Patent Application
20120003201
1. Field of the Invention
The present invention relates generally to non-viral compositions and methods useful for the cellular delivery of one or more molecules of interest. In various embodiments, vault complexes are described which comprise an agent which modifies cell adhesion, for example by inhibiting cell adhesion. Also included in the invention is the use of the compositions as cellular delivery agents for the treatment of diseases, for example chronic kidney disease.
2. Description of the Related Art
Vaults are cytoplasmic ubiquitous ribonucleoprotein particles first described in 1986 that are found in all eukaryotic cells (Kedersha et al., J Cell Biol, 103(3):699-709 (1986)). Native vaults are 12.9±1 MDa ovoid spheres with overall dimensions of approximately 40 nm in width and 70 nm in length (Kong et al., Structure, 7(4):371-379 (1999); Kedersha et al., J Cell Biol, 112(2):225-235 (1991)), present in nearly all-eukaryotic organisms with between 104 and 107 particles per cell (Suprenant, Biochemistry, 41(49):14447-14454 (2002)). Despite their cellular abundance, vault function remains elusive although they have been linked to many cellular processes, including the innate immune response, multidrug resistance in cancer cells, multifaceted signaling pathways, and intracellular transport (Berger et al., Cell Mol Life Sci, 66(1):43-61 (2009)).
Vaults are highly stable structures in vitro, and a number of studies indicate that the particles are non-immunogenic (Champion et al., PLoS One, 4(4):e5409 (2009)). Vaults can be engineered and expressed using a baculovirus expression system and heterologous proteins can be encapsulated inside of these recombinant particles using a protein-targeting domain termed INT for vault INTeraction. Several heterologous proteins have been fused to the INT domain (e.g. fluorescent and enzymatic proteins) and these fusion proteins are expressed in the recombinant vaults and retain their native characteristics, thus conferring new properties onto these vaults (Stephen et al., J Biol Chem, 276(26):23217-23220 (2001); Kickhoefer et al., Proc Natl Acad Sci USA, 102(12):4348-4352 (2005)).
Vaults are generally described in U.S. Pat. No. 7,482,319, filed on Mar. 10, 2004; U.S. application Ser. No. 12/252,200, filed on Oct. 15, 2008; International Application No. PCT/US2004/007434, filed on Mar. 10, 2004; U.S. Provisional Application No. 60/453,800, filed on Mar. 20, 2003; U.S. Pat. No. 6,156,879, filed on Jun. 3, 1998; U.S. Pat. No. 6,555,347, filed on Jun. 28, 2000; U.S. Pat. No. 6,110,740, filed on Mar. 26, 1999; International Application No. PCT/US1999/06683, filed on Mar. 26, 1999; U.S. Provisional App. No. 60/079,634, filed on Mar. 27, 1998; and International Application No. PCT/US1998/011348, filed on Jun. 3, 1998. Vault compositions for immunization against chlamydia genital infection are described in U.S. application Ser. No. 12/467,255, filed on May 15, 2009. The entire contents of these applications are incorporated by reference in their entirety for all purposes.
Cellular adhesion is the binding of a cell to a surface, extracellular matrix, or another cell using cell adhesion molecules such as integrins, selectins, cadherins, and immunoglobulin-like adhesion molecules.
Integrins are non-covalently linked heterodimers of alpha and beta subunits. They are transmembrane proteins that are constitutively expressed, but require activation in order to bind their ligands. 15 α subunits and 8 β subunits have been identified. These can combine in various ways to form different types of integrin receptors. In many cases, one β subunit combines with several different a subunits to form a subfamily of integrin receptors.
The cadherins are calcium-dependent adhesion molecules. The three most common cadherins are neural (N)-cadherin, placental (P)-cadherin, and epithelial (E)-cadherin. All three belong to the classical cadherin subfamily. There are also desmosomal cadherins and proto-cadherins. Cadherins are involved in embryonic development and tissue organization and exhibit homophilic adhesion. The extracellular domain consists of several cadherin repeats, each capable of binding a calcium ion. When calcium is bound, the extracellular domain has a rigid, rod-like structure. Following the transmembrane domain, the intracellular domain is highly conserved. The intracellular domain is capable of binding catenins. The adhesive properties of the cadherins have been shown to be dependent upon the ability of the intracellular domain to interact with cytoplasmic proteins such as the catenins.
The selectins are a family of divalent cation dependent glycoproteins. They are carbohydrate-binding proteins, binding fucosylated carbohydrates, especially, sialylated Lewis(X), and mucins. The three family members include: Endothelial (E)-selectin, leukocyte (L)-selectin, and platelet (P)-selectin. The extracellular domain of each consists of a carbohydrate recognition motif, an epidermal growth factor (EGF)-like motif, and varying numbers of a short repeated domain related to complement-regulatory proteins (CRP).
The Ig superfamily CAMs are calcium-independent transmembrane glycoproteins. Members of the Ig superfamily include the intercellular adhesion molecules (ICAMs), vascular-cell adhesion molecule (VCAM-1), platelet-endothelial-cell adhesion molecule (PECAM-1), and neural-cell adhesion molecule (NCAM). Each Ig superfamily CAM has an extracellular domain, which contains several Ig-like intrachain disulfide-bonded loops with conserved cysteine residues, a transmembrane domain, and an intracellular domain that interacts with the cytoskeleton. Typically, they bind integrins or other Ig superfamily CAMs.
Defects in cell adhesion molecules have been associated with disease states. For example, leukocyte adhesion deficiency (LAD) syndrome is associated with cell adhesion defects. LAD I is associated with mutations in the β2 integrin. In a severe form, no LFA-1 (αLβ2) is expressed. Patients with this form of LAD I usually die within a few years of birth unless they receive bone marrow transplantation. Patients with a less severe form of the disease express low levels of β2 (i.e., about 2-5% of normal levels) and have a moderate phenotype, but experience numerous types of infections.
Chronic kidney disease (CKD) is a progressive loss in renal function over a period of time. The most common causes of CKD are diabetes mellitus, hypertension, and glomerulonephritis, which cause approximately 75% of all adult cases. To date, there are few treatment options for diabetic nephropathy (DN), the primary cause of chronic kidney disease and end stage renal disease. Thus, there is a significant need in the art for innovative therapies capable of preventing or treating DN and chronic kidney disease.
In one aspect, the present invention provides a vault complex comprising a cell adhesion modifying substance. In certain embodiments, the cell adhesion modifying substance inhibits integrin binding and/or intracellular signaling. The cell adhesion modifying substance can be an RGD-containing peptide, which can be cyclic. In particular embodiments, the RGD-containing peptide is GRGDSP. In other embodiments, the cyclic RGD-containing peptide can be attached to mINT. The cyclic RGD-containing peptide can be modified. In yet further embodiments, the vault complex contains MVP or modified MVP, and can further contain VPARP or modified VPARP, or a portion of VPARP or a modified portion of VPARP.
In another aspect, the present invention provides a pharmaceutical composition for treating and/or preventing and/or causing regression of chronic kidney disease in a subject, comprising a cell adhesion modifying substance incorporated within a vault complex, and at least one pharmaceutically acceptable excipient. In certain embodiments, the cell adhesion modifying substance inhibits integrin binding and/or intracellular signaling. The cell adhesion modifying substance can be an RGD-containing peptide, which can be cyclic. In particular embodiments, the RGD-containing peptide is GRGDSP. In other embodiments, the cyclic RGD-containing peptide can be attached to mINT. The cyclic RGD-containing peptide can be modified. In yet further embodiments, the vault contains contains MVP or modified MVP, and can further contain VPARP or modified VPARP, or a portion of VARP or a modified portion of VPARP.
In a further aspect, the present invention provides a method of treating and/or preventing chronic kidney disease in a subject, by administering to the subject an effective amount of a cell adhesion modifying substance incorporated within a vault complex. The chronic kidney disease can be caused by diabetic nephropathy. In certain embodiments, the cell adhesion modifying substance inhibits integrin binding and/or intracellular signaling. The cell adhesion modifying substance can be an RGD-containing peptide, which can be cyclic. In particular embodiments, the RGD-containing peptide is GRGDSP. In other embodiments, the cyclic RGD-containing peptide can be attached to mINT. The cyclic RGD-containing peptide can be modified. In yet further embodiments, the vault complex contains MVP or modified MVP, and can further contain VPARP or modified VPARP, or a portion of VARP or a modified portion of VPARP.
These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:
The descriptions of various aspects of the invention are presented for purposes of illustration, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Persons skilled in the relevant art can appreciate that many modifications and variations are possible in light of the embodiment teachings.
It should be noted that the language used herein has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the inventive subject matter. Accordingly, the disclosure is intended to be illustrative, but not limiting, of the scope of invention.
It must be noted that, as used in the specification, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the art of the invention. Certain terms are discussed herein to provide additional guidance to the practitioner in describing the compositions, devices, methods and the like of embodiments of the invention, and how to make or use them. It will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms can be used for any one or more of the terms discussed herein. No significance is to be placed upon whether or not a term is elaborated or discussed herein. Some synonyms or substitutable methods, materials and the like are provided. Recital of one or a few synonyms or equivalents does not exclude use of other synonyms or equivalents, unless it is explicitly stated. Use of examples, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the embodiments of the invention herein.
Terms used in the claims and specification are defined as set forth below unless otherwise specified.
As used herein, the term “vault” or “vault particle” refers to a large cytoplasmic ribonucleoprotein (RNP) particle found in eukaryotic cells. The vault or vault particle is composed of MVP, VPARP, and/or TEP1 proteins and one or more untranslated vRNA molecules.
As used herein, the term “vault complex” refers to a vault or recombinant vault that encapsulates a small molecule or protein of interest. A vault complex can include all the components of a vault or vault particle or just a subset. A vault complex with just a subset of the components found in vaults or vault particles can also be termed a “vault-like particle”. Examples of vault-like particles include: 1) MVP without VPARP, TEP1 and vRNA; 2) MVP and either VPARP or a portion of VPARP, without TEP1 and vRNA; 3) MVP and TEP1 or a portion of TEP1 with or without the one or more than one vRNA, and without VPARP; 4) MVP without VPARP, TEP1 and vRNA, where the MVP is modified to attract a specific substance within the vault-like particle, or modified to attract the vault complex to a specific tissue, cell type or environmental medium, or modified both to attract a specific substance within the vault complex and to attract the vault particle to a specific tissue, cell type or environmental medium; and 5) MVP, and either VPARP or a portion of VPARP, or TEP1 or a portion of TEP1 with or without the one or more than one vRNA, or with both VPARP or a portion of VPARP, and TEP1, with or without the one or more than one vRNA, where one or more than one of the MVP, VPARP or portion of VPARP and TEP1 is modified to attract a specific substance within the vault-like particle, or modified to attract the vault particle to a specific tissue, cell type or environmental medium, or modified both to attract a specific substance within the vault complex and to attract the vault complex to a specific tissue, cell type or environmental medium. As used herein, a vault complex is sometimes referred to as a “vault nanoparticle”.
As used herein, the term “vault targeting domain” or “vault interaction domain” is a domain that is responsible for interaction or binding of a heterologous fusion protein with a vault protein, or interaction of a VPARP with a vault protein, such as a MVP. As used herein, the term “mINT domain” is a vault interaction domain from a vault poly ADP-ribose polymerase (VPARP) that is responsible for the interaction of VPARP with a major vault protein (MVP). The term “mINT domain” refers to a major vault protein (MVP) interaction domain.
As used herein, the term “MVP” is major vault protein. The term “cp-MVP” is a cysteine-rich peptide major vault protein.
The term “VPARP” refers to a vault poly ADP-ribose polymerase.
As used herein, the term “TEP-1” is a telomerase/vault associated protein 1.
As used herein, the term “vRNA” is an untranslated RNA molecule found in vaults.
As used herein, a “cell adhesion modifying substance” is an agent which alters the adhesion of a cell to a surface, extracellular matrix, or another cell. The modification can be either inhibitory (decreases cell adhesion) or stimulatory (increases cell adhesion).
As used herein, an “RGD-containing peptide” is a peptide or protein that contains the tri-peptide sequence Arginine-Glycine-Aspartic Acid.
As used herein, the term “vector” is a DNA or RNA molecule used as a vehicle to transfer foreign genetic material into a cell. The four major types of vectors are plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes. Vectors can include an origin of replication, a multi-cloning site, and a selectable marker.
As used herein, a “cell” includes eukaryotic and prokaryotic cells.
As used herein, the terms “organism”, “tissue” and “cell” include naturally occurring organisms, tissues and cells, genetically modified organisms, tissues and cells, and pathological tissues and cells, such as tumor cell lines in vitro and tumors in vivo.
As used herein, the term “extracellular environment” is the environment external to the cell.
As used herein, the term “in vivo” refers to processes that occur in a living organism.
A “subject” referred to herein can be any animal, including a mammal (e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit, primates, etc.), a farm or commercial animal (e.g., a cow, horse, goat, donkey, sheep, etc.), a domestic animal (e.g., cat, dog, ferret, etc.), an avian species, or a human.
The term “mammal” as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.
As used herein, the term “human” refers to “Homo sapiens.”
As used herein, the term “sufficient amount” is an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate cell adhesion.
As used herein, the term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease, such as chronic kidney disease.
A “prophylactically effective amount” refers to an amount that is effective for prophylaxis.
As used herein, the term “stimulating” refers to activating, increasing, or triggering a molecular, cellular or enzymatic activity or response in a cell or organism, e.g. cell adhesion.
As used herein, the term “inhibiting” refers to deactivating, decreasing, or shutting down a molecular, cellular or enzymatic activity or response in a cell or organism, e.g. cell adhesion.
As used herein, the term “administering” includes any suitable route of administration, as will be appreciated by one of ordinary skill in the art with reference to this disclosure, including direct injection into a solid organ, direct injection into a cell mass such as a tumor, inhalation, intraperitoneal injection, intravenous injection, topical application on a mucous membrane, or application to or dispersion within an environmental medium, and a combination of the preceding.
As used herein, the term “treating” or “treatment” refers to the reduction or elimination of symptoms of a disease, e.g., chronic kidney disease.
As used herein, the term “preventing” or “prevention” refers to the reduction or elimination of the onset of symptoms of a disease, e.g., chronic kidney disease.
As used herein, the term “regressing” or “regression” refers to the reduction or reversal of symptoms of a disease after its onset, e.g., improvements in chronic kidney disease.
As used in this disclosure, the term “modified” and variations of the term, such as “modification,” means one or more than one change to the naturally occurring sequence of MVP, VPARP or TEP1 selected from the group consisting of addition of a polypeptide sequence to the C-terminal, addition of a polypeptide sequence to the N-terminal, deletion of between about 1 and 100 amino acid residues from the C-terminal, deletion of between about 1 and 100 amino acid residues from the N-terminal, substitution of one or more than one amino acid residue that does not change the function of the polypeptide, as will be appreciated by one of ordinary skill in the art with reference to this disclosure, such as for example, an alanine to glycine substitution, and a combination of the preceding.
As used herein, the term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).
One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).
As used in this disclosure, the term “comprise” and variations of the term, such as “comprising” and “comprises,” are not intended to exclude other additives, components, integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
Compositions of the Invention
As described in more detail below, the invention includes compositions and methods of using vault complexes. An embodiment of the invention has recombinant vaults having a MVP and an agent, e.g., an RGD-containing peptide. The vault complex can be used for delivery of a biomolecule, e.g., a peptide, to a cell or organ or subject.
Vaults and Vault Complexes
The compositions of the invention comprise a vault complex. A vault complex is a recombinant particle that encapsulates a small molecule (drug, sensor, toxin, etc.), or a protein of interest, e.g., a peptide, or a protein, including an endogenous protein, a heterologous protein, a recombinant protein, or recombinant fusion protein. Vault complexes of the invention can include an RGD-containing peptide.
Vaults, e.g., vault particles are ubiquitous, highly conserved ribonucleoprotein particles found in nearly all eukaryotic tissues and cells, including dendritic cells (DCs), endometrium, and lung, and in phylogeny as diverse as mammals, avians, amphibians, the slime mold Dictyostelium discoideum, and the protozoan Trypanosoma brucei (Izquierdo et al., Am. J. Pathol., 148(3):877-87 (1996)). Vaults have a hollow, barrel-like structure with two protruding end caps, an invaginated waist, and regular small openings surround the vault cap. These openings are large enough to allow small molecules and ions to enter the interior of the vault. Vaults have a mass of about 12.9±1 MDa (Kedersha et al., J. Cell Biol., 112(2):225-35 (1991)) and overall dimensions of about 42×42×75 nm (Kong et al., Structure, 7(4):371-9 (1999)). The volume of the internal vault cavity is approximately 50×103 nm3, which is large enough to enclose an entire ribosomal protein.
Vaults comprise three different proteins, designated MVP, VPARP and TEP1, and comprise one or more different untranslated RNA molecules, designated vRNAs. The number of vRNA can vary. For example, the rat Rattus norvegicus has only one form of vRNA per vault, while humans have three forms of vRNA per vault. The most abundant protein, major vault protein (MVP), is a 95.8 kDa protein in Rattus norvegicus and a 99.3 kDa protein in humans which is present in 96 copies per vault and accounts for about 75% of the total protein mass of the vault particle. The two other proteins, the vault poly-ADP ribose polymerase, VPARP, a 193.3 kDa protein in humans, and the telomerase/vault associated protein 1, TEP1, a 292 kDa protein in Rattus norvegicus and a 290 kDa protein in humans, are each present in between about 2 and 16 copies per vault.
VPARP, mINT Domain, and mINT Fusion Proteins
A vault poly ADP-ribose polymerase (VPARP) includes a region of about 350 amino acids that shares 28% identity with the catalytic domain of poly ADP-ribosyl polymerase, PARP, a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. VPARP catalyzes an NAD-dependent poly ADP-ribosylation reaction, and purified vaults have poly ADP-ribosylation activity that targets MVP, as well as VPARP itself. VPARP includes a mINT domain (major vault protein (MVP) interaction domain). The mINT domain is responsible for the interaction of VPARP with a major vault protein (MVP).
A vault complex of the invention can include a mINT domain. The mINT domain is responsible for interaction of a protein of interest with a vault protein such as a MVP. In some embodiments, the mINT domain is expressed as a fusion protein with a protein of interest. Alternatively, a protein of interest can be covalently or non-covalently attached. The mINT of the vault complexes of the invention are derived from VPARP sequences. Exemplary VPARP sequences and mINT sequences can be found in Table 1. One of skill in the art understands that the mINT can have the entire naturally occurring sequence or portions of the sequence or fragments thereof. In other embodiments, the mINT has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the VPARP and/or mINT sequences disclosed in Table 1.
In one embodiment, the mINT is derived from a human VPARP, SEQ ID NO:3, GenBank accession number AAD47250, encoded by the cDNA, SEQ ID NO:5, GenBank accession number AF158255. In some embodiments, the vault targeting domain comprises or consists of the INT domain corresponding to residues 1473-1724 of human VPARP protein sequence (full human VPARP amino acid sequence is SEQ ID NO:3). In other embodiments, the vault targeting domain comprises or consists of the mINT domain comprising residues 1563-1724 (SEQ ID NO: 2) of the human VPARP protein sequence. In certain embodiments, the vault targeting domain is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or 3.
In alternative embodiments, the mINT domain is derived from TEP1 sequences. One of skill in the art understands that the mINT can have the entire naturally occurring sequence of the vault interaction domain in TEP1 or portions of the sequence or fragments thereof.
MVP
A vault complex of the invention can include an MVP. Exemplary MVP sequences can be found in Table 1. One of skill in the art understands that the MVP can have the entire naturally occurring sequence or portions of the sequence or fragments thereof. In other embodiments, the MVP has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the MVP sequences disclosed in Table 1.
In one embodiment, the MVP is human MVP, SEQ ID NO:6, GenBank accession number CAA56256, encoded by the cDNA, SEQ ID NO:7, GenBank accession number X79882. In other embodiments, the MVP is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the MVP sequences described herein.
In one embodiment, there is provided a vault complex comprising, consisting essentially of, or consisting of an MVP modified by adding a peptide to the N-terminal to create a one or more than one of heavy metal binding domains. In a preferred embodiment, the heavy metal binding domains bind a heavy metal selected from the group consisting of cadmium, copper, gold and mercury. In a preferred embodiment, the peptide added to the N-terminal is a cysteine-rich peptide (CP), such as for example, SEQ ID NO:8, the MVP is human MVP, SEQ ID NO:6, and the modification results in CP-MVP, SEQ ID NO:9, encoded by the cDNA, SEQ ID NO:10. These embodiments are particularly useful because vault particles consisting of CP-MVP are stable without the presence of other vault proteins.
Any of the vault complexes described herein can include MVPs or modified MVPs disclosed herein.
TEP1
In some embodiments, a vault complex of the invention can include a TEP1 protein. Exemplary TEP1 sequences can be found in Table 1. One of skill in the art understands that the TEP1 can have the entire naturally occurring sequence or portions of the sequence or fragments thereof. In other embodiments, the TEP1 has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the TEP1 sequences disclosed in Table 1.
The TEP1 can be human TEP1, SEQ ID NO:11, GenBank accession number AAC51107, encoded by the cDNA, SEQ ID NO:12, GenBank accession number U86136. Any of the vault complexes described herein can include TEP1 or modifications thereof.
vRNA
A vault complex of the invention can include a vRNA. Exemplary vRNA sequences can be found in Table 1. One of skill in the art understands that the vRNA can have the entire naturally occurring sequence or portions of the sequence or fragments thereof. In other embodiments, the vRNA has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the vRNA sequences disclosed in Table 1.
In one embodiment, the vRNA can be a human vRNA, SEQ ID NO:13, GenBank accession number AF045143, SEQ ID NO:14, GenBank accession number AF045144, or SEQ ID NO:15, GenBank accession number AF045145, or a combination of the preceding.
As will be appreciated by one of ordinary skill in the art with reference to this disclosure, the actual sequence of any of MVP, VPARP, TEP1 and vRNAs can be from any species suitable for the purposes disclosed in this disclosure, even though reference or examples are made to sequences from specific species. Further, as will be appreciated by one of ordinary skill in the art with reference to this disclosure, there are some intraspecies variations in the sequences of MVP, VPARP, TEP1 and vRNAs that are not relevant to the purposes of the present invention. Therefore, references to MVP, VPARP, TEP1 and vRNAs are intended to include such intraspecies variants.
Cell Adhesion Modifying Agents
As used herein a “cell adhesion modifying substance” is an agent which modifies cell adhesion mediated by cell adhesion proteins, including, but not limited, to integrins, cadherins, selectins, or Ig superfamily CAMs. A cell adhesion modifying agent may be a peptide, protein, pharmaceutical agent, drug, compound, or composition that is useful in modifying cell adhesion. The modifying agent may, e.g., stimulate or inhibit cell adhesion.
In one advantageous embodiment, the cell adhesion modifying agent may inhibit cell adhesion mediated by integrins.
Ligands for Integrins
Mammalian genomes contain 18 α subunit and 8 β subunit genes, and 24 different αβ combinations have been identified at the protein level. Integrins mediate cell adhesion to a number of ligands, including extracellular matrix proteins, such as fibronectin, laminin, collagen, thrombospondin, VCAM-1, among others. See, e.g., Humphries et al., J. Cell Sci., 119: 3901-3903 (2006) for a review.
A number of integrin binding ligands share in common an “RGD” peptide motif. All five αV integrins, two β1 integrins (α5, α8) and αIIbβ3 share the ability to recognize ligands containing an RGD tripeptide active site. Crystal structures of αVβ3 and αIIbβ3 complexed with RGD ligands have revealed an identical atomic basis for this interaction (Xiong et al., 2002; Xiao et al., 2004). RGD binds at an interface between the α and β subunits, the R residue fitting into a cleft in a β-propeller module in the α subunit, and the D coordinating a cation bound in a von Willebrand factor A domain in the β subunit. The RGD binding integrins bind the greatest variety of ligands, with β3 integrins binding to a large number of extracellular matrix and soluble vascular ligands.
α4β1, α4β7, α9β1, the four members of the β2 subfamily and αEβ7 recognize related sequences in their ligands. α4β1, α4β7 and α9β1 bind to an acidic motif, termed ‘LDV’, that is functionally related to RGD. Fibronectin contains the prototype LDV ligand in its type III connecting segment region, but other ligands (such as VCAM-1 and MAdCAM-1) employ related sequences.
It is thought that LDV peptides bind at the junction between the α and β subunits in a manner similar to RGD.
Four α subunits containing an α A domain (α1, α2, α10 and α11) combine with α1 and form a distinct laminin/collagen-binding subfamily.
Three α1 integrins (α3, α6 and α7), plus α6β4, are highly selective laminin receptors.
As disclosed herein, one class of cell adhesion modifying agents include peptides and proteins which comprise the “RGD” peptide sequence. Peptides containing the “RGD” motif have an inhibitory effect on cell adhesion. An exemplary RGD-peptide has the sequence GRGDSP. As discussed herein, peptides comprising “RGE”, e.g., GRGESP, are frequently used as negative controls. The peptides can be used in either a linear or cyclic form. However, in some embodiments, a cyclic form of the peptide is preferred for in vivo use as it has greater bioavailability.
RGD-Containing Peptides
Compositions for treating chronic kidney disease (CKD) are provided herein comprising a cyclized RGD (Arg-Gly-Asp)-containing peptide agent associated with a recombinant vault nanoparticle delivery vehicle. The RGD peptide agents prevent and/or reverse pathological glomerular lesions associated with diabetic nephropathy and other forms of CKD by inhibiting interactions between mesangial cell integrins and extracellular matrix proteins. For example, the RGD peptide agents have been shown to block α5β1 integrin-mediated primary mesangial cell adhesion to fibronectin (FN), the predominant extracellular matrix protein accumulated in DN, by ˜50% in vitro. The RGD peptide agent have also been shown to significantly reduce urinary albumin and mesangium expansion in type 2 diabetic db/db diabetic mice to levels observed in type 2 non-diabetic db/m control animals (
In some aspects, RGD-peptide agents provided herein are modified to include a free cysteine residue. For example, exemplary RGD peptide-vault nanoparticles were constructed using a modified form of the RGD peptide agent, GRGDSP, which comprises a free cysteine residue (referred to as CGRGDSP). The free cysteine was utilized to attach the RGD peptide to one or more available cysteines on the vault mINT domain. Linking RGD peptides to mINT allows the peptides to be packaged in the interior of vault nanoparticles due The exemplary RGD peptide-vault nanoparticles exhibited a consistent barrel-shaped vault structure when visualized by electron microscopy. Using a cell adhesion assay, the RGD peptide-vault nanoparticles were shown to be as efficacious as the cysteine modified RGD-peptide in inhibiting α5β1 integrin-mediated mesangial cell adhesion to FN (
Advantageously, cysteine-modified RGD peptides, such as CGRGDSP, are more efficient in preventing progression of early DN and/or causing regression of established lesions of DN than unmodified RGD peptides. Further, cysteine-modified RGD peptide-vault nanoparticle compositions can considerably enhance in vivo delivery of the modified RGD peptides.
In some aspects, vault nanoparticles provided herein may further comprise a targeting agent, such as an antibody or an antibody fragment, which binds selectively to a therapeutic target and/or a molecule in the vicinity of a therapeutic target. For example, in some aspects, RGD peptide-vault particles are targeted at or near the α5β1-fibronectin active site. Advantageously, targeted RGD-vault nanoparticles modulate α5β1 integrin-FN signaling and/or halt progressive albuminuria in db/db mice to a similar or greater degree than the corresponding free RGD peptides.
In some aspects, mINT can be modified to contain one or more additional cysteine residues to increase binding of the cyclic RGD peptide, allowing a higher concentration of RGD peptide to be packaged inside of the vault. For example, the vault particle, CP-MVP, contains extra cysteine residues at the N-terminus. In further aspects, cyclic RGD peptides can be linked to vault particles directly via one or more cysteine residues and/or other moieties.
In some aspects, polymers of RGD sequences are engineered into mINT and/or MVP that contain flanking sequences that would allow for the cyclization following translation to form cyclic peptides. For example, flanking zinc finger motifs would cyclize in the presence of zinc ions, or flanking poly-histidine residues would cyclize in the presence of nickel. The advantage of this approach would be that the peptides would not have to be chemically synthesized thus the therapeutic vault easier to produce for therapeutic applications.
Other cell adhesion modifying agents which are useful in the practice of aspects of the invention are known in the art, as disclosed, e.g., in Horton, Exp. Nephrology, 7: 178-184 (1999). Such agents include naturally occurring protein inhibitors and derivatives (e.g., RGD-containing snake toxins), blocking antibodies to adhesion molecules, RGD-peptides and chemical derivatives, oligosaccharide analogues (e.g., for selectin inhibition), receptor-immunoglobulin chimeras, non-peptidic mimetics, antisense and siRNA nucleic acids, among others.
Isolated Nucleic Acids and Vectors
Suitable expression vectors generally include DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of expression vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
Plasmids expressing a nucleic acid sequence can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g., EPV and EBV vectors. Constructs for the recombinant expression of a nucleic acid encoding a fusion protein will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the fusion nucleic acid in target cells. Other aspects to consider for vectors and constructs are further described below.
Vectors useful for the delivery of a nucleic acid can include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the nucleic acid in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression. A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the transgene.
In a specific embodiment, viral vectors that contain the recombinant gene can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding a fusion protein are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.
Adenoviruses are also contemplated for use in delivery of isolated nucleic acids encoding fusion proteins into a cell. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia or for use in adenovirus-based delivery systems such as delivery to the liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing a nucleic acid molecule featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
Use of Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
Another preferred viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.
The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
Examples of additional expression vectors that can be used in the invention include pFASTBAC expression vectors and E. coli pET28a expression vectors.
Generally, recombinant vectors capable of expressing genes for recombinant fusion proteins are delivered into and persist in target cells. The vectors or plasmids can be transfected into target cells by a transfection agent, such as Lipofectamine. Examples of cells useful for expressing the nucleic acids encoding the fusion proteins of the invention include Sf9 cells or insect larvae cells. Recombinant vaults based on expression of the MVP protein alone can be produced in insect cells. Stephen, A. G. et al. (2001). J. Biol. Chem. 276:23217:23220; Poderycki, M. J., et al. (2006). Biochemistry (Mosc). 45: 12184-12193.
Pharmaceutical Compositions of the Invention
In one embodiment, the invention provides methods using pharmaceutical compositions comprising the vault complexes of the invention. These compositions can comprise, in addition to one or more of the vault complexes, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
In certain embodiments, the pharmaceutical compositions that are injected intra-tumorally comprise an isotonic or other suitable carrier fluid or solution.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included, as required.
In other embodiments, pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.
In some embodiments, administration of the pharmaceutical compositions may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration. Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.
Methods of Use
Vault complexes described herein can be used to deliver a protein of interest to a cell, a tissue, an environment outside a cell, a tumor, an organism or a subject. In one embodiment, the vault complex comprises an RGD-containing peptide, and the vault complex is introduced to the cell, tissue, or tumor. In some embodiments, the vault complex is introduced into the extracellular environment surrounding the cell. In other embodiments, the vault complex is introduced into an organism or subject. Delivery of the vault complex of the invention can include administering the vault complex to a specific tissue, specific cells, an environmental medium, or to the organism.
The methods of the invention comprise delivering a biomolecule to a cell by contacting the cell with any of the vault complexes described herein. Cells of the invention can include, but are not limited to, any eukaryotic cell, mammalian cell, or human cells, including tumor cells.
Methods of the invention include delivery of the vault complex to a subject. The delivery of a vault complex to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a vault complex to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the vault complex or components of the vault complex. In one embodiment, the vault complex is administered to a mammal, such as a mouse or rat. In another embodiment, the vault complex is administered to a human.
In another embodiment, the methods of delivery of the invention include systemic injection of vault.
Methods of Treatment
The invention features a method of treating or managing disease, such as chronic kidney disease, by administering the vault complex of the invention to a subject (e.g., patient). In some embodiments, the method of the invention comprises treating or managing chronic kidney disease in a subject in need of such treatment or management, comprising administering to the subject a therapeutically effective amount of the vault complexes described herein.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the vault complex. Such information can be used to more accurately determine useful doses in humans.
The pharmaceutical composition according to the present invention to be given to a subject, administration is preferably in a “therapeutically effective amount” or “prophylactically effective amount” (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980. A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
In certain embodiments, the dosage of vault complexes is between about 0.1 and 10,000 micrograms per kilogram of body weight or environmental medium. In another embodiment, the dosage of vault complexes is between about 1 and 1,000 micrograms per kilogram of body weight or environmental medium. In another embodiment, the dosage of vault complexes is between about 10 and 1,000 micrograms per kilogram of body weight or environmental medium. For intravenous injection and intraperitoneal injection, the dosage is preferably administered in a final volume of between about 0.1 and 10 ml. For inhalation the dosage is preferably administered in a final volume of between about 0.01 and 1 ml. As will be appreciated by one of ordinary skill in the art with reference to this disclosure, the dose can be repeated a one or multiple times as needed using the same parameters to effect the purposes disclosed in this disclosure.
For instance, the pharmaceutical composition may be administered once to a subject, or the vault complex may be administered as two, three, or more sub-doses or injections at appropriate intervals. In that case, the vault complexes can be injected in sub-doses in order to achieve the total required dosage.
The vault complexes featured in the invention can be administered in combination with other known agents effective in treatment of chronic kidney disease. An administering physician can adjust the amount and timing of vault complex administration or injection on the basis of results observed using standard measures of efficacy known in the art or described herein. The skilled artisan will also appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
Methods of Preparing Vault Complexes
The methods of the invention include preparing the vault complexes described herein.
In one embodiment, the vault complexes are derived or purified from natural sources, such as mammalian liver or spleen tissue, using methods known to those with skill in the art, such as for example tissue homogenization, differential centrifugation, discontinuous sucrose gradient fractionation and cesium chloride gradient fractionation. In another embodiment, the vault complexes are made using recombinant technology.
In some embodiments, a target of interest, i.e., protein of interest, is selected for packaging in the vault complexes. The target of interest may be selected from the group consisting of an enzyme, a pharmaceutical agent, a plasmid, a polynucleotide, a polypeptide, a sensor and a combination of the preceding. In a preferred embodiment, the target of interest is a recombinant protein, e.g., a cell adhesion modifying substance, e.g., an RGD-containing peptide.
Preferably, if the target of interest is a recombinant protein, the polynucleotide sequences encoding the recombinant protein are used to generate a bacmid DNA, which is used to generate a baculovirus comprising the sequence. The baculovirus is then used to infect insect cells for protein production using an in situ assembly system, such as the baculovirus protein expression system, according to standard techniques, as will be appreciated by one of ordinary skill in the art with reference to this disclosure. Advantageously, the baculovirus protein expression system can be used to produce milligram quantities of vault complexes, and this system can be scaled up to allow production of gram quantities of vault complexes according to the present invention.
In another embodiment, the target of interest is incorporated into the provided vaults. In one embodiment, incorporation is accomplished by incubating the vaults with the target of interest at an appropriate temperature and for an appropriate time, as will be appreciated by one of ordinary skill in the art with reference to this disclosure. The vaults containing the protein of interest are then purified, such as, for example sucrose gradient fractionation, as will be appreciated by one of ordinary skill in the art with reference to this disclosure.
In other embodiments, the vaults comprising the target of interest are administered to an organism, to a specific tissue, to specific cells, or to an environmental medium. Administration is accomplished using any suitable route, as will be appreciated by one of ordinary skill in the art with reference to this disclosure.
In one embodiment, the method comprises preparing the composition of the invention by a) mixing a fusion protein comprising a RGD-containing peptide fused to a mINT generated in Sf9 cells with a rat MVP generated in Sf9 cells to generate a mixture; b) incubating the mixture for a sufficient period of time to allow packaging of the fusion protein inside of vault complexes, thereby generating the composition. Sf9 cells are infected with pVI-MVP encoding recombinant baculoviruses. Lysates containing recombinant RGD-peptide-INT and rat MVP generated in Sf-9 cells can be mixed to allow the formation of a macromolecular vault complex containing the RGD-peptide-INT fusion protein.
In another embodiment, the composition is prepared by a) mixing a fusion protein comprising an RGD-peptide fused to a mINT generated in insect larvae cells with a rat MVP generated in insect larvae cells to generate a mixture; b) incubating the mixture for a sufficient period of time to allow packaging of the fusion protein inside of vault complexes.
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B(1992).
We investigated the ability of cyclic GRGDSP vs. GRGESP in a pilot study to prevent accumulation of glomerular lesions in early DN in 20 week old diabetic type 2 db/db vs non-diabetic db/m and diabetic type 1 Ins2Akita+ vs. non-diabetic Ins2+/+ mice with GRGESP and GRGESP (400-2400 μg/kg) and observed up to 52% reduction in albuminuria in the type 2 diabetic mice (
We investigated the ability of cyclic GRGDSP vs. GRGESP in a pilot study to reverse established DN and treated 21-week-old diabetic db/db mice with GRGDSP and GRGESP (2400-4800 μg/kg) and observed up to 71% reduction in albuminuria (
Vaults are self-assembled from 96 copies of the major vault protein (MVP) to provide a dynamic, accessible internal volume (5×107 Å) with a cysteine-rich 162aa sequence (INT-domain) on the C-terminus of the vault poly ADP-ribose polymerase, which interacts with MVP. The vault dimension is 72.5×41 nm. In order to design RGD-containing-vault nanocapsules, cyclic GRGDSP- and GRGESP-control peptides were modified to incorporate a free cysteine residue to allow formation of disulfide bonds between the peptides and one or more free cysteine residues on the INT-domain (22 kDa) of vault MVP. Incorporation of the cyclic GRGDSP- and GRGESP-control peptides into vault nanocapsules was monitored during generation and dialysis-purification steps by detecting free SH groups using Ellman's reagent and a cysteine standard based on molar absorbance at 412 nm. D-peptide was incubated with INT and GL-INT (which is a variant with fluorescent green lantern (GL) protein fused to the INT-domain to facilitate vault visualization in vitro and in vivo) in binding buffer containing glutathione redox pairs ×1 h and dialyzed (MWCO 12-14 kDa).
The CGRGDSP peptide was more potent in inhibiting α5β1 integrin receptor binding to FN than the GRGDSP peptide at comparable dose. In particular, CGRGDSP showed a 2.4-fold and 2.8 fold enhanced potency of D- vs. E-control peptide (200 μg/ml and 400 μg/ml, respectively) in inhibiting MC adhesion to fibronectin-coated plates compared to GRGDSP (
Naturally occurring vaults (MVP) were found to be present in primary mesangial cells and renal tissues (
Active RGD vault nanocapsules were observed by immunofluorescence to be primarily localized to the cell surface of primary mesangial cells (
CGRGDSP peptide D-vault and E-vault (control) samples were generated by incubation of peptide-INT with purified vault (96MVP's; ˜100 kDa) and dialyzed (MWCO 50 kDa) until no free SH groups were detected. Adhesion assays demonstrated that D-vault inhibited attachment of MC to fibronectin 1.7-fold compared to untreated MC, p<0.05, a level of inhibition comparable to that observed with GRGDSP. There was no inhibition of adhesion with the empty vault or E-vault controls (
The above results demonstrate that when the RGD inhibitor is inserted, it will attach to the integrin, thereby blocking the fibronectin from adhesion. Preferably, the synthetic analog inhibitors should have higher affinity to the integrin than the RGD loop of fibronectin. Regarding the potential antibody selection, since the six-member amino acid cyc-RGDSPG has been proved to be a successful candidate of inhibitors, it was used as a standard reference structure for the design of potential inhibitors. It was determined that new compounds that are structurally similar to cyc-RGDSPG can also act as efficient inhibitors. For example, two additional RGD peptides (cyc-RGDSPCG and cyc-RGDSPSG) have been confirmed to be efficient inhibitors. These two cyclic peptides were also used as reference structures to construct new inhibitors.
The investigations began with the predictions of characteristics of cyc-RGDSPG, cyc-RGDSPCG and cyc-RGDSPSG (which are six-member and seven-member amino acids related to the cyclic pattern RGD inhibitors). Various conformers of the above three peptides were studied. The calculations included optimizations of molecular structures, predictions of electrostatic potentials, and computations of charge density distribution.
As the next step, the five-member amino acid of cyc-RGDSG were studied to check if the amino acid P has any influence on the properties of the RGD fragment of the cyc-RGDSPG. Also, cyc-RGDGPS and cyc-RGDGS, which differ by the positions of G and S from the known inhibitor, were evaluated. Through comparison of the properties of cyc-RGDGPS, cyc-RGDSPG, cyc-RGDGS, and cyc-RGDSG, it was possible to reveal how the differences of the molecular structures of considered compounds influence their properties.
Based on the results from the previous step, analogous compounds having high activity were designed. Various structures were tested, including the cyclic types derived from the five-member amino acid RGD inhibitors, six-member amino acid RGD inhibitors, seven-member amino acid RGD inhibitors, and linear-type inhibitors which are constructed by 3-7 amino acids, including the RGD fragment.
Nonempirical—reliable Density Functional Theory at the B3LYP/6-31G(d,p) level is used in this study. Molecular geometries of various components have been fully optimized. So far, six different conformers of the linear RGD chain have been located. The geometrical parameters, stabilities, electronic energies, charge distribution and other properties of these conformers were analyzed and compared.
According to the experimentally-verified results, one energy minima structure has been located for each cyc-RGDSPG, cyc-RGDSPCG, and cyc-RGDSPSG complex. Also different conformers of these cyclic RGD inhibitors were located on their respective potential energy surfaces. These results were compared with the RGD chain conformers to establish the most favorable conformer structure for the inhibition.
Combining the experimental results with the data obtained from our calculations we were able to predict the efficiency of the various inhibitors and to determine which structure is the most favorable fragment with regard to integrin binding energy.
While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.
All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.
This application claims the benefit of U.S. Provisional Application No. 61/326,518, filed Apr. 21, 2010, the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes.
This invention was made with support from the Government under Grant No. K08DK059343 awarded by the National Institutes of Health/National Institute of Diabetes and Digestive Kidney Diseases. The Government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 61326518 | Apr 2010 | US |
