EP-4740954-A2 - COMPOSITIONS AND METHODS FOR TREATMENT OF NETHERTON SYNDROME WITH LEKTI EXPRESSING RECOMBINANT MICROBES

Abstract

The present disclosure provides, inter alia, engineered microbes expressing recombinant LEKTI domains that are effective to treat or ameliorate the symptoms of Netherton Syndrome. In certain embodiments, compositions, methods, and kits are provided comprising LEKTI domain expressing microbes.

Inventors

• WHITFILL, Travis, Michael
• MUNIVAR, Azim, Momin

Assignees

• AZITRA, INC.

Dates

Publication Date

20260513

Application Date

20180615

Claims (15)

1. A composition for the treatment of a skin disease, wherein the skin disease is Netherton Syndrome, comprising: a microbe genetically modified to express and provide one or more LEKTI protein domains onto the skin of a mammal, wherein the microbe is selected from the group consisting of Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, and mixtures thereof, wherein at least one of the LEKTI protein domains comprises LEKTI Domain 6; wherein the one or more LEKTI protein domains are effective to penetrate one or more layers of the mammal's skin and effective to inhibit serine protease activity of at least one serine protease in or on the mammal's skin, and wherein the LEKTI protein domains are effective to ameliorate the symptoms of Netherton Syndrome.
2. The composition of claim 1, wherein the microbe is adapted to live for a controlled duration on the surface of the mammal's skin to provide a continuous supply of LEKTI protein domains; and/or wherein the microbe is genetically modified by transfection/transformation with a recombinant DNA plasmid encoding the LEKTI protein domains.
3. The composition of any one of the previous claims, wherein the LEKTI domains are operably linked to one or more recombinant protein domains that are effective to enhance secretion from the microbe and/or penetration of the mammal's skin.
4. The composition of any one of the previous claims, wherein at least one LEKTI domain is operably linked to a SecA domain; and/or wherein at least one LEKTI domain is operably linked to an RMR domain; and/or wherein at least one LEKTI domain comprises an amino acid sequence according to SEQ ID NO: 1.
5. The composition of any one of the previous claims, wherein the microbe is adapted to multiply on the skin of the mammal.
6. The composition of any one of the previous claims, wherein expression of at least one LEKTI domain is controlled by an operon and the amount of LEKTI provided to the mammal's skin is proportional to the availability of an extrinsic factor; and/or wherein the expression of at least one LEKTI domain is controlled by a promoter that is constitutively active.
7. The composition of any one of the previous claims, wherein the microbe has been genetically modified by transfection/transformation with a recombinant DNA plasmid encoding the one or more LEKTI protein domains and one or more antibiotic resistance genes.
8. The composition of claim 1 for use in a method of treating or ameliorating the effects of a skin disease of a mammal in need thereof, said method comprising: providing onto a surface of the skin of the mammal the composition of claim 1 comprising a microbe genetically modified to express one or more LEKTI protein domains, wherein the LEKTI protein domains are effective to penetrate one or more layers of the mammal's skin and effective to inhibit activity of at least one serine protease in or on the mammal's skin.
9. The composition according to claim 8, wherein the microbe is adapted to live for a controlled duration on the surface of the mammal's skin to provide a continuous supply of LEKTI protein domains.
10. A kit for the treatment or amelioration of the effects of a skin disease of a mammal in need thereof comprising: (1) a composition comprising a microbe that is genetically modified to express one or more LEKTI protein domains, wherein at least one of the LEKTI protein domains comprises LEKTI Domain 6, wherein the microbe is selected from the group consisting of Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, and mixtures thereof, wherein the LEKTI protein domains are effective to penetrate one or more layers of the mammal's skin and effective to inhibit serine protease activity of at least one serine protease in or on the mammal's skin; and (2) reagents for applying the composition to the skin of the mammal.
11. The kit according to claim 10, wherein the microbes are adapted to live for a controlled duration on the surface of the mammal's skin to provide a continuous supply of LEKTI protein domains.
12. A composition for the treatment of skin disease comprising a microbe comprising pJB38-LEKTI-domain 6 complete plasmid construct, wherein LEKTI domain 6 comprises SEQ ID NO: 109, optionally wherein the microbe is selected from the group consisting of Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, and mixtures thereof.
13. A composition comprising pJB38-LEKTI-complete plasmid construct.
14. The composition according to claim 13, wherein the pJB38-LEKTI-complete plasmid construct is expressed in a microbe selected from the group consisting of Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Pediococcus, Leuconostoc, or Oenococcus, and mixtures thereof.
15. A recombinant microorganism capable of secreting a polypeptide, wherein the recombinant microorganism comprises an expression vector comprising a first coding sequence comprising a gene capable of expressing the polypeptide and a second coding sequence comprising a gene capable of expressing a cell penetrating peptide, wherein the polypeptide is LEKTI Domain 6 and wherein the cell penetrating peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 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, and 102.

Description

RELATED APPLICATIONS This application claims priority to U.S. Provisional Application 62/521,050, filed on June 16, 2017, the entire contents of which are incorporated by reference in its entirety herein. FIELD OF THE DISCLOSURE The present disclosure relates to methods, kits, and compositions for treating or ameliorating the effects of Netherton Syndrome using one or more recombinant microorganisms that are genetically modified to express one or more therapeutic LEKTI domains on the skin of a subject. BACKGROUND OF THE INVENTION The epidermis, the squamous stratified epithelium of the skin, consists of multiple sublayers and is one of the most important barriers of the body against the outside world. The stratum corneum is the outermost layer of the epidermis and develops as a result of the final anucleated step in keratinocyte differentiation from the cells in nucleated epidermal layers. Although the stratum corneum is recognized as the most important physical barrier, the nucleated epidermal layers are also significant in barrier function (Proksch, Brandner et al. 2008). Together, the skin barrier protects against extensive water loss in one direction (inside-outside barrier) and against the invasion of harmful substances from the environment (outside-inside barrier) (Proksch, Brandner et al, 2008). The maintenance of the barrier is also important for balanced proliferation in the basal layer and preservation of the calcium ion gradient and thus proper epidermal differentiation (Lee, Jeong et al. 2006). A number of current limitations exist in the treatment of skin. Many treatments, such as topical corticosteroids or biologics, do not treat the underlying issues of deficient intrinsic protein in the epidermis or imbalances in the microbial diversity in the skin. While recombinant proteins represent a promising group of therapeutic agents in the treatment of skin disease, several problems accompany their use in the context of the skin. Traditional methods purify and concentrate recombinant proteins that are extracted from bacterial systems, and then incorporate such preparations into a delivery system. The purification of recombinant proteins is often a very costly method of obtaining protein. Moreover, a number of problems are associated with these traditional methods, including proteolytic degradation, inefficient delivery, and the need for repeated application overtime to achieve therapeutic effect. One skin disease that would benefit from improved treatment modalities is Netherton Syndrome (NS). NS is a rare autosomal skin disease manifested as severe skin inflammation and scaling, hair shaft defects, constant allergic symptoms, and immune system problems. Newborns with NS often have red and scaly skin that may leak fluid, which creates a risk of dehydration and infections of the skin or throughout the body. Affected children may also fail to grow at a normal rate. The health of older children and adults with NS typically improves, but those individuals are often underweight and of short stature. Most people with NS also have immune system problems such as food allergies, hay fever, asthma, or eczema. NS is caused by a loss-of-function defect in the gene SPINK5 (serine protease inhibitor of kazal type 5), which encodes lymphoepithelial kazal type related inhibitor type 5 (LEKTI) protein. LEKTI is a multi-domain serine protease inhibitor that is normally expressed in all stratified epithelial cells and the Hassal corpuscules of the thymus. The SPINK5 gene encoding LEKTI is located on chromosome 5 among a cluster of other SPINK genes (e.g. SPINK6 and SPINK9), and comprises 33 exons encoding 15 inhibitory domains separated by linker regions. SPINK5 stands out among the other SPINK genes for the large number of inhibitory domains it encodes. Additionally, the SPINK5 gene is transcribed into three different transcripts, resulting in three different LEKTI proteins that differ in the C-terminal region; i.e. a 145 kDa full length protein having inhibitory domains D1-D15, a 125 kDa (short) protein having inhibitory domains D1-D12, and a 148 kDa (long) protein having an extended linker region 13. The LEKTI protein is a Kazal-type-related inhibitor. The Kazal motif is defined by the presence of six cysteine residues positioned at specific distances to allow formation of three disulfide bonds in a 1-5, 2-4, and 3-6 pattern. Two of the domains of LEKTI (D2 and D5) form this six cysteine motif, while other domains share four cysteine residues, which produce a rigid inhibitory loop believed to mimic the substrate of target proteases and inactivate the target protease catalytic site. The LEKTI protein requires proteolytic cleavage for activation of its inhibitory function against many proteases. Specifically, the full length protein is cleaved into domains D1-D5 and D6-D15. The D6-D15 domains are then further cleaved in multiple steps into D6-D9 and D10-D15, → D6 and D7-D9 → D7 and D8-D9 → D8. This process results in