptide having a specified degree of sequence identity to one of SEQ ID No.s 5 and 6 to an RNA transcript of any one of these sequences, to a fragment of any one of the preceding sequences or to the complementary sequence of any one of these sequences or fragments. Alternatively a Cardosin nucleic acid may be one that hybridises to one of these sequence under high or very high stringency conditions. The specified degree of sequence identity may be from at least 60% to 100% sequence identity. More preferably, the specified degree of sequence identity may be one of at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
In this specification, a Cardosin polypeptide may be any peptide, polypeptide or protein having an amino acid sequence having a specified degree of sequence identity to one of SEQ ID NO.s 1, 2, 5 or 6 or to a fragment of one of these sequences. The cardosin may be, or have a specified degree of sequence identity to, cardosin A as deposited at GenBank under accession number Q9XFX3.1 (GI: 75267434). The cardosin may be, or have a specified degree of sequence identity to, cardosin B as deposited at GenBank under accession number Q9XFX4.1 (GI: 75338567).
The specified degree of sequence identity may be from at least 60% to 100% sequence identity. More preferably, the specified degree of sequence identity may be one of at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
Sequence Identity
Percentage (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with residues in the given listed sequence after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of the respective sequences.
Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalW 1.82. T-coffee or Megalign (DNASTAR) software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used. The default parameters of ClustalW 1.82 are: Protein Gap Open Penalty=10.0, Protein Gap Extension Penalty=0.2, Protein matrix=Gonnet, Protein/DNA ENDGAP=−1, Protein/DNA GAPDIST=4.
In certain aspects the invention concerns compounds which are isolated peptides/polypeptides comprising an amino acid sequence having a sequence identity of at least 60% with a given sequence. Alternatively, this identity may be any of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence Identity.
Identity of nucleic acid sequences may be determined in a similar manner involving aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and calculating sequence identity over the entire length of the respective sequences.
In certain aspects the invention concerns compounds which are isolated nucleic acids comprising a nucleotide sequence having a sequence identity of at least 60% with a given sequence. Alternatively, this identity may be any of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity.
Certain aspects of the invention relate to complete plant aspartic proteases (i.e. comprising substantially all domains present in the wild-type protein). For example, Cardosin A may have an amino acid sequence having a specified degree of sequence identity to SEQ ID NO: 3, or Cardosin B may have an amino acid sequence having a specified degree of sequence identity to SEQ ID NO: 4.
Preferably, the Cardosins of the invention lack a functional PSI domain. For example, Cardosin B may have an amino acid sequence having a specified degree of sequence identity to SEQ ID NO: 1, and Cardosin A may have an amino acid sequence having a specified degree of sequence identity to SEQ ID NO: 2.
In certain aspects of the invention, the compound of the invention may a be a protein fragment which retains the properties and/or therapeutic or cosmetic effects of the plant aspartic proteases described herein. For example, the compound may be a fragment of Cardosin A or B which has protease activity and/or induces skin desquamation.
A fragment may comprise a nucleotide or amino acid sequence encoding a portion of the corresponding full length sequence. In this specification the corresponding full length sequence may be one of SEQ ID No.s 1, 2, 5, or 6. Said portion may be of defined length and may have a defined minimum and/or maximum length.
Accordingly, the fragment may comprise at least, i.e. have a minimum length of, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% of the corresponding full length sequence. The fragment may have a maximum length, i.e. be no longer than, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% of the corresponding full length sequence. The fragment may have a length anywhere between the said minimum and maximum length.
The fragment may comprise at least, i.e. have a minimum length of, at least 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 377, 380, 383, 400, 420, 440, 460, 480 or 500 amino acids. The fragment may have a maximum length, i.e. be no longer than, 220, 240, 260, 280, 300, 320, 340, 360, 377, 380, 383, 400, 420, 440, 460, 480 or 483 amino acids.
Although wild type plant aspartic proteases will be useful in the therapeutic and cosmetic applications described herein, in some embodiments the plant aspartic protease may be a mutant or modified plant aspartic protease, such as a mutant or modified Cardosin. The plant aspartic protease may be mutated relative to the wild-type or genomic plant aspartic protease, carrying one or more alterations to the nucleic acid encoding the plant aspartic protease and/or to the amino acid sequence of the plant aspartic protease. The alteration may take the form of an addition, insertion, substitution or deletion.
In some embodiments of the invention the plant aspartic protease is mutated such that it does not have a functional PSI domain. In some cases, the PSI domain is entirely or substantially absent. In others at least one mutation is included in the protein and/or nucleic acid sequence such that the PSI domain of the aspartic protease is not fully transcribed, is incorrectly transcribed, or is otherwise non functional. Mutations may be point mutations or larger mutations, wherein one or more base pairs of the nucleic acid sequence encoding the aspartic protease are added, substituted, deleted or inserted. In some cases, the mutation is one that causes the subsequent nucleic acids to be transcribed out of frame, thereby producing a non-functional protein product. In other cases, mutation of a single base pair causes an alteration in the protein sequence such that the protein product is non functional. Where the mutation causes subsequent nucleic acids to be transcribed out of frame it may be necessary to include a further change downstream of the first mutation in order to restore transcription of a subsequent part of the protein, e.g. after some or all of the PSI domain, back into frame.
Methods for introducing mutations are known in the art, and the skilled person will readily appreciate suitable methods for creating a modified or mutant plant aspartic protease according to the invention. Preferably, mutations are introduced by site directed mutagenesis, for example through PCR mutagenesis. PCR mutagenesis is a method for generating point mutations on a double stranded plasmid and involves the use of two synthetic oligonucleotide primers containing the desired mutation, each complementary to the opposite strands of a vector containing the plant aspartic protease to be mutated.
Methods of Producing a Plant Aspartic Protease
Plant aspartic proteases may be produced according to any method known in the art, such as microbial fermentation, plant, insect or mammalian cell culture.
Certain methods according to the invention involve expressing a plant aspartic protease that lacks a functional PSI domain in a cell. The method optionally further comprises the step of collecting plant aspartic protease that has been secreted from the cell.
Molecular biology techniques suitable for the producing plant aspartic proteases according to the invention in cells are well known in the art, such as those set out in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989
The plant aspartic protease may be expressed from a nucleotide sequence encoding the plant aspartic protease. The nucleotide sequence may be contained in a vector present in the cell, or may be incorporated into the genome of the cell.
A “vector” as used herein is an oligonucleotide molecule (DNA or RNA) used as a vehicle to transfer foreign genetic material into a cell. The vector may be an expression vector for expression of the foreign genetic material in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the gene sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express plant aspartic proteases from a vector according to the invention. Suitable vectors include plasmids, binary vectors, viral vectors and artificial chromosomes. (e.g. yeast artificial chromosomes).
In this specification the term “operably linked” may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence (e.g. promoter and/or enhancer) are covalently linked in such a way as to place the expression of the nucleotide sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus a regulatory sequence is operably linked to the selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of the nucleotide sequence. Where appropriate, the resulting transcript may then be translated into a desired protein or polypeptide.
Any cell suitable for the expression of polypeptides may be used for producing plant aspartic proteases according to the invention. The cell may be a prokaryote or eukaryote. Preferably the cell is a eukaryotic cell such as a yeast cell, a plant cell, insect cell or a mammalian cell. In some cases the cell is not a prokaryotic cell because some prokaryotic cells do not allow for the same post-translational modifications as eukaryotes. In addition, very high expression levels are possible in eukaryotes and proteins can be easier to purify from eukaryotes using appropriate tags. Specific plasmids may also be utilised which enhance secretion of the protein into the media.
In some embodiments the cell is not a plant cell, or a plant protoplast cell.
In some preferred embodiments the cell is a fungi (including yeasts and molds) or microbial eukaryote, or single coil eukaryote, preferably a yeast of the genus Kluyveromyces, Rhizomucor, Endothia, Aspergillus or Saccharomyces. 
Suitable yeast cells include Kluyveromyces lactis, Kluyveromyces marxianus, Rhizomucor meihei, Endothia parasitica, Rizomucor pusillus, Pichia pastoris, Aspergillus niger, Aspergillus oryzae and Saccharomyces cerevisae. The yeast may be a GRAS (Generally Regarded As Safe) yeast, i.e. a yeast that has GRAS status from the Food and Drug Administration (FDA).
Methods of producing the plant aspartic protease may involve culture or fermentation of a eukaryotic cell modified to express the plant aspartic protease. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air/oxygen and/or growth factors. Secreted proteins can be collected by partitioning culture media/fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted aspartic protease. Culture, fermentation and separation techniques are well known to those of skill in the art.
Bioreactors include one or more vessels in which cells may be cultured. Culture in the bioreactor may occur continuously, with a continuous flow of reactants into, and a continuous flow of cultured cells from, the reactor. Alternatively, the culture may occur in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.
Following culture of cells that express a plant aspartic protease, the plant aspartic protease is preferably isolated. Any suitable method for separating proteins from cell culture known in the art may be used. In order to isolate a protein of interest from a culture, it may be necessary to first separate the cultured cells from media containing the protein of interest. If the protein of interest is secreted from the cells, the cells may be separated from the culture media that contains the secreted protein by centrifugation. If the protein of interest collects within the cell, for example in the vacuole of the cell, it will be necessary to disrupt the cells prior to centrifugation, for example using sonification, rapid freeze-thaw or osmotic lysis. Centrifugation will produce a pellet containing the cultured cells, or cell debris of the cultured cells, and a supernatant containing culture medium and the protein of interest.
It may than be desirable to isolate the protein of interest from the supernatant or culture medium, which may contain other protein and non-protein components. A common approach to separating protein components from a supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of precipitating agent such as ammonium sulfate. For example, at low concentrations of precipitating agent, water soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitating agent, proteins of different solubilities may be distinguished. Dialysis may be subsequently used to remove ammonium sulfate from the separated proteins.
Other methods for distinguishing different proteins are known in the art, for example ion exchange chromatography and size chromatography. These may be used as an alternative to precipitation, or may be performed subsequently to precipitation.
Once the protein of interest has been isolated from culture it may be necessary to concentrate the protein. A number of methods for concentrating a protein of interest are known in the art, such as ultrafiltration or lyophilisation.
A plant aspartic protease that has been isolated from a cell may be mixed with a carrier, adjuvant or diluent to form a product comprising a composition containing the plant aspartic protease. The product formed may be of any kind, e.g. liquid, solid, powder, cream and may be suitable for at least any of the following uses: as a cosmetic or therapeutic preparation, as a detergent or washing powder, as a food modifier, as a meat tenderiser, as a stain remover, as a leather softener, as a rennet substitute.
Methods for Promoting Accumulation of a Polypeptide of Interest
The invention also provides methods for promoting the accumulation of a polypeptide of interest in the vacuole of a cell, particularly a plant cell. Such methods involve expressing a polypeptide construct in the cell, the construct comprising the amino acid sequence of the polypeptide of interest and the amino acid sequence of a PSI domain. The amino acid sequences are preferably covalently linked to form a single contiguous amino acid sequence forming the polypeptide construct. As such, in some embodiments, the polypeptide construct may be a fusion protein.
The PSI domain may be included in the amino acid sequence of the construct at any position. In some embodiments the PSI domain may be added at any one of the N-terminus, C-terminus or a position between the N- and C-termini.
The polypeptide of interest can be any polypeptide, but is preferably not a polypeptide that normally (i.e. in the wild type sequence) encodes a PSI domain. For example, in some embodiments the polypeptide of interest is not an aspartic protease, and in some embodiments the polypeptide of interest is not a plant aspartic protease.
The polypeptide of interest is preferably a polypeptide that forms a protein having a measurable activity, e.g. binding to another molecule, or enzyme activity. The polypeptide construct preferably retains such a measurable activity, although the level of activity may be reduced or increased compared to the wild type polypeptide of interest. As such, the polypeptide will typically have a minimum length of at least about 50 amino acids, and more preferably one of about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 amino acids.
In some other embodiments the polypeptide of interest may be a small peptide, and may have a length of less than about 50 amino acids.
The polypeptide construct may be expressed from a nucleotide sequence encoding the polypeptide construct. The nucleotide sequence may be contained in a vector present in the cell, or may be incorporated into the genome of the cell.
Molecular biology techniques suitable km the producing plant aspartic protcascs according to the invention in cells are well known in the art, such as those set out in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989
The details of one or more embodiments of the invention are set forth in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.
EXAMPLES
Example 1—Synthesis of Cardosin B in K. lactis 
Strains and Growth Conditions
All plasmid constructions and propagations were performed using the Escherichia coli strain Top10F′ (Invitrogen). The bacterial cells were grown at 37° C. in LB (Miller's Formulation—Invitrogen) liquid and solid (1.5% agar) medium, supplemented with ampicillin at 100 μg/ml (GE-Healthcare). The Kluyveromyces lactis GG799 strain was purchased from New England Biolabs and used as host strain to the recombinant protein expression studies. K. lactis cells were grown and maintained in YPD media (2% bactopeptone, 1% yeast extract, 2% glucose) whereas the expression experiments were performed in YPGal (2% bactopeptone, 1% yeast extract, 4% galactose) as culture media, both at 30° C. with shaking. The recombinant K. lactis cells were selected on solid Yeast Carbon Base (New England Biolabs) supplemented with 5 mM acetamide (New England Biolabs) plates.
proCardosinBΔPSI pKLAC1 Sub Cloning
The cloning and subcloning procedures were performed according to the manufacturers' instructions and using standard molecular biology cloning techniques. The construct proCardosinB lacking the PSI region (pCBΔPSI, also referred to herein as Bwo) was amplified by PCR, using the construct pCBΔPSI/TA as template, in order to introduce upstream and downstream of the cDNA the restriction sites XhoI and SalI, respectively. The pair of oligonucleotides used in the PCR reaction were:









pCB-XhoI



(CTCGAGAAAAGAATGGTCTCCAACGGCGGATTGCTTC 



[SEQ ID NO: 7])



and



 



pCB-SalI



(GTCGACTCAAACTGCTTCTGCAAATCCCACTCGTAAC 



[SEQ ID NO:8]).






After amplification the PCR product was cloned into pGEM (Promega) cloning vector and afterwards subcloned into the integrative expression pKLAC1 (NEB). The subcloning process was performed by cleavage/ligation at the XhoI/SalI restriction sites, resulting in pCBΔPSI cloning in frame with the α-mating factor secretion leader sequence.
K. lactis Recombinant Strains Construction
The recombinant plasmid pCBΔPSI/pKLAC1 was linearized by SaclI (NEB) digestion, in order to obtain the insertion cassette fragments that were afterwards used in K. lactis transformation step. A total of 2 μg DNA was used in K. lactis GG799 transformation. This process was performed by electroporation with a “Gene Pulser” (BioRad) apparatus, using the following electroporation conditions: 1.5 KV, 25 mF and 200 Ohm. The positive transformants were selected based on their ability to growth on YCB acetamide media, and the multi-integrants clones selected by whole-cell PCR, following the instructions described on the “K. lactis expression Kit” protocols (NEB).
Heterologous pCBΔPSI Mutants Expression and Purification
An integrative recombinant K. lactis clone was selected for pCBΔPSI construct and was grown in YPD media, at 30° C. with shaking, for 16 h. The cultures were diluted to an OD600 nm of 0.3 in YPGal media and incubated at 30° C., with shaking for and 4 days. Thereafter the cultures were centrifuged and the supernatants sequentially filtered through 0.8 μm, 0.45 μm and 0.2 μm filters. The samples were concentrated and activated by dilution 1:10 with 0.5M sodium acetate buffer pH4.0, at 37° C. A size exclusion chromatography was the first purification step. The samples were applied to a S200 26/60 column (GE-Healthcare) and the proteins were eluted with buffer 20 mM Tris-HCl pH7.5, 0.1M NaCl at a flow rate of 1 ml/min. Fractions were pooled and applied to an ionic exchange on a Mono Q, using the buffer 20 mM Tris-HCl pH7.5. The proteins were then eluted with a linear gradient of 0-0.5M NaCl, at a flow rate of 0.75 ml/min. Both expression and purification procedures were followed by SDS-PAGE analysis (see FIG. 1).
This expression and purification method results in the production of a plant origin-based enzyme in considerable amounts (3 mg/L) and with a high purity level.
Example 2—pAP Induces Desquamation
3D Skin Model (“EpiSkin”-SkinEthic (L'Oreal®), and “Epidermal Skin Test” (EST-1000-CellSystems))
Two rounds of experiments were performed using skin models. In both experiments the enzyme sample (hereby named pAP) was applied to model surface and each model was incubated for different incubation times at 37° C., in an atmosphere of 5% CO2. After incubation, each sample was collected from the top of the model, centrifuged and the detached cells were counted on a hemocytometer. The models surface was washed with PBS, and the models were transferred to a new plate with fresh culture medium and incubated at the same conditions.
In the first round of experiments the desquamation activity was tested for three incubation times and two different pH values. The pAP was tested in a final formulation of 0.1% (w/v), in 0.1M buffer sodium citrate pH 4.0 and pH 5.0. The sample Trypsin/EDTA (0.025%) was used as positive control and a sample of buffer pH 4.0 as negative control. The results are shown in FIG. 2.
In the second round of experiments, the protein was tested for the concentrations 1 mg/ml (0.1%) and 5 mg/ml (0.5%) and for different incubation times. The pH dependence of desquamation activity was also studied. The model cell viability was determined by the MTT assay method, 42 h after the enzymatic incubation. The results are shown in FIGS. 3 and 4.
MTT Assay
The MTT test was performed in order to test the enzyme effect on skin model cellular viability. After the exposure to enzymatic action, each model was incubated in fresh culture medium at 37° C. atmosphere with 5% CO2, for 24 h. The medium was changed and the models were incubated for another 18 h time period at the same conditions. After this recovery time, the models were incubated with the MTT solution for 3 h and the converted dye was solubilized by using isopropanol. The absorbance was measured at 570 nm. See FIG. 5.
The pAP enzyme was shown to have an epidermal cell desquamation activity. The results indicate that pAP is approximately 13 times more effective at causing cellular shedding than a control of buffer solution and approximately 3 times more effective at causing cellular shedding than the positive control Trypsin/EDTA (0.025% w/v), at a pH of 4 and at a concentration of 0.1% (w/v), in 0.1 M buffer sodium citrate.
The MTT viability assay demonstrates that across a range of pH's and concentrations, the enzyme is well tolerated and could therefore be utilised at a range of concentrations for different therapeutic indications and non-therapeutic applications without damaging the remaining un-desquamated cells.
The pAP enzyme has greater desquamation capacity than either Renin or Cathepsin D (a mammalian aspartic protease involved in the natural skin desquamation process).
Example 3—Comparison of pAP with Two Different Aspartic Proteases
This set of experiments had as major goal the comparison between the enzymatic desquamation activity of two different aspartic proteases (Cathepsin D and Renin) and Cardosin B (pAP) activity. Additionally, the desquamation enzymatic activity pH dependence experiments were performed, for a shorter incubation time, in order to determine the effect of the normal skin neutralization process
Cardosin B, Cathepsin D and Renin Enzymatic Desquamation Activity (“EpiSkin”-SkinEthic (L'Oreal®))
One round of experiments was performed for each proteolytic enzyme, using Episkin models. As described above, the enzyme samples were applied to model surface and the models were incubated for 4 h, at 37° C., in an atmosphere of 5% CO2. After incubation, each sample was collected from the top of the model, centrifuged and the detached squames were counted on a hemocytometer. The enzymes (130 HUT/mg) were tested using about 10 HUT enzyme units, in 0.1M citrate buffer pH 5.0. Renin was tested at its optimal pH-pH 6.0, and the citrate buffer pH 5.0 was used as negative control. See FIG. 6.
Enzymo Dosquamation Activity pH Dependence
The pH dependence of desquamation activity was studied, testing a shorter incubation period. After an injury, the skin recovery time is about 30 minutes to 1 h, if the agent is a non-toxic agent. In these assays, the pAP was incubated for 1 h, in citrate buffer pH 4.0, pH5.0, and in PBS pH7.0, at 37° C. in an atmosphere of 5% CO2. See FIG. 7. Although displaying desquamation activity at pH 7, pAP activity is greatly enhanced at pH 4.0 and pH 5.0 what is consistent with the optimum pH of cardosins (ref 10)
REFERENCES


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## Claims

1. An ointment, cream or emulsion composition comprising an isolated plant aspartic protease, wherein the isolated plant aspartic protease is a Cardosin, and wherein the isolated plant aspartic protease has at least 70% sequence identity to the full length of SEQ ID NO: 1 or SEQ ID NO: 2.

2. The ointment, cream or emulsion composition according to claim 1 wherein the plant specific insert (PSI) domain of the plant aspartic protease is entirely or partially deleted.

3. The ointment, cream or emulsion composition according to claim 1 wherein the composition additionally comprises a pharmaceutically or cosmetically acceptable carrier, adjuvant or diluent.

4. A method of medical treatment comprising administering a plant aspartic protease to the skin of a subject to induce skin desquamation in the subject, wherein the plant aspartic protease is a Cardosin, and wherein the plant aspartic protease has at least 70% sequence identity to the full length of SEQ ID NO: 1 or SEQ ID NO: 2.

5. The method of medical treatment according to claim 4 wherein the subject is suffering from a skin disorder or disease.

6. The method of medical treatment according to claim 4 wherein the subject is suffering from at least one of xerosis, eczema, acne, psoriasis or ichthyosis, and the method involves the treatment of at least one of xerosis, eczema, acne, psoriasis or ichthyosis.

7. The method of medical treatment according to claim 4 wherein the plant aspartic protease is Cardosin B or Cardosin A.

8. The method of medical treatment according to claim 4 wherein the plant specific insert (PSI) domain of the plant aspartic protease is entirely or partially deleted.

9. A cosmetic method for improving the appearance of the skin comprising administering a plant aspartic protease to the skin of a human, wherein the plant aspartic protease is a Cardosin, and wherein the plant aspartic protease has at least 70% sequence identity to the full length of SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the plant aspartic protease induces skin desquamation in the human.

10. The cosmetic method according to claim 9, wherein the plant aspartic protease is Cardosin B or Cardosin A.

11. The cosmetic method according to claim 9, wherein the plant specific insert (PSI) domain of the plant aspartic protease is entirely or partially deleted.