Patent Application
20230374471
Self-assembly is the spontaneous organization of molecules into a precise supramolecular architecture without any external guidance. Throughout nature, biomolecule self-assembly gives rise to various functional biomaterials that can perform complex tasks, such as molecular sensing and recognition, chemical synthesis, motility, and compartmentalization, as well as multi-scale hierarchical organization. There is increasing interest in biomolecule self-assembly for bottom-up fabrication of biomaterials for various technological applications.
Aspects of the disclosure relate to methods and compositions for delivering a uricase protein (also referred to as “a uricase” herein) to a subject. In some embodiments, a uricase protein is fused to a self-assembling peptide, for example one or more of a set of charge complementary self-assembling peptides.
Aspects of the disclosure relate to methods for treating diseases or disorders related to the formation of monosodium urate (MSU) crystals. In some embodiments, a disease or disorder related to the formation of monosodium urate (MSU) crystals is an acute inflammatory condition. In some embodiments, a disease or disorder related to the formation of monosodium urate (MSU) crystals is gout.
Described herein are sets of charge complementary self-assembling peptides that can be associated with (e.g., fused to) a therapeutic cargo (e.g., a uricase) and that can contain a positive peptide that can contain at least 3 amino acids (A1-A3, as set forth sequentially from C to N terminus), wherein A1, A2, and A3 can be each independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A3 can be a positively charged amino acid and at least one amino acid of A1-A3 can be a hydrophobic amino acid; and a negative peptide can include at least 3 amino acids (B1-B3), wherein B1, B2, and B3 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B3 can be a negatively charged amino acid and at least one amino acid of B1-B3 can be a hydrophobic amino acid, and wherein the positive and the negative peptide can be configured to self-assemble when mixed under a stimulating condition.
In some embodiments, the positive peptide can have at least 4 amino acids (A1-A4, as set forth sequentially from C to N terminus), wherein A1, A2, A3, and A4, are each independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A4 can be a positively charged amino acid and at least one amino acid of A1-A4 can be a hydrophobic amino acid; and wherein the negative peptide comprises at least 4 amino acids (B1-B4), wherein B1, B2, B3, and B4 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B4 can be a negatively charged amino acid and at least one amino acid of B1-B4 can be a hydrophobic amino acid, wherein the positive and the negative peptide can be configured to self-assemble when mixed under a stimulating condition.
In some embodiments, the positive peptide and the negative peptide each comprise an additional amino acid such that the positive peptide can include 5 amino acids (A1-A5 as set forth sequentially from C to N terminus), wherein A1, A2, A3, A4, and A5, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A5 can be a positively charged amino acid and at least one amino acid of A1-A5 can be a hydrophobic amino acid; and the negative peptide can include 5 amino acids (B1-B5 as set forth sequentially from C to N terminus), wherein B1, B2, B3, B4, and B5 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B5 can be a negatively charged amino acid and at least one amino acid of B1-B5 can be a hydrophobic amino acid.
In some embodiments, the positive peptide and the negative peptide can each include three additional amino acids such that the positive peptide can include: 7 amino acids (A1-A7 as set forth sequentially from C to N terminus), wherein A1, A2, A3, A4, A5, A6, and A7, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A7 can be a positively charged amino acid and at least one amino acid of A1-A7 can be a hydrophobic amino acid; and the negative peptide can include: 7 amino acids (A1-A7 as set forth sequentially from C to N terminus), wherein A1, A2, A3, A4, A5, A6, and A7, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A7 can be a positively charged amino acid and at least one amino acid of A1-A7 can be a hydrophobic amino acid. In some embodiments, the positive peptide can include at least 11 amino acids (A1-A11 as set forth sequentially from C to N terminus), wherein A1, A9, and A11 can each be a polar or a cationic amino acid, wherein A2 can be a polar amino acid, wherein A3 can be a cationic or a polar amino acid, wherein A4, A6, and A8 can each be a hydrophobic amino acid. wherein As and A7 can each be a cationic amino acid or a polar amino acid, wherein Aio can be a polar amino acid, and wherein the negative peptide can include at least 11 amino acids amino acids (B1-B11 as set forth sequentially from C to N terminus), wherein B1 and B11 can each be a polar or an anionic amino acid, wherein B2 and B10, can each be a polar amino acid, wherein B3, B5, B7, and B9, can each be an anionic amino acid or a polar amino acid, and wherein B4, B6, and B8, can each be a hydrophobic amino acid. In some embodiments, the positive peptide can have a sequence of SEQ ID NO: 1, 3, 5, 6, or 8. In some embodiments, the negative peptide can have a sequence of SEQ ID NO: 2, 4, or 7. In some embodiments, the stimulating condition can be an aqueous solution having a pH ranging from about 6.5 to about 8.5. In some embodiments, the positive peptide, the negative peptide, or the positive and the negative peptide can each further include one or more cargo polypeptides (e.g., uricase proteins) coupled to the N-terminus, the C terminus, or both the N-terminus and the C-terminus of the positive peptide, the negative peptide, or both the positive and the negative peptide. The positively charged amino acids can each be independently selected from the group consisting of: lysine, histidine, and arginine. In some embodiments, the negatively charged amino acids can each be independently selected from the group consisting of: aspartate and glutamate. In some embodiments, the hydrophobic amino acids can each be independently selected from the group consisting of: glycine, alanine, valine, leucine, methionine, isoleucine, phenylalanine, tyrosine, and tryptophan.
Also described herein are supramolecular structures that can include a positive peptide that can include at least 4 amino acids (A1-A4), as set forth sequentially from C to N terminus), wherein A1, A2, A3, and A4, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A4 can be a positively charged amino acid and at least one amino acid of A1-A4 can be a hydrophobic amino acid; and a negative peptide that can be at least 4 amino acids (B1-B4), wherein B1, B2, B3, and B4 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B4 can be a negatively charged amino acid and at least one amino acid of B1-B4 can be a hydrophobic amino acid, wherein the positive and the negative peptide can be attached to each other via electrostatic interactions. In some embodiments of a supramolecular structure, the positive peptide and the negative peptide each can include an additional amino acid such that the positive peptide can include: at least 5 amino acids (A1-A5 as set forth sequentially from C to N terminus), wherein A1, A2, A3, A4, and A5, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A5 can be a positively charged amino acid and at least one amino acid of A1-A5 can be a hydrophobic amino acid; and the negative peptide can include: at least 5 amino acids (B1-B5 as set forth sequentially from C to N terminus), wherein B1, B2, B3, B4, and Bs can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B5 can be a negatively charged amino acid and at least one amino acid of B1-B5 can be a hydrophobic amino acid. In some embodiments of a supramolecular, the positive peptide and the negative peptide can each include three additional amino acids such that the positive peptide contains: at least 7 amino acids (A1-A7 as set forth sequentially from C to N terminus), wherein A1, A2, A3, A4, A5, A6, and A7, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A7 can be a positively charged amino acid and at least one amino acid of A1-A7 can be a hydrophobic amino acid; and the negative peptide can include: at least 7 amino acids (A1-A7 as set forth sequentially from C to N terminus), wherein A1, A2, A3, A4, A5, A6, and A7, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A7 can be a positively charged amino acid and at least one amino acid of A1-A7 can be a hydrophobic amino acid. In some embodiments of a supramolecular structure, the positive peptide can include at least 11 amino acids (A1-A11 as set forth sequentially from C- to N-terminus), wherein A1, A9, and A11 are each a polar or a cationic amino acid, wherein A2 can be a polar amino acid, wherein A3 can be a cationic or a polar amino acid, wherein A4, A6, and A8 can each be a hydrophobic amino acid, wherein A5 and A7 can each be a cationic amino acid or a polar amino acid, wherein A10 can be a polar amino acid, and wherein the negative peptide can include at least 11 amino acids amino acids (B1-B11 as set forth sequentially from C to N terminus), wherein B 1 and B11 can each be a polar or an anionic amino acid, wherein B2 and B10, can each be polar amino acids, wherein B3, B5, B7, and B9, can each an anionic amino acid or a polar amino acid, and wherein B4, B6, and B8, can each be a hydrophobic amino acid. In some embodiments of the supramolecular structure, the positive peptide, the negative peptide, or the positive and the negative peptide can each further include one or more cargo polypeptides (e.g., uricase proteins) coupled to the N-terminus, the C terminus, or both the N-terminus and the C-terminus of the positive peptide, the negative peptide, or both the positive and the negative peptide.
Also provided herein are methods that can include the steps of mixing a positive peptide and a negative peptide in a solution at a pH ranging from about 6.5 to about 8.5, at least 3 amino acids (A1-A3, as set forth sequentially from C to N terminus), wherein A1, A2, and A3 can be each independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A3 can be a positively charged amino acid and at least one amino acid of A1-A3 can be a hydrophobic amino acid; and a negative peptide can include at least 3 amino acids (B1-B3), wherein B1, B2, and B3 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B3 can be a negatively charged amino acid and at least one amino acid of B1-B3 can be a hydrophobic amino acid.
In some embodiments, the positive peptide can include at least 4 amino acids (A1-A4, as set forth sequentially from C to N terminus), wherein A1, A2, A3, and A4, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A4 can be a positively charged amino acid and at least one amino acid of A1-A4 can be a hydrophobic amino acid; and wherein the negative peptide comprises at least 4 amino acids (B1-B4), wherein B1, B2, B3, and B4 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B4 can be a negatively charged amino acid and at least one amino acid of B1-B4 can be a hydrophobic amino acid. The positive peptide and the negative amino acid can each include an additional amino acid such that the positive peptide can include at least 5 amino acids (A1-A5 as set forth sequentially from C to N terminus), wherein A1, A2, A3, A4, and A5, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A5 can be a positively charged amino acid and at least one amino acid of A1-A5 can be a hydrophobic amino acid; and wherein the negative peptide can include: at least 5 amino acids (B1-B5 as set forth sequentially from C to N terminus), wherein B1, B2, B3, B4, and Bs can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B5 can be a negatively charged amino acid and at least one amino acid of B1-B5 can be a hydrophobic amino acid.
In some embodiments, the positive peptide and the negative amino acid can each include three additional amino acids such that the positive peptide includes: at least 7 amino acids (A1-A7 as set forth sequentially from C to N terminus), wherein A1, A2, A3, A4, A5, A6, and A7, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A7 can be a positively charged amino acid and at least one amino acid of A1-A7 can be a hydrophobic amino acid; and the negative peptide can include: at least 7 amino acids (A1-A7 as set forth sequentially from C to N terminus), wherein A1, A2, A3, A4, A5, A6, and A7, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A7 can be a positively charged amino acid and at least one amino acid of A1-A7 can be a hydrophobic amino acid.
In some embodiments, the positive peptide can include at least 11 amino acids (A1-A11 as set forth sequentially from C- to N-terminus), wherein A1, A9, and A11 can each be a polar or a cationic amino acid, wherein A2 can be a polar amino acid, wherein A3 can be a cationic or a polar amino acid, wherein A4, A6, and A8 can each be a hydrophobic amino acid, wherein A5 and A7 are can each be independently selected from a cationic amino acid or a polar amino acid, wherein A10 can be a polar amino acid, and wherein the negative peptide can include at least 11 amino acids amino acids (B1-B11 as set forth sequentially from C to N terminus), wherein B1 and B11 can each be independently selected from a polar or an anionic amino acid wherein B2 and B10, can each be polar amino acids, wherein B3, B5, B7, and B9, can each be independently selected from an anionic amino acid or a polar amino acid, and wherein B4, B6, and B8, can each be a hydrophobic amino acid.
In some embodiments, any of the peptides herein can comprise any of the sequences as outlined herein without histidine tags (e.g., with poly histidine tags omitted).
In some embodiments, a cargo polypeptide comprises an enzyme. In some embodiments, a cargo polypeptide comprises a uricase enzyme (e.g., a uricase component of a fusion protein), or a fragment thereof. In some embodiments, a uricase cargo polypeptide is a wild-type uricase. In some embodiments, a uricase cargo polypeptide is an animal uricase. In some embodiments, a uricase cargo polypeptide is a mammalian uricase. In some embodiments, a uricase cargo polypeptide is a human uricase.
In some embodiments, a uricase cargo polypeptide comprises an amino acid sequence with at least 70% identity to SEQ ID NO: 9 (
In some embodiments, the uricase component has an amino acid sequence that is 80-85%, 85-90%, 90-95%, or 95-100% identical to the amino sequence of SEQ ID NO: 9 or the sequence encoded by SEQ ID NO: 10. In some embodiments, the uricase component has an amino acid sequence that differs from the sequence of SEQ ID NO: 9 or the sequence encoded by SEQ ID NO: 10 by 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, and/or 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, and/or 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid deletions.
In some embodiments, a uricase cargo polypeptide is attached to a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached to a negative peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached to both a positive and negative peptide as described herein.
In some embodiments, a uricase cargo polypeptide is attached to the N-terminus of a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached to the C-terminus of a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached between the N and C termini of a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached to more than one point on a positive peptide as described herein (e.g., N-terminus, C-terminus, point in between N and C termini, or any combination thereof). In some embodiments, a uricase cargo polypeptide is attached to the N-terminus of a negative peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached to the C-terminus of a negative peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached between the N and C termini of a negative peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached to more than one point on a negative peptide as described herein (e.g., N-terminus, C-terminus, point in between N and C termini, or any combination thereof).
In some embodiments, a uricase cargo polypeptide is attached through its N-terminus to a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached through its C-terminus to a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached through a point between its N and C termini to a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached through its N-terminus to a negative peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached through its C-terminus to a negative peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached between its N and C termini to a negative peptide as described herein.
In some embodiments, a uricase cargo polypeptide is attached to a positive peptide as described herein by a linker. In some embodiments, a uricase cargo polypeptide is attached to a negative peptide as described herein by a linker.
In some embodiments, a positive and negative peptide as described herein are fused with a uricase cargo polypeptide. In some embodiments, the fusion protein comprises a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the fusion protein comprises a sequence of SEQ ID NO: 9. In some embodiments, a positive and negative peptide as described herein are fused with a uricase cargo polypeptide. In some embodiments, the fusion protein comprises a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 9 without histidine tags (e.g., with poly histidine tags omitted). In some embodiments, the fusion protein comprises a sequence of SEQ ID NO: 9 without histidine tags (e.g., with poly histidine tags omitted). In some embodiments, a positive and negative peptide as described herein are fused with a uricase cargo polypeptide. In some embodiments, the fusion protein comprises an amino acid sequence encoded by a nucleic acid sequence with at least 70% identity to the sequence of SEQ ID NO: 10. In some embodiments, the fusion protein comprises an amino acids sequence encoded by a nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the fusion protein comprises an amino acid sequence encoded by a nucleic acid sequence with at least 70% identity to the sequence of SEQ ID NO: 10 without histidine tags (e.g., with poly histidine tags omitted). In some embodiments, the fusion protein comprises an amino acid sequence encoded by a nucleic acid sequence of SEQ ID NO: 10 without histidine tags (e.g., with poly histidine tags omitted).
In some embodiments, a positive and negative peptide as described herein are fused with a uricase cargo polypeptide. In some embodiments, the fusion protein comprises an amino acid sequence encoded by a nucleic acid sequence with at least 70% identity to the sequence of SEQ ID NO: 10. In some embodiments, the fusion protein comprises a sequence encoded by SEQ ID NO: 10.
In some embodiments, the uricase component of the fusion protein has an amino acid sequence that is 80-85%, 85-90%, 90-95%, or 95-100% identical to the amino sequence of the uricase enzyme sequence of SEQ ID NO: 9 or encoded by SEQ ID NO: 10. In some embodiments, the uricase component of the fusion protein has an amino acid sequence that differs from the uricase enzyme sequence of SEQ ID NO: 9 or encoded by SEQ ID NO: 10 by 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, and/or 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, and/or 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid deletions.
In some embodiments, the peptides (e.g., positive, negative, fusion peptides) of the present disclosure are useful in treating diseases and/or disorders. In some embodiments, a disease and/or disorder is related to uric acid. In some embodiments, a disease or disorder is related to high levels of uric acid. In some embodiments, the peptides (e.g., positive, negative, fusion peptides) of the present disclosure are useful for modulating uric acid levels. In some embodiments, the disease or disorder is gout.
In some aspect, the disclosure relates to method of modulating the uric acid levels in a subject. In some embodiments, the method comprises administering any of the peptides of the present disclosure. In some embodiments, the peptides of the present disclosure reduce the levels of uric acid in a subject.
In some embodiments, the disclosure relates to methods of modulating serum levels of uric acid in a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is human. In some embodiments, the subject has, is at risk of having, or is suspected of having, gout.
Further aspects of the present disclosure will be readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
All publications and patents cited in this specification are cited to disclose and describe the methods and/or materials in connection with which the publications are cited. All such publications and patents are herein incorporated by references as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and/or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definition in the publications and patents cited that is not also expressly repeated in the instant application should not be treated as such and should not be read as defining any terms appearing in the accompanying claims. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of molecular biology, microbiology, nanotechnology, organic chemistry, biochemistry, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
Definitions
As used herein, As used herein, “about,” “approximately,” and the like, when used in connection with a numerical variable, can generally refers to the value of the variable and to all values of the variable that are within the experimental error (e.g., within the 95% confidence interval for the mean) or within +/−10% of the indicated value, whichever is greater.
As used herein, “antibody” can refer to a glycoprotein containing at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region and a light chain constant region. The VH and VL regions retain the binding specificity to the antigen and can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR). The CDRs are interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four framework regions, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
As used herein, “anti-infective” can refer to compounds or molecules that can either kill an infectious agent or inhibit it from spreading. Anti-infectives include, but are not limited to, antibiotics, antibacterials, antifungals, antivirals, and antiprotozoans.
As used herein, “aptamer” can refer to single-stranded DNA or RNA molecules that can bind to pre-selected targets including proteins with high affinity and specificity. Their specificity and characteristics are not directly determined by their primary sequence, but instead by their tertiary structure.
As used herein, “attached,” “attachment” and the like can refer to the formation of a covalent or non-covalent association (e.g. a bond) between two or more molecules or conjugation of two or more molecules. As used herein, “attached,” “attachment” and the like can refer to direct association of two or more molecules together with no intermediate molecules between those that are attached together or to the indirect attachment of two or more molecules together that is mediated via one or more linkers. Where the association is non-covalent, this can encompass charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, pi-pi stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and/or combinations thereof. Where the association is covalent, this can encompass bonds where a pair of electrons is shared between one or more atoms in each molecule involved.
As used herein, “cargo polypeptide (e.g., a uricase protein)” can refer to any peptide polypeptide that can be coupled to the N- and/or the C-terminus of a positive or negative peptide segment as provided herein. The cargo polypeptide (e.g., a uricase protein) can be coupled to the positive or negative peptide segment using standard molecular biology and recombinant DNA technology techniques. For example, a fusion peptide segment containing the cargo polypeptide (e.g., a uricase protein), can be produced from a recombinant DNA construct containing DNA encoding the negative or positive peptide segment operatively coupled with DNA encoding the cargo polypeptide (e.g., a uricase protein) and any optional linker. The DNA encoding the negative or positive peptide segment can be operatively coupled to the cargo polypeptide (e.g., a uricase protein) and any optional linker such that the cargo polypeptide (e.g., a uricase protein) is translated in-frame with negative or positive peptide segment. The cargo polypeptide (e.g., a uricase protein) can be a reporter protein (e.g. a fluorescent protein), a pharmaceutically relevant protein (a protein that can be effective to prevent or treat a disease or symptom thereof in a subject), a cell- or tissue-targeting protein, an antibody or fragment thereof, an antigen, an enzyme, a growth factor, a cytokine, a chemokine, an extracellular matrix protein or fragment thereof, a transmembrane receptor or fragment thereof, a toxin or a fragment thereof, and a transcription factor or fragment thereof.
As used herein, “cDNA” can refer to a DNA sequence that is complementary to a RNA transcript in a cell. It is a man-made molecule. Typically, cDNA is made in vitro by an enzyme called reverse-transcriptase using RNA transcripts as templates.
As used herein, “chemotherapeutic agent” or “chemotherapeutic” can refer to a therapeutic agent utilized to prevent or treat a cancer.
As used herein, “coupled” can refer to the direct or indirect (e.g. via a linker) attachment of two or more molecules and/or compounds.
As used herein, “concentrated” can refer to a molecule or population thereof, including but not limited to a polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, that is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than that of its naturally occurring counterpart.
As used herein, “control” can refer to an alternative subject or sample used in an experiment for comparison purpose and included to minimize or distinguish the effect of variables other than an independent variable.
As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” can generally refer to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. RNA may be in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), or ribozymes.
As used herein, “DNA molecule” can include nucleic acids/polynucleotides that are made of DNA. As used herein, “expression” can refer to the process by which polynucleotides are transcribed into RNA transcripts. In the context of mRNA and other translated RNA species, “expression” also refers to the process or processes by which the transcribed RNA is subsequently translated into peptides, polypeptides, or proteins. Techniques and methods appropriate for determining an amount of expression will be instantly appreciated by those of ordinary skill in the art and include, but are not limited to, western blotting for the transcribed protein, pyro-sequencing, polymerase chain reaction (PCR) based methods (e.g. reverse transcription PCR and quantitative real-time PCR), and mass-spectrometric based analysis.
As used herein, the term “encode” can refer to principle that DNA can be transcribed into RNA, which can then be translated into amino acid sequences that can form proteins.
As used herein, the term “enzyme” can refer to any protein that can catalyze a chemical or biochemical reaction.
As used herein, the term “fluorescent protein” can refer to any protein that can produce fluorescence when excited by the proper excitation wavelength or by other resonance transfer of energy, e.g. FRET. The term “fluorescent protein” includes, but is not limited to, all types and variants of current fluorescent proteins known in the art, include but not limited to green fluorescent proteins, yellow fluorescent proteins, blue fluorescent proteins, cyan fluorescent proteins, red fluorescent proteins, etc. The term “fluorescent protein” include, without limitation, all forms of such proteins as they are routinely modified, derivitized, and generally known to those of ordinary skill in the art. For example “green fluorescent protein” includes, without limitation, enhanced green fluorescent protein (eGFP), redox sensitive GFP (roGFP), and all color mutants. The amino acid sequences and other characteristics of suitable fluorescent proteins will be known to those of skill in the art and are within the scope of this disclosure.
As used herein, “gene” can refer to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism. The term gene can refer to both translated and untranslated regions of a subject's genome.
As used herein, “identity,” “identical to”, and the like can refer to the relationship between two or more nucleotide or polypeptide sequences, as determined by comparing the sequences. In the art, “identity” also refers to the degree of sequence relatedness between nucleotide or polypeptide as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math. 1988, 48: 1073. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 1970, 48: 443-453,) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present disclosure, unless stated otherwise.
As used herein, “immunomodulator,” can refer to an agent, such as a therapeutic agent, which is capable of modulating or regulating one or more immune function or response.
As used herein, “isolated” can mean separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. A non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, do not require “isolation” to distinguish it from its naturally occurring counterpart.
As used herein, “linker” can refer to any amino acid or peptide that can be optionally included between a positive or negative peptide segment and a cargo protein. The linker can range in length from about 1 to about 60 amino acids in length. The linker can be composed of any of the 20 naturally occurring amino acids and be present in any arrangement that does not otherwise perturb the peptide segment assembly or cargo protein activity.
As used herein, “mammal,” for the purposes of treatments, can refer to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as, but not limited to, dogs, horses, cats, and cows.
The term “molecular weight”, as used herein, can generally refer to the mass or average mass of a material. If a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in various ways including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (Mw) as opposed to the number-average molecular weight (Mn). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.
As used herein, “negative control” can refer to a “control” that is designed to produce no effect or result, provided that all reagents are functioning properly and that the experiment is properly conducted. Other terms that are interchangeable with “negative control” include “sham,” “placebo,” and “mock.”
As used herein, “nucleic acid” and “polynucleotide” generally refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. “Polynucleotide” and “nucleic acids” also encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. For instance, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. “Polynucleotide” and “nucleic acids” also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, but contain the same bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or “polynucleotide” as that term is intended herein. As used herein, “nucleic acid sequence” and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined above.
As used herein, “operatively linked” can indicate that the regulatory sequences useful for expression of the coding sequences of a nucleic acid are placed in the nucleic acid molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same term can be applied to the arrangement of coding sequences and/or transcription control elements (e.g. promoters, enhancers, and termination elements), and/or selectable markers in an expression vector. “Operatively linked” can also refer to an indirect attachment (i.e. not a direct fusion) of two or more polynucleotide sequences or polypeptides to each other via a linking molecule (also referred to herein as a linker).
As used herein, “nucleic acid sequence” and “oligonucleotide” can also encompass a nucleic acid and polynucleotide as defined above.
As used herein, “overexpressed” or “overexpression” can refer to an increased expression level of an RNA or protein product encoded by a gene as compared to the level of expression of the same or different RNA or protein product in the same cell type or population thereof or a different cell type, normal cell, and/or control cell or any population thereof. Typically, expression is stated as an increase in the fold-change when determined relatively. The amount of overexpression can also be expressed as a quantitative amount. Techniques are known to those of ordinary skill in the art that can determine the absolute number of molecules of RNA or protein product. Therefore, overexpression can be stated as an amount of the RNA molecule or protein product of interest as compared to a control RNA or protein product (e.g. GAPDH, beta-actin) in the same cell or population thereof, or a different cell or population there of (e.g. different cell type, normal cell, and/or control cell or any population thereof). In the case of overexpression the absolute amount of the RNA molecule or protein product of interest will be less than that of the other RNA or protein product in the same or different cell type that is being used for comparison.
As used herein “peptide” refers to chains of at least 2 amino acids that are short, relative to a protein or polypeptide.
As used herein, “pharmaceutical formulation” refers to the combination of an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo. As used herein, “active ingredient” refers to a component of a composition that effects a therapeutic function (e.g., reducing acid uric levels, or dissolving uric acid crystals)
As used herein, “pharmaceutically acceptable carrier or excipient” can refer to a carrier or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, non-toxic, and is neither biologically or otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier or excipient” as used in the specification and claims includes both one and more than one such carrier or excipient.
As used herein, “pharmaceutically acceptable salt” can refer to any acid or base addition salt whose counter-ions are non-toxic to the subject to which they are administered in pharmaceutical doses of the salts.
As used herein, “plasmid” as used herein can refer to a non-chromosomal double-stranded DNA sequence including an intact “replicon” such that the plasmid is replicated in a host cell.
As used herein, “positive control” can refer to a “control” that is designed to produce the desired result, provided that all reagents are functioning properly and that the experiment is properly conducted.
As used herein, “protein” as used herein can refer to a molecule composed of one or more chains of amino acids in a specific order. The term protein is used interchangeable with “polypeptide.” The order is determined by the base sequence of nucleotides in the gene coding for the protein. Proteins are required for the structure, function, and regulation of the body's cells, tissues, and organs.
As used herein, “preventative” and “prevent” can refer to hindering or stopping a disease or condition before it occurs, even if undiagnosed, or while the disease or condition is still in the sub-clinical phase.
As used herein, “purified” or “purify” can be used in reference to a nucleic acid sequence, peptide, or polypeptide that has increased purity relative to the natural environment.
As used herein, the term “recombinant” or “engineered” can generally refer to a non-naturally occurring nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and/or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a fusion protein (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g., a nucleic acid and a constitutive promoter), etc. Recombinant or engineered can also refer to the polypeptide encoded by the recombinant nucleic acid. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.
As used herein, “separated” can refer to the state of being physically divided from the original source or population such that the separated compound, agent, particle, or molecule can no longer be considered part of the original source or population.
As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human).
As used herein, “substantially pure” can mean an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises about 50 percent of all species present. Generally, a substantially pure composition will comprise more than about 80 percent of all species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single species.
As used herein, “therapeutic” can refer to treating, healing, and/or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect. A “therapeutically effective amount” can therefore refer to an amount of a compound that can yield a therapeutic effect.
As used herein, the terms “treating” and “treatment” can refer generally to obtaining a desired pharmacological and/or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term “treatment” as used herein covers any treatment of a disease, in a mammal, particularly a human, and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, e.g., arresting its development; and (c) relieving the disease, e.g., mitigating or ameliorating the disease and/or its symptoms or conditions. The term “treatment” as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and/or those in which the disorder is to be prevented.
As used herein, “underexpressed” or “underexpression” can refer to a decreased expression level of an RNA or protein product encoded by a gene as compared to the level of expression of the same or different RNA or protein product in the same cell type or population thereof or a different cell type, normal cell, and/or control cell or any population thereof. Typically, expression is stated as an increase in the fold-change when determined relatively. The amount of underexpression can also be expressed as a quantitative amount. Techniques are known to those of ordinary skill in the art that can determine the absolute number of molecules of RNA or protein product. Therefore, underexpression can be stated as an amount of the RNA molecule or protein product of interest as compared to a control RNA or protein product (e.g. GAPDH, beta-actin) in the same cell or population thereof, or a different cell or population there of (e.g. different cell type, normal cell, and/or control cell or any population thereof). In the case of underexpression the absolute amount of the RNA molecule or protein product of interest will be less than that of the other RNA or protein product in the same or different cell type that is being used for comparison.
As used herein, “variant” refers to a polypeptide that differs from a reference polypeptide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. “Variant” can also include functional and structural variants.
As used herein, the term “vector” or is used in reference to a vehicle used to introduce an exogenous nucleic acid sequence into a cell. A vector may include a DNA molecule, linear or circular (e.g. plasmids), which includes a segment encoding a polypeptide of interest operatively linked to additional segments that provide for its transcription and translation upon introduction into a host cell or host cell organelles. Such additional segments may include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from yeast or bacterial genomic or plasmid DNA, or viral DNA, or may contain elements of both.
Aspects of the present disclosure relate to methods and compositions for delivering a uricase to a subject. In some embodiments, a uricase protein is fused to a self-assembling peptide, for example one or more of a set of charge complementary self-assembling peptides.
Aspects of the disclosure relate to methods comprising the administration of a uricase (e.g., in the form of a fusion protein comprising a self-assembling peptide) for treating diseases or disorders related to the formation or deposition of monosodium urate (MSU) crystals. MSU or uric acid is the etiological agent in gout. In some embodiments, a disease or disorder related to the formation or deposition of monosodium urate (MSU) crystals is an acute inflammatory condition. In some embodiments, a disease or disorder related to the formation or deposition of monosodium urate (MSU) crystals is gout. In some embodiments, the disease or disorder is hyperuricemia. Accordingly, in some embodiments, a uricase (e.g., in the form of a fusion protein comprising a self-assembling peptide) is administered to a subject (e.g., a human) in need thereof (e.g., a subject having a predisposition to, having one or more symptoms of, and/or diagnosed as having a disease or condition associated with the formation of MSU crystals) in an amount sufficient to prevent, reduce, delay the onset or progression of, and/or alleviate one or more symptoms of the disease or condition. In some embodiments, a composition (e.g., a composition administered to a subject) comprises a uricase (e.g., in the form of a fusion protein comprising a self-assembling peptide) in a pharmaceutically acceptable formulation. In some embodiments, a composition comprises a solution of a uricase (e.g., of a uricase fused to a self-assembling peptide), for example in a pharmaceutically acceptable buffer and/or with one or more pharmaceutically acceptable salts or other agents.
Gout, or gouty arthritis, is the most common arthropathy associated with crystal formation, and the most common inflammatory arthritis overall. In gout, deposition of MSU crystals within joints and connective tissue engenders highly inflammatory but localized responses. The susceptibility to form MSU crystals is a consequence of excessive blood levels of soluble urate, one of the final products of the metabolic breakdown of purine nucleotides. Hyperuricemia is typically defined as occurring above the saturation point of MSU, at which point the risk of crystallization increases. Notably, it has been shown that MSU crystals induce the release of inflammatory cytokines such as interleukin (IL)-1β. MSU crystals were shown to activate caspase-1, which is required for IL-1β maturation, through theNLRP3 (also known as NALP3 or cryopyrin) inflammasome. The NLRP3 inflammasome is an intracellular multi-protein caspase-1-activating complex comprising the NLR protein NLRP3 and the adaptor ASC, and plays a central role in innate immunity. In some embodiments, uricase (e.g., in the form of a fusion protein comprising a self-assembling peptide) can be administered to a subject (e.g., a human subject) to treat one or more of these symptoms.
In some embodiments, a uricase enzyme or protein is fused to a self-assembling peptide. Self-assembly is the spontaneous organization of molecules into a precise supramolecular architecture without any external guidance. Throughout nature, biomolecule self-assembly gives rise to various functional biomaterials that can perform complex tasks, such as molecular sensing and recognition, chemical synthesis, motility, and compartmentalization, as well as multi-scale hierarchical organization. Thus, there is increasing interest in biomolecule self-assembly for bottom-up fabrication of biomaterials for medical and technological applications.
Peptides and peptide analogs that can form elongated nanofibers by adopting β-sheet secondary structures can be advantageous for creating supramolecular biomaterials. Peptide self-assembly into β-sheet nanofibers has been demonstrated to occur under mild aqueous conditions with the resultant nanofibers often undergoing sol-gel transition into macroscopic, physically-crosslinked hydrated structures (i.e. “hydrogels”) above a critical concentration. The amino acid sequence of β-sheet fibrillizing peptides can be systematically modified to tailor nanofiber morphology, gelation properties, and stimuli-responsiveness according to the intended application. In addition, β-sheet peptide nanofibers can be fabricated into micron-sized gels using various conventional polymer-processing techniques. Finally, β-sheet peptide nanofibers are well-suited for use in biomedical applications because they are composed of natural amino acids that can be metabolized, they are often biocompatible with cells and tissues, and they typically elicit minimal inflammation and weak or no adaptive immunity directed against the peptide itself, despite being foreign to the host.
To be useful for medical or technological applications, supramolecular biomaterials must often demonstrate functional capabilities beyond robust self-assembly and biocompability. Since synthetic peptides designed to self-assemble typically lack any additional functional features, ligands, fluorophores, antigens, drugs, or other bioactive compounds are often installed into supramolecular biomaterials to impart functional capabilities. Supramolecular biomaterials can be functionalized post-assembly via various covalent and non-covalent grafting approaches, However, current supramolecular biomaterials suffer from several deficiencies. They often rely on complex, inefficient, or poorly reproducible reactions and may only provide transient functionality within complex biological environments.
Alternatively, functional capabilities can be installed directly into supramolecular biomaterials via covalent fusion of a functional molecule and a self-assembling peptide in the pre-assembled state (i.e. “fusion peptides”). Installing functional molecules with more sophisticated properties, such as catalysis, selective molecular recognition, tunable visible light fluorescence, or precise antigen conformation, could impart unprecedented functional capabilities into supramolecular biomaterials already finding use in biomedical and biotechnological applications. Current self-assembling peptides rely on covalent grafting of folded proteins to the self-assembled peptides. Covalent fusions of folded proteins and self-assembling can provide many advantages over covalent grafting, particularly from a material fabrication perspective.
Despite the clear advantages of a covalent fusion approach, there are significant challenges inherent to covalent fusions from a synthesis perspective that have hindered the development and adoption of this approach in the context of self-assembling peptides. In particular, conventional peptide synthesis and purification cannot be adapted to produce protein-peptide fusions. Instead, fusion proteins are typically expressed from recombinant DNA using translational machinery housed within microbes or other cellular hosts. The environment within the cytoplasm of expression hosts is often favorable for β-sheet peptide self-assembly, however, which can hinder recovery of soluble, bioactive fusion proteins by inducing aggregation or misfolding. Self-assembly domains with slow fibrillization kinetics or pH-responsive assembly can address this limitation. However, the former requires assembly kinetics that are well-behaved under various expression conditions and with a wide variety of fusion protein partners, while the latter necessitates proteins with folded conformations that are not pH-sensitive.
With the deficiencies in the current technologies in mind, described herein are self-assembling peptides that can be engineered to resist assembly until acted upon by a stimulus that can be applied under neutral, near-physiological conditions that support maintaining protein folding and function. In some embodiments, charge-complementary peptides can contain one or more ionizable residues that renders the self-assembly of the charge-complementary peptides energetically unfavorable until the two peptides are combined in solution.
Other compositions, compounds, methods, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.
Co-Assembly Peptides and Nanostructures
Charge Complementary Peptide Segments
As shown in
A positive peptide can include at least 3 amino acids (A1-A3 as set forth sequentially from C to N terminus), wherein A1, A2, and A3 can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A3 can be a positively charged amino acid and at least one amino acid of A1-A3 can be a hydrophobic amino acid. A negative peptide can include at least 3 amino acids (B1-B3 as set forth sequentially from C to N terminus), wherein B1, B2, and B3 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B3 can be a negatively charged amino acid and at least one amino acid of B1-B3 can be a hydrophobic amino acid. When one or more positive and one or more negative peptide segments are mixed together under stimulating conditions, they can self-assemble to form supramolecular structures. The 3 amino acids (A1-A3) can form a core of a larger peptide and can have additional amino acids at the N- and/or C-terminus of the 3 amino acid (A1-A3) core. The additional amino acids can range in number from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more. The additional amino acids can each be selected from natural and unnatural amino acids, a cationic amino acid, an anionic amino acid, a hydrophobic amino acid, or a polar amino acid. In some embodiments the amino acids can be modified, such as iodinated, fluorinated, or otherwise labeled.
A positive peptide can include at least 4 amino acids (A1-A4 as set forth sequentially from C to N terminus), wherein A1, A2, A3, and A4, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A4 can be a positively charged amino acid and at least one amino acid of A1-A4 can be a hydrophobic amino acid. A negative peptide can include at least 4 amino acids (B1-B4 as set forth sequentially from C to N terminus), wherein B1, B2, B3, and B4 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B4 can be a negatively charged amino acid and at least one amino acid of B1-B4 can be a hydrophobic amino acid. When one or more positive and one or more negative peptide segments are mixed together under stimulating conditions, they can self-assemble to form supramolecular structures. The 4 amino acids (A1-A4) can form a core of a larger peptide and can have additional amino acids at the N- and/or C-terminus of the 4 amino acid (A1-A4) core. The additional amino acids can range in number from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more. The additional amino acids can each be selected from natural and unnatural amino acids, a cationic amino acid, an anionic amino acid, a hydrophobic amino acid, or a polar amino acid. In some embodiments the amino acids can be modified, such as iodinated, fluorinated, or otherwise labeled.
A positive peptide can include at least 5 amino acids (A1-A5 as set forth sequentially from C to N terminus), wherein A1, A2, A3, A4, and A5, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A5 can be a positively charged amino acid and at least one amino acid of A1-A5 can be a hydrophobic amino acid. A negative peptide can include at least 5 amino acids (B1-B5 as set forth sequentially from C to N terminus), wherein B1, B2, B3, B4, and B5 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B5 can be a negatively charged amino acid and at least one amino acid of B1-B5 can be a hydrophobic amino acid. When one or more positive and one or more negative peptide segments are mixed together under stimulating conditions, they can self-assemble to form supramolecular structures. The 5 amino acids (A1-A5) can form a core of a larger peptide and can have additional amino acids at the N- and/or C- terminus of the 5 amino acid (A1-A5) core. The additional amino acids can range in number from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more. The additional amino acids can each be selected from natural and unnatural amino acids, a cationic amino acid, an anionic amino acid, a hydrophobic amino acid, 10 or a polar amino acid. In some embodiments the amino acids can be modified, such as iodinated, fluorinated, or otherwise labeled.
A positive peptide can include at least 7 amino acids (A1-A7 as set forth sequentially from C to N terminus), wherein A1, A2, A3, A4, A5, A6, and A7, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A7 can be a positively charged amino acid and at least one amino acid of A1-A7 can be a hydrophobic amino acid. A negative peptide can include at least 7 amino acids (B1-B7 as set forth sequentially from C to N terminus), wherein B1, B2, B3, B4, B5, B6, and B7 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B7 can be a negatively charged amino acid and at least one amino acid of B1-B7 can be a hydrophobic amino acid. When one or more positive and one or more negative peptide segments are mixed together under stimulating conditions, they can self-assemble to form supramolecular structures. The 7 amino acids (A1-A7) can form a core of a larger peptide and can have additional amino acids at the N- and/or C-terminus of the 5 amino acid (A1-A7) core. The additional amino acids can range in number from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more. The additional amino acids can each be selected from natural and unnatural amino acids, a cationic amino acid, an anionic amino acid, a hydrophobic amino acid, or a polar amino acid. In some embodiments the amino acids can be modified, such as iodinated, fluorinated, or otherwise labeled.
The positively charged amino acids (cationic amino acids) can each be independently selected from the group of lysine, histidine, and arginine. The negatively charged amino acids (anionic amino acids) can each be independently selected from the group of aspartate and glutamate. The hydrophobic amino acids can each be independently selected from the group consisting of: glycine, alanine, valine, leucine, methionine, isoleucine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the positive and/or negative peptide segment can further include a cargo polypeptide (e.g., a uricase protein) coupled to the N and/or the C terminus of the peptide. Cargo polypeptides (e.g., uricase proteins) are discussed in greater detail elsewhere herein.
In some embodiments, the peptide having a net positive charge can have at least 11 amino acids (A1-A11 as set forth sequentially from C to N terminus), wherein A1, A9, and A11 can each be a polar or a cationic amino acid, wherein A2 can be a polar amino acid, wherein A3 can be a cationic or a polar amino acid, wherein A4, A6, and A8 can each be a hydrophobic amino acid, wherein A5 and A7 can each be a cationic amino acid or a polar amino acid, and wherein A10 can be a polar amino acid.
In some embodiments, the negative peptide can be composed of at least 11 amino acids (B1-B11 as set forth sequentially from C to N terminus), wherein B1 and B11 can each be a polar or an anionic amino acid, wherein B2 and B10, can each be polar amino acids, wherein B3, B5, B7, and B9, can each be an anionic or a polar amino acid, and wherein B4, B6, and B8, can each be hydrophobic amino acids.
Suitable cationic amino acids can have positively charged side (or “R” groups) groups and can include, but are not limited to, lysine, arginine, and histidine. Suitable anionic amino acids can have negatively charged side groups and can include, but are not limited to, aspartate and glutamate. Suitable polar amino acids can have polar, uncharged side groups, and can include, but are not limited to, serine, threonine, cysteine, proline, asparagine, and glutamine. Suitable hydrophobic amino acids can have nonpolar, aliphatic or aromatic side groups and can include, but are not limited to, glycine, alanine, valine, leucine, methionine, isoleucine, phenylalanine, tyrosine, and tryptophan. All sequences resulting from employing the general scheme shown in
In some embodiments, including but not limited to those shown in e.g.
The positive and negative peptide segments can be produced from DNA that can encode the positive and negative peptide segments. Based on the amino acid sequences provided herein, one of ordinary skill in the art will know techniques and methods that will enable them to generate suitable coding DNA sequences for the peptide segments. In some embodiments, the DNA that can encode the positive and negative peptide segments can be codon optimized for expression in a particular cell type, such as E. coli. Codon optimization techniques will be appreciated by those of skill in the art. The peptide segment encoding DNA can be included in a suitable expression vector. Suitable expression vectors will be appreciated by those of ordinary skill in the art. In some embodiments, the expression vector can also express genes that can result in more efficient and/or accurate protein folding and other post-translation modifications. Such expression vectors will be appreciated by those of ordinary skill in the art. The expression vectors can be introduced into a suitable cells and the polypeptides can be produced by expression in the cells and harvested using techniques generally known in the art.
Cargo Polypeptide Fusion Charge Complementary Peptide Segments
As shown in
The cargo polypeptide (e.g., a uricase protein) can be coupled to the positive or negative peptide segment using standard molecular biology and recombinant DNA technology techniques. The cargo polypeptide (e.g., a uricase protein) fusion charged complementary peptide segments can be produced from DNA that can encode the cargo polypeptide (e.g., a uricase protein) charge complementary peptide segments. Based on the amino acid sequences provided herein, one of ordinary skill in the art will know techniques and methods that will enable them to generate suitable coding DNA sequences for the cargo polypeptide (e.g., a uricase protein) fusion charged complementary peptide segments. In some embodiments, the DNA that can encode the cargo polypeptide (e.g., a uricase protein) fusion charged complementary peptide segments can be codon optimized for expression in a particular cell type, such as E. coli. Codon optimization techniques will be appreciated by those of skill in the art. The cargo polypeptide (e.g., a uricase protein) fusion charged complementary peptide segment encoding DNA can be included in a suitable expression vector. Suitable expression vectors will be appreciated by those of ordinary skill in the art. In some embodiments, the expression vector can also express genes that can result in more efficient and/or accurate protein folding and other post-translation modifications. Such expression vectors will be appreciated by those of ordinary skill in the art. The expression vectors can be introduced into a suitable cells and the polypeptides can be produced by expression in the cells and harvested using techniques generally known in the art.
For example a fusion peptide segment containing the cargo polypeptide (e.g., a uricase protein), can be produced from a recombinant DNA construct containing DNA encoding the negative or positive peptide segment operatively coupled with DNA encoding the cargo polypeptide (e.g., a uricase protein) and any optional linker. The DNA encoding the negative or positive peptide segment can be operatively coupled to the cargo polypeptide (e.g., a uricase protein) and any optional linker such that the cargo polypeptide (e.g., a uricase protein) is translated in-frame with negative or positive peptide segment.
The cargo polypeptide (e.g., a uricase protein) can be a reporter protein (e.g. a fluorescent protein), a pharmaceutically relevant protein (a protein that can be effective to prevent or treat a disease or symptom thereof in a subject), a cell- or tissue-targeting protein, an antibody or fragment thereof, enzyme, growth factor, cytokine, chemokine, extracellular matrix protein or fragment thereof, structural protein or fragment thereof, a transmembrane protein or fragment thereof, a transcription factor or fragment thereof, and/or an antigen. The amino acid sequences and/or coding DNA sequences of the cargo polypeptides (e.g., uricase proteins) can be appreciated by those of skill in the art.
In some embodiments, a cargo polypeptide comprises an enzyme. In some embodiments, a cargo polypeptide comprises a uricase enzyme (e.g., a uricase component of a fusion protein), or a fragment thereof. In some embodiments, a uricase cargo polypeptide is a wild-type uricase. In some embodiments, a uricase cargo polypeptide is an animal uricase. In some embodiments, a uricase cargo polypeptide is a mammalian uricase. In some embodiments, a uricase cargo polypeptide is a human uricase.
In some embodiments, a positive and negative peptide as described herein are fused with a uricase cargo polypeptide. In some embodiments, the fusion protein comprises a sequence with at least 70% (e.g., 70-80%, 80-90%, 90-95%. 95-100%) identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the fusion protein comprises a sequence of SEQ ID NO: 9. In some embodiments, a positive and negative peptide as described herein are fused with a uricase cargo polypeptide. In some embodiments, the fusion protein comprises a sequence with at least 70% (e.g., 70-80%, 80-90%, 90-95%. 95-100%) identity to the amino acid sequence of SEQ ID NO: 9 without histidine tags (e.g., with poly histidine tags omitted). In some embodiments, the fusion protein comprises a sequence of SEQ ID NO: 9 without histidine tags (e.g., with poly histidine tags omitted).
In some embodiments, a positive and negative peptide as described herein are fused with a uricase cargo polypeptide. In some embodiments, the fusion protein comprises an amino acid sequence encoded by a nucleic acid sequence with at least 70% (e.g., 70-80%, 80-90%, 90-95%. 95-100%) identity to the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the fusion protein comprises an amino acid sequence encoded by SEQ ID NO: 10. In some embodiments, a positive and negative peptide as described herein are fused with a uricase cargo polypeptide. In some embodiments, the fusion protein comprises an amino acid sequence encoded by a nucleic acid sequence with at least 70% (e.g., 70-80%, 80-90%, 90-95%. 95-100%) identity to the nucleic acid sequence of SEQ ID NO: 10 without histidine tags coding sequences (e.g., with poly histidine tags coding sequences omitted). In some embodiments, the fusion protein comprises a sequence encoded by SEQ ID NO: 10 without histidine tags (e.g., with poly histidine tags omitted).
In some embodiments, the uricase component of the fusion protein has an amino acid sequence that is 80-85%, 85-90%, 90-95%, or 95-100% identical to the amino sequence of the uricase portion of SEQ ID NO: 9 or encoded by SEQ ID NO: 10. In some embodiments, the uricase component of the fusion protein has an amino acid sequence that differs from the sequence of the uricase portion of SEQ ID NO: 9 or encoded by SEQ ID NO: 10 by 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, and/or 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, and/or 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid deletions. In some embodiments, the uricase component of the fusion protein has the amino acid sequence of the uricase portion of SEQ ID NO: 9 or encoded by SEQ ID NO: 10.
In some embodiments, a uricase cargo polypeptide is attached to a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached to a negative peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached to both a positive and negative peptide as described herein.
In some embodiments, a uricase cargo polypeptide is attached to the N-terminus of a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached to the C-terminus of a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached between the N and C termini of a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached to more than one point on a positive peptide as described herein (e.g., N-terminus, C-terminus, point in between N and C termini, or any combination thereof). In some embodiments, a uricase cargo polypeptide is attached to the N-terminus of a negative peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached to the C-terminus of a negative peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached between the N and C termini of a negative peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached to more than one point on a negative peptide as described herein (e.g., N-terminus, C-terminus, point in between N and C termini, or any combination thereof).
In some embodiments, a uricase cargo polypeptide is attached through its N-terminus to a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached through its C-terminus to a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached through a point between its N and C termini to a positive peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached through its N-terminus to a negative peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached through its C-terminus to a negative peptide as described herein. In some embodiments, a uricase cargo polypeptide is attached between its N and C termini to a negative peptide as described herein.
In some embodiments, a uricase is attached to a positive peptide as described herein by a linker. In some embodiments, a uricase cargo polypeptide is attached to a negative peptide as described herein by a linker.
Co-Assembly Peptide Nanostructures
Generally, the charge complementary segments do not self-assemble until both the positive and the negative charge complementary segments are present together under stimulating conditions. In some embodiments, the stimulating conditions can be incubation and/or placement in a solutions (e.g. an aqueous solution) at about a neutral or near physiological pH. In some embodiments, the pH of the solution can range from about 6.5 to about 8.5. Once stimulated, the ionizable amino acids are ionized and the charge complementary segments self-assemble into beta sheets with alternating positive and negative segments. The charge complementary peptides (with and/or without a cargo polypeptide (e.g., a uricase protein)) can self-assemble into structures, such as nanofibrillar hydrogels, nanofibers, microparticles, or nanoparticles depending on the concentration of the complementary charged peptide segments with no cargo polypeptide (e.g., a uricase protein) and concentration of positive and negative co-assembly cargo polypeptide (e.g., a uricase protein) fused peptide segments present in the mixture prior to self-assembly.
The co-assembly peptides and resulting nanostructures can be used to endow other supramolecular materials with functional properties of the cargo polypeptides (e.g., uricase proteins) that can be coupled to the co-assembly peptides. The nanostructures can be optionally incorporated into other supermolecular biomaterials and compositions including, but not limited to, hydrogels, synthetic polymer matrices or network, natural polymer matrices or networks, composite networks of natural and synthetic polymers, polymer nanoparticles, and/or polymer microparticles.
Provided herein are supramolecular structures that can be composed of one or more positive peptides and one or more negative peptides that can be attached to each other via e.g. an electrostatic interactions. A positive peptide can include at least 3 amino acids (A1-A3 as set forth sequentially from C to N terminus), wherein A1, A2, and A3 can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A3 can be a positively charged amino acid and at least one amino acid of A1-A3 can be a hydrophobic amino acid. A negative peptide can include at least 3 amino acids (B1-B3 as set forth sequentially from C to N terminus), wherein B1, B2, and B3 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B3 can be a negatively charged amino acid and at least one amino acid of B1-B3 can be a hydrophobic amino acid. When one or more positive and one or more negative peptide segments are mixed together under stimulating conditions, they can self-assemble to form supramolecular structures. The 3 amino acids (A1-A3) can form a core of a larger peptide and can have additional amino acids at the N- and/or C-terminus of the 4 amino acid (A1-A3) core. The additional amino acids can range in number from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more. The additional amino acids can each be selected from natural and unnatural amino acids, a cationic amino acid, an anionic amino acid, a hydrophobic amino acid, or a polar amino acid. In some embodiments the amino acids can be modified, such as iodinated, fluorinated, or otherwise labeled.
A positive peptide can include at least 4 amino acids (A1-A4 as set forth sequentially from C to N terminus), wherein A1, A2, A3, and A4, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A4 can be a positively charged amino acid and at least one amino acid of A1-A4 can be a hydrophobic amino acid. A negative peptide can include at least 4 amino acids (B1-B4 as set forth sequentially from C to N terminus), wherein B1, B2, B3, and B4 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B4 can be a negatively charged amino acid and at least one amino acid of B1-B4 can be a hydrophobic amino acid. When one or more positive and one or more negative peptide segments are mixed together under stimulating conditions, they can self-assemble to form supramolecular structures. The 4 amino acids (A1-A4) can form a core of a larger peptide and can have additional amino acids at the N- and/or C-terminus of the 4 amino acid (A1-A4) core. The additional amino acids can range in number from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more. The additional amino acids can each be selected from natural and unnatural amino acids, a cationic amino acid, an anionic amino acid, a hydrophobic amino acid, or a polar amino acid. In some embodiments the amino acids can be modified, such as iodinated, fluorinated, or otherwise labeled.
A positive peptide can include at least 5 amino acids (A1-A5 as set forth sequentially from C to N terminus), wherein A1, A2, A3, A4, and A5, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A5 can be a positively charged amino acid and at least one amino acid of A1-A5 can be a hydrophobic amino acid. A negative peptide can include at least 5 amino acids (B1-B5 as set forth sequentially from C to N terminus), wherein B1, B2, B3, B4, and B5 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B5 can be a negatively charged amino acid and at least one amino acid of B1-B5 can be a hydrophobic amino acid. When one or more positive and one or more negative peptide segments are mixed together under stimulating conditions, they can self-assemble to form supramolecular structures. The 5 amino acids (A1-A5) can form a core of a larger peptide and can have additional amino acids at the N- and/or C-terminus of the 5 amino acid (A1-A5) core. The additional amino acids can range in number from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more. The additional amino acids can each be selected from natural and unnatural amino acids, a cationic amino acid, an anionic amino acid, a hydrophobic amino acid, or a polar amino acid. In some embodiments the amino acids can be modified, such as iodinated, fluorinated, or otherwise labeled.
A positive peptide can include at least 7 amino acids (A1-A7 as set forth sequentially from C to N terminus), wherein A1, A2, A3, A4, A5, A6, and A7, can each be independently selected from a positively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of A1-A7 can be a positively charged amino acid and at least one amino acid of A1-A7 can be a hydrophobic amino acid. A negative peptide can include at least 7 amino acids (B1-B7 as set forth sequentially from C to N terminus), wherein B1, B2, B3, B4, B5, B6, and B7 can each be independently selected from a negatively charged amino acid and a hydrophobic amino acid, and wherein at least one amino acid of B1-B7 can be a negatively charged amino acid and at least one amino acid of B1-B7 can be a hydrophobic amino acid. When one or more positive and one or more negative peptide segments are mixed together under stimulating conditions, they can self-assemble to form supramolecular structures. The 7 amino acids (A1-A7) can form a core of a larger peptide and can have additional amino acids at the N- and/or C-terminus of the 5 amino acid (A1-A7) core. The additional amino acids can range in number from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more. The additional amino acids can each be selected from natural and unnatural amino acids, a cationic amino acid, an anionic amino acid, a hydrophobic amino acid, or a polar amino acid. In some embodiments the amino acids can be modified, such as iodinated, fluorinated, or otherwise labeled.
The positively charged amino acids (cationic amino acids) can each be independently selected from the group of lysine, histidine, and arginine. The negatively charged amino acids (anionic amino acids) can each be independently selected from the group of aspartate and glutamate. The hydrophobic amino acids can each be independently selected from the group consisting of: glycine, alanine, valine, leucine, methionine, isoleucine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the positive and/or negative peptide segment can further include a cargo polypeptide (e.g., a uricase protein) coupled to the N and/or the C terminus of the peptide. Cargo polypeptides (e.g., uricase proteins) are discussed in greater detail elsewhere herein.
In some embodiments, the peptide having a net positive charge can have at least 11 amino acids (A1-A11 as set forth sequentially from C to N terminus), wherein A1, A9, and A11 can each be a polar or a cationic amino acid, wherein A2 can be a polar amino acid, wherein A3 can be a cationic or a polar amino acid, wherein A4, A6, and A8 can each be a hydrophobic amino acid, wherein A5 and A7 can each be a cationic amino acid or a polar amino acid, and wherein A10 can be a polar amino acid.
In some embodiments, the negative peptide can be composed at least 11 amino acids amino acids (B1-B11 as set forth sequentially from C to N terminus), wherein B1 and B11 can each be a polar or an anionic amino acid, wherein B2 and B10, can each be polar amino acids, wherein B3, B5, B7, and B9, can each be an anionic or a polar amino acid, and wherein B4, B6, and B8, can each be hydrophobic amino acids.
Suitable cationic amino acids can have positively charged side (or “R” groups) groups and can include, but are not limited to, lysine, arginine, and histidine. Suitable anionic amino acids can have negatively charged side groups and can include, but are not limited to, aspartate and glutamate. Suitable polar amino acids can have polar, uncharged side groups, and can include, but are not limited to, serine, threonine, cysteine, proline, asparagine, and glutamine. Suitable hydrophobic amino acids can have nonpolar, aliphatic or aromatic side groups and can include, but are not limited to, glycine, alanine, valine, leucine, methionine, isoleucine, phenylalanine, tyrosine, and tryptophan. All sequences resulting from employing the general scheme shown in
The positive peptides and negative peptides can be synthesized using de novo peptide synthesis techniques generally known in the art.
In other embodiments, the peptides can be produced via recombinant protein expression techniques based on expression of a peptide or protein sequence from a suitable expression vector. A cartoon of one embodiment of such an expression system is shown in
The DNA can further include one or more segments that each encode for a purification tag. Suitable purification tags are generally known in the art and can include, but are not limited to, FLAG, His tag, maltose binding protein, and glutathione-S-transferase. When the polypeptide is expressed from the vector, it can be recovered from the host production cell by any suitable method. After recovery, the produced polypeptide can be purified using a suitable purification technique, including but not limited to, affinity purification or other suitable separation technique (e.g. immunoseparation). After recovery and/or purification, the polypeptide can be exposed to a protease to cleave the polypeptide to release the individual peptide segments. The protease itself can contain an affinity purification tag such that the protease can be removed after cleaving the produced polypeptide via a suitable affinity purification technique, that can include affinity purification based on a protein tag and immunopurification techniques. In other embodiments, the protease can be made inactive by contacting the protease with a protease inhibitor.
In other embodiments a synthetic gene can be generated that encodes a larger carrier protein that is operatively coupled to a positive or negative peptide provided herein. The carrier protein and the positive or negative peptide can be separated by a protease cleavage site. As described above. The synthetic gene can be included in a suitable expression vector. After production of the polypeptide from the synthetic gene, the positive or negative peptide can be separated from the carrier protein by exposing the polypeptide to a protease capable of cleaving at the protease cleavage site in the polypeptide. Suitable carrier proteins can include, without limitation, any protein ranging from about 100 amino acids to about 600 amino acids in length such as, but not limited to, kerosteroid isomerase, maltose binding protein, and glutathione S-transferase.
As such, also provided herein are DNA segments that can encode a positive and/or negative peptide described herein. One of ordinary skill in the art, based upon a peptide or polypeptide sequence, will be able to construct suitable coding sequences for the peptides and polypeptides described herein. Also provided herein are DNA vectors that can include one or more DNA segments that can encode one or more positive and/or negative peptides described herein. The DNA segment that can encode one or more positive and/or negative peptides described herein can be operatively coupled to a suitable promoter that can drive expression of the peptide or polypeptide that is encoded by the DNA segment.
The positive and negative peptides (with or without cargo molecules) and/or vectors that can be used to produce them can be provided as a kit. The kits can contain the positive and negative peptides separate from one another in appropriate containers. The peptides can be solubilized in a carrier solution, lyophilized, or otherwise provided. The kits can also include other reagents such as a solution at a pH ranging from 6.5 to about 8.5. The solution can be used to supply the stimulating condition upon which the positive and negative peptides can self-assemble.
Uses of the Co-Assembly Peptides and Nanostructures
The co-assembly peptides and resulting nanostructures can be used to endow other supramolecular materials with functional properties of the cargo polypeptides (e.g., uricase proteins) that can be coupled to the co-assembly peptides. As discussed above, depending on the concentration of the co-assembly tag peptides mixed and stimulated, different nanostructures can be formed. In embodiments, a method of using the co-assembly peptides provided herein can include the step of mixing at least one engineered peptide having a net positive charge with at least one engineered peptide having a net negative charge in an aqueous solution having a pH ranging from about 6.5 to about 8.5. The mixture can be a mixture containing one or more types of positive peptide (s) and one or more types of negative peptide(s). It will be appreciated that multiple types of positive and negative peptides can be mixed together, where the “type” refers to the cargo molecule attached (none or one present). To put this in context and as a non-limiting example, three types of negative peptides can be a negative peptide having no cargo polypeptide (e.g., a uricase protein) (a first type), a negative peptide having a first cargo polypeptide (e.g., a uricase protein) (a second type), and a negative peptide having a second cargo polypeptide (e.g., a uricase protein) (a third type), where the first cargo polypeptide (e.g., a uricase protein) and the second cargo polypeptide (e.g., a uricase protein) are not the same polypeptide. The number of types of peptides that can be present in a mixture can be at least greater than 2. In some embodiments, the number of types of peptides that can be present in a mixture can range from 2 to 50 or more.
The concentration of any charge complementary peptide in any mixture can range from about 1 0.1 nM to about 1000 mM or more. In some binary mixtures (e.g., mixtures containing one type of positive peptide and one type of negative peptide), the concentration of the positive peptide can be equal to the concentration of the negative peptide. In other binary mixtures, the concentration of the positive peptide can be greater than the concentration of the negative peptide. In further binary mixtures, the concentration of the positive peptide can be less that the concentration of the negative peptide. In some tertiary mixtures (e.g., mixtures containing 3 different types of peptides but at least one type of positive peptide and at least one type of negative peptide), the concentration of each type of peptide can be equal or at least one or at least two types of peptides can be present a concentration that is greater or less than the third type or peptide. In a ternary mixture (e.g., mixtures containing 4 different types of peptides but at least one type of positive peptide and at least one type of negative peptide), the concentrations of each peptide type can be equal or 1, 2, or 3 peptide types can be present at a concentration greater than or less than that of the fourth peptide type present. In view of this description, it will be appreciated how the concentration of the multiple different types of peptides can be varied no matter what the number of types of peptides used.
In some embodiments, the peptides (e.g., positive, negative, fusion peptides) of the present disclosure are useful in treating diseases and/or disorders. In some embodiments, a disease and/or disorder is related to uric acid. In some embodiments, a disease or disorder is related to high levels of uric acid. In some embodiments, the peptides (e.g., positive, negative, fusion peptides, e.g., positive and/or negative peptides attached to a uricase, or fragment thereof) of the present disclosure are useful for modulating uric acid levels. In some embodiments, the disease or disorder is gout.
In some aspect, the disclosure relates to method of modulating the uric acid levels in a subject. In some embodiments, the method comprises administering any of the peptides of the present disclosure. In some embodiments, the peptides (e.g., positive, negative, fusion peptides, e.g., positive and/or negative peptides attached to a uricase, or fragment thereof) present disclosure reduce the levels of uric acid in a subject. In some embodiments, the disclosure relates to methods of modulating serum levels of uric acid in a subject.
Administration routes for the peptides and compositions described herein (e.g., comprising one or more uricase protein fusions) include, but are not limited to, direct injection into a joint or tissue, intravenous injection, intramuscular injection, oral ingestion. In some embodiments, a peptide or composition described herein is directly injected into the big toe of a subject. In some embodiments, a peptide or composition described herein is directly injected into the knee, the ankle, the hip, the wrist, the neck, the shoulder, the elbow, or a digital articulation of a subject.
In some embodiments, a dose of a composition described herein and administered to a subject comprises between 1 ug and 100 ug of active ingredient (e.g., a uricase protein, optionally in the form of a fusion protein comprising a self-assembling peptide). In some embodiments, the active ingredient is combined, fused or complexed to the self-assembling peptides of the present invention. In some embodiments, the administered dose comprises between 1 ug and 10 ug of active ingredient. In some embodiments, the administered dose comprises between 10 ug and 20 ug of active ingredient. In some embodiments, the administered dose comprises 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 ug of active ingredient. In some embodiments, the administered dose comprises between 2 ug and 10 ug of active ingredient (e.g., a uricase protein) combined to the peptides of the present invention. In some embodiments, the administered dose comprises about 2, 3, 4, 5, 6, 7, 8, 9, or 10 ug of active ingredient (e.g., a uricase protein) combined to the peptides of the present invention. In some embodiments, the dose is administered to a subject on a daily basis. In some embodiments, the dose is administered to a subject on a weekly, monthly, quarterly, semi-annual or yearly basis.
In some aspects, the disclosure provides methods and compositions for delivering a uricase (e.g., in the form of a fusion protein comprising a self-assembling peptide) to a subject with high levels of uric acid. Hyperuricemia is generally defined as occurring when serum urate levels are above 6.8 mg/dL (Burns CM, WR . Disorders of purine and pyramidine metabolism. In: Longo FADL, Kasper D L, Hauser S L, Jameson J L, Loscalzo J, editors. Harrison's priciples of internal medicine. McGraw-Hill; New York: 2012). Some references, define hyperuricemia as occurring when serum urate levels are above 6.8 mg/dL. In some embodiments, a uricase protein is fused to a self-assembling peptide, for example one or more of a set of charge complementary self-assembling peptides.
Aspects of the disclosure relate to methods for treating diseases or disorders related to high levels of uric acid in a subject. In some aspects, the disclosure provides methods of using a uricase (e.g., in the form of a fusion protein comprising a self-assembling peptide) for treating tumor lysis syndrome and graft versus host disease.
Aspects of the disclosure relate to methods for treating diseases or disorders related to the formation or deposition of monosodium urate (MSU) crystals in joints and/or tissue of a subject. In some embodiments, a disease or disorder related to the formation or deposition of monosodium urate (MSU) crystals is an acute inflammatory condition. In some embodiments, a disease or disorder related to the formation or deposition of monosodium urate (MSU) crystals is gout. Gout can affect anyone, but generally occurs earlier in men than women. Men are more likely to develop gout because they have higher levels of uric acid most of their lives. Women are more likely to develop gout after menopause as their uric acid levels increase at this stage of their lives. Risk factors for gout include obesity, congestive heart failure, diabetes, family history of gout, hypertension, and kidney disease.
Aspects of the disclosure relate to methods for alleviating symptoms and/or slowing (e.g., preventing) the progression of diseases or disorders related to high levels of uric acid and/or the formation (or deposition) of MSU crystals in a subject. Such symptoms may include pain (e.g., intense pain attacks), swelling, redness, burning sensation, stiffness, or tenderness in one or more joints, tissues, or organs of the subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is human. In some embodiments, the subject has, is at risk of having, or is suspected of having, gout.
Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.
Self-assembly is the spontaneous organization of molecules into a precise supramolecular architecture without any external guidance.49 Throughout nature, biomolecule self-assembly gives rise to various functional biomaterials that can perform complex tasks, such as molecular sensing and recognition, chemical synthesis, motility, and compartmentalization, as well as multi-scale hierarchical organization.34 There is increasing interest in biomolecule self-assembly for bottom-up fabrication of biomaterials for medical and technological applications.48 Among the various synthetic biomolecules capable of self-assembly,11 peptides and peptide analogs that form elongated nanofibers by adopting β-sheet secondary structures provide numerous advantages for creating supramolecular biomaterials. In particular, β-sheet fibrillizing peptides can be synthesized and purified in high yield using conventional methods. Peptide self-assembly into β-sheet nanofibers occurs under mild aqueous conditions, with the resultant nanofibers often undergoing sol-gel transition into macroscopic, physically-crosslinked hydrated structures (i.e. “hydrogels”) above a critical concentration.20 The amino acid sequence of β-sheet fibrillizing peptides can be systematically modified to tailor nanofiber morphology, gelation properties, and stimuli-responsiveness according to the intended application.4 In addition, β-sheet peptide nanofibers can be fabricated into micron-sized gels using various conventional polymer-processing techniques.12,46 Finally, β-sheet peptide nanofibers are well-suited for use in biomedical applications because they are composed of natural amino acids that can be metabolized, they are often biocompatible with cells and tissues, and they typically elicit minimal inflammation and weak or no adaptive immunity directed against the peptide itself, despite being foreign to the host.5,10,14,15,17,18,22
To be useful for medical or technological applications, supramolecular biomaterials must often demonstrate functional capabilities beyond robust self-assembly and biocompability. Since synthetic peptides designed to self-assemble typically lack any additional functional features, ligands, fluorophores, antigens, drugs, or other bioactive compounds are often installed into supramolecular biomaterials to impart functional capabilities. Supramolecular biomaterials can be functionalized post-assembly via various covalent and non-covalent grafting approaches,13,23,29,31,32,35,38,42-44 However, the former often rely on complex, inefficient, or poorly reproducible reactions, while the latter may only provide transient functionality within complex biological environments.
Alternatively, functional capabilities can be installed directly into supramolecular biomaterials via covalent fusion of a functional molecule and a self-assembling peptide in the pre-assembled state (i.e. “fusion peptides”), which provides many advantages over post-assembly modification. In particular, diverse libraries of fusion peptides modified with functional molecules that are stable in organic solvents (e.g. peptides, sugars, or small organic compounds) can be prepared in high yield and purity by adapting conventional solid-phase peptide synthesis methods.20,30,39 The conditions that promote self-assembly of fusion peptides are often similar to those of the unmodified peptide. The concentration of functional molecule integrated into single-component supramolecular assemblies via fusion peptides is well defined and highly reproducible, yet multi-component supramolecular biomaterials with modular and tunable functional capabilities can also be fabricated via simple mixing of two or more fusion peptide variants in the pre-assembled state.21 Finally, functional molecules fused to a self-assembling peptide are often stably integrated into supramolecular biomaterials because dissociation of peptides inserted into β-sheet nanofibers is energetically unfavorable.19 As a result, fusion peptides have led to a broad assortment of supramolecular biomaterials with functional capabilities that are suitable for use in various biomedical and biotechnological applications, such as promoting cell adhesion for tissue engineering and regenerative medicine,8,30 enabling controlled-release of therapeutics,6,9,12,26,27,45 and presenting antigens and immunomodulatory signals to elicit robust adaptive immunity.3,37,41
Installing functional molecules with more sophisticated properties, such as catalysis, selective molecular recognition, tunable visible light fluorescence, or precise antigen conformation, could impart unprecedented functional capabilities into supramolecular biomaterials already finding use in biomedical and biotechnological applications. Owing to the diversity of specialized functional capabilities afforded by folded proteins when compared to unfolded peptides and small molecules, there is growing interest in installing folded proteins into supramolecular biomaterials. Covalent fusions of folded proteins and self-assembling peptides provide many advantages over covalent grafting from a material fabrication perspective, yet there are many challenges inherent to covalent fusions from a synthesis perspective. In particular, conventional peptide synthesis and purification cannot be adapted to produce protein-peptide fusions. Instead, fusion proteins are typically expressed from recombinant DNA using translational machinery housed within microbes or other cellular hosts. The environment within the cytoplasm of expression hosts is often favorable for β-sheet peptide self-assembly, however, which can hinder recovery of soluble, bioactive fusion proteins by inducing aggregation or misfolding.24,50 Self-assembly domains with slow fibrillization kinetics or pH-responsive assembly can address this limitation,1,2,19,28,33 however the former requires assembly kinetics that are well-behaved under various expression conditions and with a wide variety of fusion protein partners, while the latter necessitates proteins with folded conformations that are not pH-sensitive. An under-explored alternative is a peptide that is designed to resist assembly until acted upon by a stimulus that can be applied under neutral, near-physiological conditions that are ideal for maintaining protein folding and function.
In this Example, an approach to install a folded protein that can only assemble into β-sheet nanofibers at neutral pH in the presence of a second charge-complementary peptide (
Electrostatic control of assembly is expected to enable optimal E. coli expression of a soluble, monomeric fusion of CATCH(−) and a folded protein by reducing the propensity for fusion protein aggregation or misfolding due to pre-mature peptide self-assembly. In turn, the CATCH fusion protein can integrate into supramolecular biomaterials upon addition of excess CATCH(+) and CATCH(−) (
Materials and Methods
Peptide Synthesis and Purification. The two complimentary peptides, Ac-EQEFEFEFEQE-Am [CATCH(−)] (SEQ ID NO: 2) and Ac-QQKFKFKFKQQ-Am [CATCH(+)] (SEQ ID NO: 1), and their respective mutants, Ac-EQEPEPEPEQE-Am [mCATCH(−)] and Ac-QQKPKPKPKQQ-Am [mCATCH(+)], were synthesized using standard Fmoc solid-phase peptide synthesis on a CS336X automated peptide synthesizer (CS Bio), according to established methods.12 All reagents for peptide synthesis were purchased from Novabiochem, unless stated otherwise. Following final Fmoc deprotection, peptides were acetylated at their N-termini with acetic anhydride (Sigma) (10% acetic anhydride (Sigma), 80% dimethylformamide (DMF) (Fisher), and 10% N,N-Diisopropylethylamine (DIEA) (Fisher)). Synthesis resin was collected, washed with acetone (Fisher), and dried in vacuo overnight. Peptides were cleaved and deprotected with a cocktail of 9.5:0.25:0.25 trifluoroacetic acid (TFA) (Fisher):triisopropylsilane (TIS) (Sigma):water. Peptides were then precipitated with cold diethyl ether (Fisher), collected via centrifugation, washed, and dried in vacuo overnight. Peptides were dissolved in water, frozen, and freeze-dried with a FreeZone 1 lyophilizer (Labconco).
Peptides were purified to greater than 90% purity by reverse phase high-performance liquid chromatography (RP-HPLC) using a DionexTM Ultimate 3000TM System (Thermo Scientific) equipped with a C-18 column (Thermo Scientific) (
MALDI-Time-of-Flight Mass Spectrometry (MALDI-TOF-MS). For MALDI-TOF-MS analysis (
Thioflavin T (ThT) Assay. A ThT stock solution containing 0.8 mg/mL of thioflavin T (Acros) in 1x phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.4) was filtered through a 0.22 μm syringe filter (Millex), and further diluted 1:10 with 1x PBS to create a working solution. Peptide samples were mixed with the ThT working solution at a 1:10 (v/v) ratio, added to a black 96-well plate (Corning), and analyzed with a Molecular Devices SpectraMax M3 spectrophotometer (excitation 450, emission 482 nm). All samples were run in triplicate, with the mean and standard deviation of these samples reported.
Circular Dichroism. Circular Dichroism was performed on an AVIV 202 spectrometer. Peptides were prepared in 1x PBS at 500 μM, unless stated otherwise. Samples were run 3 times, averaged, and converted to mean residue ellipticity. All data reported was for dynode values <500 V.
Expression and Purification of Peptide Fusion Proteins. DNA encoding fusion proteins consisting of each CATCH peptide or mutant CATCH peptide linked to superfolder GFP by a serine-glycine linker was inserted into a pET-21d vector between the NcoI site and XhoI site (
Origami B (DE3) clones harboring plasmids encoding CATCH fusion proteins were grown overnight in 5 mL of 2xTY protein expression media (16 g tryptone, 10 g yeast, 5 g NaCl) with 0.1 mg/mL ampicillin and 0.05 mg/mL kanamycin in an orbital shaker at 225 rpm and 37° C. Origami B (DE3) cultures were then sub-cultured into 1L 2xTY media with 0.1 mg/mL ampicillin and 0.05 mg/mL kanamycin, and maintained in an orbital shaker at 225 RPM and 37° C. until an OD (λ=600 nm) of 0.6-0.8 was reached. At the specified OD, 1 mL of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added into the media, and cultures were maintained in an orbital shaker at 225 RPM at 18° C. for 18 hours. Bacteria were pelleted by centrifugation (24600×g at 4° C. for 10 minutes) and re-suspended/washed in 1x PBS three times. Bacteria were then lysed by the addition of 4 mL of B-PER (Thermo) per gram of bacteria pellet. 2 μL of DNAse (2500 U/mL) and 2 μL lysozyme (50 mg/mL) were added for each 1 mL of B-PER added. 1 Pierce protease inhibitor tablet (Thermo) was added to the lysis cocktail. The bacteria pellet was manually separated using a metal spatula, and then incubated at room temperature on a rocker for 20 minutes. Lysate was centrifuged at 47700×g for 15 minutes at 4° C. to remove insoluble material, and the supernatant was collected by decanting. Fusion proteins were purified using metal-affinity chromatography with HisPur cobalt resin (Thermo) and elution with a step-gradient of 5-250 mM imidazole in 1x PBS, similar to established methods.19 Molecular weight of all fusion proteins was verified with SDS-PAGE.
Protein Fluorimetry. Fluorescence spectra of both CATCH(−)GFP and mCATCH(−)GFP were acquired with a Molecular Devices SpectraMax M3 spectrophotometer. 10 μM CATCH(−) GFP or mCATCH(−)GFP in 1x PBS was excited with 485 nm light (cutoff=495 nm) and emission was recorded over the range of 495-600 nm. Black 96-well clear bottom plates (Corning) were used to minimize background fluorescence. Spectra were collected in triplicate, with the average reported.
Size Exclusion Chromatography. Aggregation of CATCH(−)GFP and mCATCH(−)GFP was analyzed using size exclusion chromatography on an AKTA Pure fast protein liquid chromatography system equipped with a GE Healthcare Superdex 200 Increase 10/300 column. 200 μL of 10 μM CATCH(−)GFP or mCATCH(−)GFP in 1x PBS was injected into the sample loop. The sample loop was subsequently cleared with 5 mL of 1x PBS to ensure the entire sample was loaded onto the column, and then proteins were eluted with 2.0 column volumes of 1x PBS. Proteins were detected by absorbance at 280 nm.
Nanofiber preparation. Stock solutions of CATCH(−) and CATCH(+) were prepared by dissolving lyophilized peptides in 1x PBS to a final concentration of 500 μM. Peptide concentration was confirmed using Phe absorbance (λ=260 nm). CATCH peptides were then mixed at a 1:1 (v/v) ratio and incubated overnight for formation of nanofibers, unless stated otherwise.
Nanofibers with integrated fusion proteins were prepared by mixing stock solutions of CATCH(−)GFP, CATCH(−), and CATCH(+) at final concentrations of 0.75 μM, 187.5 μM, and 200 μM, respectively, unless stated otherwise. Although not explicitly studied, we expected that CATCH(+) and CATCH(−) would co-assemble most efficiently at a nearly equimolar ratio. Thus, here a slight molar excess of CATCH(+) relative to total CATCH(−) (i.e. [CATCH(−) peptide]+[CATCH(−)GFP]) was expected to maximize CATCH(−)GFP integration into nanofibers by preventing competition between CATCH(−) and CATCH(−)GFP during co-assembly.
Transmission Electron Microscopy. Formvar-carbon coated 400 mesh copper grids (FCF400-CU-UB, EMS) were floated on top of 20 μL of 1x PBS solutions containing CATCH peptides with or without CATCH(−)GFP, and then dried by tilting the grid on a Kimwipe. Samples were negatively stained with 2% uranyl acetate in water, and analyzed using a Hitachi H-7000.
Fluorescence Microscopy. Microscopic morphology of CATCH peptide or peptide-fusion protein mixtures was investigated with a Zeiss Axio Observer inverted epifluorescent microscope. For samples containing only CATCH peptides, ThT was mixed with samples at a 1:1 (v/v) ratio, 5 μL of each sample was placed onto a glass microscope slide, samples were covered with a cover slip, and samples were viewed using a GFP filter set. For samples containing CATCH peptides and CATCH(−)GFP, 0.75 μM CATCH(−)GFP was mixed with 200 μM CATCH(+) and 187.5 μM CATCH(−), unless otherwise specified. 5 μL of each sample was placed onto a glass microscope slide, samples were covered with a cover slip, and samples were viewed using a GFP filter set. For all samples, images were collected using an exposure time of 4 s.
Particle Analysis. Size distribution of microscopic materials formed in solutions of 0.75 μM CATCH(−)GFP and 200 μM CATCH(+) under various conditions was determined from fluorescent photomicrographs using the Analyze Particle function in ImageJ software (NIH). Particle range was set from 2 to infinity for all samples, and 8-bit images were set with threshold values below 0.36% to minimize the halo around large particles, yet allow for small particles to be detected. Commercially-available fluorescent polymeric microspheres with known diameters of 1, 2, 4, and 9.9 μm were used for calibration of image analysis, as supplied by the manufacturer (Life Technologies). A minimum of 140 particles was analyzed per condition.
Dynamic light scattering (DLS) was measured on a Brookhaven 90Plus Particle Size Analyzer (Brookhaven Instruments Inc., NY) with BIC Particle Sizing Software. 0.75 μM CATCH(−)GFP+200 μM CATCH(+) samples were prepared in 1x PBS and either incubated for about 15 hours without mixing (i.e. “static”) or stirred for 10 and 60 minutes. All samples were run 10 times for a period of about 30 seconds. Particle size histograms are reported in terms of intensity (%).
Zeta Potential. Zeta potential was analyzed on a Brookhaven Zeta Potential Analyzer (Brookhaven Instruments Inc., NY) with PALS Zeta Potential Analyzer Software. Measurements were obtained from samples of 1x PBS solutions containing 0.75 μM CATCH(−)GFP+200 μM CATCH(+) stirred for 1 hour. The sample was analyzed 10 times and only readings below 0.02 relative residual were used to calculate mean zeta potential.
Macroscopic Hydrogel Formation. A 12 mM stock solution of CATCH(−) was prepared by dissolving dry peptide in 1x PBS and adjusting the pH to ˜7.6 with ammonium bicarbonate (Fisher). A 12 mM stock solution of CATCH(+) was prepared by dissolving dry peptide in 1X PBS and adjusting the pH to ˜7.2 with hydrochloric acid (0.5 N Fisher). CATCH peptide stock solutions were combined at a 1:1 (v/v) ratio in a 0.2 mL glass vial (Thermo), and the vial was gently swirled for 2 min until the hydrogel formed. The hydrogel was stained with red food coloring to aid visualization. Hydrogel images were recorded using a digital still camera.
Protein Incorporation into Macroscopic Hydrogels. Hydrogels were prepared as described above, except 1.5 μM CATCH(−)GFP or mCATCH(−)GFP was added to the 12 mM CATCH(−) solution. Immediately after hydrogel formation, fluorescence was visualized by placing the glass vials on a SafeImager 2.0 transilluminator (Invitrogen) and recording images with a digital still camera. 800 μL of PBS was then carefully overlaid on top of hydrogel. The samples were then incubated for 48 hours to evaluate the protein incorporation within the hydrogel. At various time points, fluorescence in the buffer supernatant was visualized by placing the glass vials on a SafeImager 2.0 transilluminator (Invitrogen) and recording images with a digital still camera. At the end-point, buffer supernatant was carefully removed by decanting and fluorescence was visualized by placing the glass vials on a SafeImager 2.0 transilluminator (Invitrogen) and recording images with a digital still camera.
Results
CATCH peptide co-assembly into β-sheet nanofibers. First, CATCH(+) and CATCH(−) co-assembly was characterized using ThT fluorimetry, transmission electron microscopy (TEM), circular dichroism (CD), and fluorescence microscopy (
Elongated structures having widths of about 5 nm and spanning hundreds of nm in length were observed in transmission electron micrographs of solutions containing an equimolar mixture of CATCH peptides (
Finally, fluorescent flocculates were observed in fluorescence photomicrographs of solutions containing ThT and an equimolar mixture of CATCH peptides (
Expression and recovery of a fluorescent CATCH fusion protein. The expression and recovery of a recombinant fusion of CATCH(−) and superfolder green fluorescent protein (GFP) herein referred to as “CATCH(−)GFP”, from E. coli hosts was characterized using fluorimetry, native polyacrylamide gel electrophoresis (PAGE), and size exclusion chromatography (SEC) (
Co-assembly of CATCH(−)GFP and CATCH peptides. The co-assembly of CATCH(−) GFP with CATCH peptides was characterized under various conditions using fluorescence microscopy and transmission electron microscopy (
Micron-sized fluorescent particles (i.e. “microparticles”) were observed in a binary mixture of CATCH(−)GFP and CATCH(+) (
Understanding the extent and kinetics of CATCH fusion protein and CATCH peptide di-assembly could also be advantageous from a practical perspective, as supramolecular microparticles with integrated functional proteins could be useful for various medical and biotechnological applications. It was envisioned that fusing CATCH(−) to GFP could introduce a significant diffusion limitation that would diminish reaction kinetics and in turn reduce the extent of reaction observed in real-time. Thus, we used fluorescence microscopy to assess the influence of incubation time and stirring on microparticle number, a qualitative measure of the extent of reaction, and microparticle size (
Finally, we used fluorescence microscopy and TEM to investigate the relationship between the composition of CATCH(−)GFP, CATCH(+), and CATCH(−) mixtures and the transition from di-assembly to tri-assembly (
Macroscopic CATCH hydrogels with an integrated folded protein component. To demonstrate the potential of CATCH for creating functional supramolecular biomaterials with an integrated folded protein component, we characterized the formation of self-supporting macroscopic fluorescent hydrogels from ternary mixtures of CATCH(+), CATCH(−), and CATCH(−)GFP (
In this Example, at least a pair of synthetic peptides that can co-assemble into β-sheet nanofibers in aqueous media via electrostatic complementarity was demonstrate and is referred to as CATCH. CATCH(+) and CATCH(−) peptides are cationic and anionic variants of Q11, a zwitterionic synthetic peptide that self-assembles into β-sheet nanofibers in aqueous media.7 CATCH peptides rapidly co-assemble into β-sheet nanofibers when combined in aqueous media above a critical concentration, yet cannot self-assemble under identical conditions, likely due to electrostatic repulsion resulting from their net charge at neutral pH. Fibrillization of CATCH(+) and CATCH(−) was mediated by their core sequence of alternating phenylalanine residues and hydrophilic amino acids, analogous to the parent peptide Q11.7,40 The co-assembly behavior of CATCH peptides was similar to that of the charge-complementary synthetic peptides P11-13 and P11-14 developed by Aggeli et al,25 which only assembled into elongated β-sheet nanofibers when combined. Together, these observations suggest that the common primary sequence motif shared by CATCH peptides and the charge-complementary P11 peptides, in which charged and polar amino acids flank a core of alternating hydrophobic and hydrophilic residues, is likely to be broadly applicable for designing synthetic peptides that selectively co-assemble into β-sheet nanofibers. However, inserting charged residues within the primary sequence may also introduce an energetic penalty for peptide β-sheet fibrillization, given that the critical concentration for CATCH peptide co-assembly was more than an order of magnitude higher than that of Q11, which self-assembles into β-sheet nanofibers at a concentration of ˜10 μM.7 Future efforts could systematically investigate CATCH primary sequences to identify motifs that confer robust electrostatic control of assembly, yet decrease the critical fibrillization concentration.
The CATCH system can create a versatile recombinant fusion tag to install folded proteins into supramolecular biomaterials to endow them with specific functional features. For a fusion tag to be broadly useful, it should not drive protein aggregation or disrupt protein folding during expression or purification, a noted limitation for recombinant tags having high propensity for self-assembly into β-sheet nanofibers.24,50 Proteins that self-assemble into nanofibers in response to specific stimuli, such as pH, can address this limitation.1,2,28,33 However, the functional protein domain appended onto the assembly domain must have a conformation that is insensitive to the assembly stimulus. Additionally, the size of the functional domain may need to be limited to enable accurate translation and high yield expression if the assembly domain is large. Therefore, key features of more broadly useful recombinant tags to install folded proteins into supramolecular biomaterials would include selective assembly in response to a mild external stimulus that is unlikely to perturb protein conformation, as well as a relatively low molecular weight to enable co-expression with fusion partners having a range of molecular weights. CATCH peptides were ideal candidates in this regard, given their relatively low molecular weight (about 1.5 kDa) and tightly controlled mixing-induced assembly in aqueous media at neutral pH.
In this report it was demonstrated that a fusion of CATCH(−) and GFP was expressed and recovered as a soluble monomer from E.coli hosts in mg/L yields that were often as good or better than those for an mCATCH(−)GFP variant having a mutated fusion tag that cannot assemble into β-sheet nanofibers (
Beyond facilitating fusion protein expression, CATCH also met the criteria for a recombinant fusion tag that can endow supramolecular biomaterials with the functional properties of folded proteins. Herein, we demonstrated that simply combining CATCH(−)GFP with CATCH peptides in aqueous media yielded fluorescent supramolecular biomaterials via integration of GFP into the resulting assemblies in a CATCH-dependent manner. Interestingly, the structural features of the resulting fluorescent supramolecular biomaterials could be varied by altering the concentration or composition of CATCH peptides added to solutions containing CATCH(−)GFP. For example, ternary mixtures of CATCH(−)GFP and both CATCH peptides produced soluble fluorescent nanofibers or macroscopic nanofibrillar hydrogels depending on the peptide concentration, whereas binary mixtures of CATCH(−)GFP and its complementary CATCH(+) peptide yielded micron-sized particles. We speculate that supramolecular di-assembly of microparticles in binary mixtures may be due to β-sheet fibrillization of CATCH(−) GFP and CATCH(+), analogous to the tri-assembly of fluorescent nanofibers from ternary mixtures. However, given that CATCH peptides adopted a primarily random-coil conformation at concentrations of 50 μM, observing CATCH(−)GFP and CATCH(+) co-assembly into microparticles at concentrations that were well below the critical fibrillization concentration of CATCH peptides was unexpected. One possible explanation is arrived at by rationalizing that the magnitude of the system free energy change is likely primarily governed by differences in the entropic penalty of organizing CATCH(−) or CATCH(−)GFP into a supramolecular structure. It can be assumed that any entropic gain due to release of water molecules from the surface of CATCH(−) is similar for peptides and fusion proteins (i.e. that the protein domain does not alter the ordering of water molecules around CATCH(−), or any hydrophobic effect is independent of the protein domain). Second, it can be assumed that the entropic penalty for organizing CATCH(+) into a supramolecular structure is similar regardless of whether the peptide is binding to CATCH(−) peptide or a CATCH(−) fusion protein. Third, it can be assumed that enthalpic contributions from binding of CATCH(−) and CATCH(+) are also similar for peptides and fusion proteins (i.e. that protein-protein or protein-peptide interactions are negligible). Finally, it can be inferred that fusing CATCH(−) to a large protein molecule will reduce the translational and rotational entropy of the peptide, analogous to immobilizing the peptide onto a solid phase. In other words, the entropic penalty for organizing freely diffusible CATCH(−) into a supramolecular structure is much greater than that for CATCH(−)GFP. Therefore, the magnitude of the free energy change for co-assembly of CATCH(+) and CATCH(−)GFP is much greater, and thus more favorable, than that for CATCH(+) and CATCH(−). It can be expected that this same relationship would hold for co-assembly of CATCH(−) peptides with a CATCH(+) fusion protein, while co-assembly of CATCH(−) and CATCH(+) fusion proteins may be even more energetically favorable.
Beyond informing a mechanistic basis for CATCH(−)GFP and CATCH(+) di-assembly into supramolecular microparticles, considering the behavior of the system as it approached equilibrium could also be advantageous from a practical perspective. In particular, a near-quantitative co-assembly reaction could ultimately be useful for fabricating functional supramolecular microparticles for various medical and biotechnological applications. Here, we observed that the kinetics and extent of di-assembly of CATCH(−)GFP and CATCH(+) into microparticles were increased by mixing, as stirring for 10-60 minutes qualitatively increased the number of microparticles observed within the binary mixtures. Interestingly, however, it was noted that mixing seemed to preferentially induce nucleation of new microparticles, rather than propagating growth of larger microparticles, as particle size distribution narrowed and centered about 700 nm with increasing stirring time. Thus, it remains to be determined if the di-assembly process and resultant features of supramolecular microparticles, such as the kinetics, extent of reaction, and size distribution, are influenced by the folded protein domain or net charge of the CATCH tag. Toward that end, future work will move toward characterizing microparticle fabrication with CATCH fusions consisting of folded proteins having functional properties that are relevant for biomedical or biotechnological applications.
Interesting CATCH assembly behaviors were observed in the transition from binary to ternary mixtures. In particular, low concentrations of CATCH(−) peptide in ternary mixtures yielded fluorescent microparticles similar to those observed in binary mixtures, whereas increasing the CATCH(−) concentration induced a transition to fluorescent nanofibers whose relative density increased with CATCH(−) concentration. Unexpectedly, CATCH(−) and CATCH(+) peptides co-assembled into fluorescent nanofibers in the presence of CATCH(−)GFP at peptide concentrations that were well below the critical concentration for peptide co-assembly in the absence of a fusion protein. Building from the thermodynamic considerations of di-assembly discussed above, the lower concentrations required for peptide co-assembly in the presence of the fusion protein may be explained by rapid, energetically favorable formation of stable CATCH(−)GFP and CATCH(+) nuclei that propagate co-assembly of CATCH(−) and CATCH(+) peptides, analogous to seeds in samples of amyloid-β.16 Thus, assembly of CATCH fusion proteins into supramolecular biomaterials is not governed exclusively by a binary or ternary mixture, but rather by the relative concentration of each molecular component within the system. This flexibility in supramolecular assembly structural features, coupled with the versatility of CATCH tags for installing a wide variety of functional proteins, suggests the enormous potential of the CATCH system for creating new biomaterials for a broad assortment of applications.
Supramolecular assembly is receiving increasing attention for fabricating functional biomaterials for various biomedical and biotechnological applications, including tissue engineering, regenerative medicine, immunology, biosensors, and chemical synthesis. To be useful for these applications, however, supramolecular biomaterials must often demonstrate functional capabilities, such as molecular recognition, antigen display, tunable drug release, catalysis, or fluorescence. Installing molecules having particular functional properties is often required to endow supramolecular biomaterials with the desired functional capabilities. Toward this end, we created a pair of anionic and cationic synthetic peptides, referred to as “CATCH” or Co-Assembly Tags based on CHarge complementarity, to install folded proteins into supramolecular biomaterials. CATCH peptides were designed to co-assemble into β-sheet nanofibers when combined, yet resist self-assembly due to electrostatic repulsion. This electrostatically controlled assembly enabled high yield production of soluble CATCH(−)GFP by E. coli. Binary mixtures of CATCH(−)GFP and its charge-complementary peptide assembled into fluorescent microparticles, whereas ternary mixtures of CATCH(−)GFP and both CATCH peptides yielded fluorescent nanofibers and macroscopic hydrogels. A reliable approach for stable integration of folded proteins into supramolecular assemblies is likely to be broadly useful for creating biomaterials with more sophisticated functional capabilities that greatly advance the state-of-the-art. Thus, the CATCH system can be useful in addressing the impending functionality bottleneck imposed by continued reliance on unfolded peptides and small organic compounds as functional components within biomaterials.
25. S Kyle, Felton S H, McPherson M J, Aggeli A, Ingham E. Rational molecular design of complementary self-assembling peptide hydrogels. Adv Healthc Mater. 2012;1(5):640-5.
37. R R Pompano, Chen J, Verbus E A, Han H, Fridman A, McNeely T, Collier J H, Chong A S. Titrating T-cell epitopes within self-assembled vaccines optimizes CD4+ helper T cell and antibody outputs. Adv Healthc Mater. 2014;3(11):1898-908.
Multiple CATCH peptides can be produced from a single synthetic gene that encodes a polypeptide containing multiple CATCH peptides that separated from each other by a flanking protease cleavage amino acid sequence. The synthetic gene can include DNA segments that each encode a CATCH peptide that are separated in the synthetic gene by DNA segments that can each encode a protease cleavage sequence. This can allow for scalable production of the CATCH peptides.
Experiments were carried out to evaluate the retention of enzymatic activity of uricase cargo polypeptides when fused with the peptides of the present disclosure. Gout was used as a model for investigation to remove/reduce uric acid levels using a uricase fused to the peptides and immobilized in a hydrogel after self-assembly. A schematic depicting gout: inflammatory arthritis caused by uric acid crystallization within the joint space is shown in
CATCH(+)/(−) peptides were prepared to self-assembly into nanofibers (
In vitro Assessment of the Activity of Soluble and Gel Forms of CATCH-U
The enzymatic activity of CATCH-U in soluble and gel form was evaluated by measuring uric acid depletion over time via absorbance at 293 nm and 37° C. for varying concentrations of uric acid substrate. Hydrogels were left to incubate at 37° C. for 4 days with substrate removed and replenished daily to determine if the gels remain active over this timeframe. Gels were also incubated with monosodium urate (MSU) crystals.
Enzymatic activity of the uricase fusion protein immobilized in the peptide nanofiber hydrogels is retained (
Soluble CATCH-U activity in dissolving MSU crystals was assessed by incubating 12.6 ug soluble CATCH-U with 0.5 mg MSU crystals and 2250 U/mL catalase for 96 hours (
The hydrogel creates a concentration gradient sufficient to dissolve crystallized uric acid in the form of monosodium urate (MSU) crystals (
Crystallized uric acid leads to the pathology of gout. These data indicate that the uricase-loaded gel is able to deplete uric acid crystals in vitro.
Assessment of CATCH-U Gel Efficacy in an in vivo Model of Gout
Mice were subcutaneously injected in the top of the hind paw with either 40 uL blank gels (0 ug) or CATCH-uricase (12.6 ug) in a 40 uL gel formulation at t=−48 hours (
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be illustrative examples, and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g, “comprising”) are also contemplated, in alternative embodiments, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.
This application claims the benefit of the filing date of U.S. Provisional Application No. 63/091,267, filed Nov. Oct.13, 2020, the entire contents of which are incorporated herein by reference.
This invention was made with government support under grant number EB024762 awarded by the National Institutes of Health and grant number 1743432 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/054869 | 10/13/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63091267 | Oct 2020 | US |
