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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate
  • C12N2310/3513—Protein; Peptide

Definitions

• the present invention relates to conjugates between p97 (melanotransferrin) and polynucleotides such as small interfering RNA (siRNA) molecules, and related compositions and methods of use thereof, for instance, to facilitate delivery of siRNA agents across the blood-brain barrier (BBB) and/or improve their tissue penetration in CNS and/or peripheral tissues, and thereby treat and/or diagnose various diseases, including those having a central nervous system (CNS) component.
• p97 melanotransferrin
• siRNA small interfering RNA
• RNA interference is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. It may be possible to exploit RNA interference in therapy. Although it is difficult to introduce long dsRNA strands into mammalian cells due to the interferon response, the use of short interfering RNA (siRNA) has been more successful.
• BBB blood-brain barrier
• Em bodiments of the present invention include conjugates between p97 (melanotransferrin) and polynucleotides such as small interfering NA (siRNA) molecules, and related compositions and methods of use thereof.
• certain em bodiments include p97 conjugates, comprising a p97 polypeptide that is covalently linked to a first polynucleotide of a bout 15-40 nucleotides in length with an optional linker (L) in between, a nd a second polynucleotide of a bout 15-40 nucleotides in length which is su bstantially complementary to and hybridized to the first polynucleotide, where the first or second polynucleotide is an antisense strand that is su bstantially complementary to a target mRNA sequence.
• L optional linker
• the first and second polynucleotides form 1-3 nucleotide overhang(s) at the 3' end of the antisense strand, the 5' end of the antisense strand, or both.
• the first polynucleotide is the antisense strand and the second polynucleotide is a sense strand.
• the second polynucleotide is the antisense strand and the first polynucleotide is a sense strand.
• the first a nd second polynucleotides are a bout 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.
• the antisense strand is su bstantially or fully complementary to a target mRNA sequence of a gene selected from Table 2.
• the gene is a human NOX gene.
• the human NOX gene is one or more of NOXl, NOX2, or NOX4.
• the human NOX gene is NOX4 and where the first and second polynucleotides are selected from 5'-A UGU UCA CAA AGU CAG GUC TT-3' (SEQ I D NO:31) and 5'-GAC CUG ACU UUG UGA ACA UTT-3' (SEQ I D NO:32).
• the p97 polypeptide comprises (a) an amino acid sequence set forth in SEQ I D NO: 1-30 or Table 1 or Table B; (b) an amino acid sequence at least 90% identical to a sequence of (a); (c) or an amino acid sequence that differs from a sequence of (a) by addition, su bstitution, insertion, or deletion of a bout 1-50 amino acids.
• the p97 polypeptide is a bout 10-50 amino acids in length.
• the p97 polypeptide is a bout 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length.
• the p97 polypeptide comprises, consists, or consists essentially of DSSHAFTLDELR (SEQ ID NO:14) or DSSHAFTLDELRYC (SEQ ID NO:29) or DSSHAFTLDELRC (SEQ I D NO:30).
• the p97 polypeptide has a cysteine residue at the C-terminus, the N-terminus, or both.
• the conjugate comprises the linker (L) in between.
• pharmaceutical compositions comprising a pharmaceutically-acceptable carrier and a p97 conjugate described herein, where the pharmaceutical composition is sterile and non-pyrogenic.
• the target gene is a human NOX gene, optionally NOX1, NOX2, or NOX4, and where the subject has a NOX-associated disease or condition of the central nervous system (CNS).
• the NOX-associated disease or condition of the CNS is a neurodegenerative disease, a psychiatric disease, ischemia, stroke, CNS trauma, or neuropathic pain.
• the p97 conjugate or pharmaceutical composition is administered by intravenous (IV) infusion or subcutaneous injection.
• Figures 1A-1L illustrate the general structure of exemplary conjugates having a p97
• (melanotransferrin) polypeptide that is covalently linked least one strand of an siRNA molecule or an shRNA molecule, and optionally a linker (L).
• Figure 2 illustrates the structure of an exemplary p97-siRNA conjugate (see Example 1).
• Figure 3 shows the location of the four fields of view in the brain sections analyzed (see Example 1).
• Figures 4A-4D show immunofluorescence imaging of mice brain tissues following intravenous administration of test agents.
• Control test agents PBS only (4A) and unconjugated siRNA (4B) show no AF680 (red) staining in brain tissues.
• p97-siRNA conjugates (4C-4D) show significant AF680 (red) staining in brain tissues, evidencing that conjugation to p97 polypeptides can enhance delivery of siRNA molecules into CNS tissues such as the brain parenchyma.
• Figures 5A-5B show exemplary modifications to the sugar (5A) and nucleobase (5B) chemistries of siRNA molecules.
• Figure 6 illustrates a reaction scheme for conjugating a p97 polypeptide to a polynucleotide strand of an siRNA molecule to create a p97-siRNA conjugate.
• Figure 7 shows the volume fractions of the siRNA and p97-siRNA conjugate in brain tissues relative to the PBS control.
• Figure 8 shows the RT-PCR results for the seven different mice tested (one PBS control and three for each siRNA test molecule, as indicated). Levels of NOX4 mRNA are shown relative to GAPDH control.
• Figure 9A shows that NOX4 mRNA is significantly down-regulated in brain tissues from mice treated with p97-siRNA conjugate relative to controls (PBS and unconjugated siRNA).
• Figure 9B shows the percentage reduction in endogenous NOX4 expression in brain tissues from mice treated with p97-siRNA conjugate relative to unconjugated siRNA.
• Figure 9C shows the fold-reduction ( ⁇ 1.8-fold) in NOX4 expression in brain tissues from mice treated with p97-siRNA conjugate relative to unconjugated siRNA.
• Figure 10 shows that mice pretreated with p97-siRNA conjugate (MTfp-siRNA) and subject to ischemic stroke induction by middle cerebral artery occlusion (MCAO) had significantly smaller infarct areas relative to the PBS and siRNA-only groups.
• MTfp-siRNA p97-siRNA conjugate
• MCAO middle cerebral artery occlusion
• Figure 11 shows that pretreatment with MTfp-siRNA conjugates improved the neurological deficit in the animals at 0.5 hours post-stroke induction relative to pretreatment with PBS or siRNA- only.
• Figures 12A-12B show the results of RT-PCR analysis of NOX4 mRNA levels following pretreatment and stroke induction.
• AACT (CT(Nox4,PBS-sham)-CT(B-actin, PBS-sham))-(CT(Nox4, MTfp-siRNA-stroke)-CT(B-actin, MTfp- siRNA-stroke)).
• Oligonucleotide Synthesis (N. Gait, ed., 1984); Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed., 2004); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Nucleic Acid Hybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Freshney, R.I. (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th Ed.
• an element means one element or more than one element.
• amino acid is intended to mean both naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics.
• Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example.
• Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art.
• Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids.
• Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid.
• Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics Arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid.
• Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.
• conjugate is intended to refer to the entity formed as a result of covalent or non- covalent attachment or linkage of an agent or other molecule, e.g., a biologically active molecule, to a p97 polypeptide.
• the terms “function” and “functional” and the like refer to a biological, enzymatic, or therapeutic function.
• Homology refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al., Nucleic Acids Research. 12, 387-395, 1984), which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
• isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
• an "isolated peptide” or an “isolated polypeptide” and the like, as used herein, includes the in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell; i.e., it is not significantly associated with in vivo substances.
• modulating and “altering” include “increasing,” “enhancing” or “stimulating,” as well as “decreasing” or “reducing,” typically in a statistically significant or a physiologically significant amount or degree relative to a control.
• An “increased,” “stimulated” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and a bove 1, e.g., 1.5, 1.6, 1.7.
• the amount produced by no composition e.g., the a bsence of a conjugate of the invention
• a control composition sample or test su bject.
• a "decreased” or “reduced” amount is typically a "statistically significant” amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in the amount produced by no composition or a control composition, including all integers in between.
• a control could compare the activity, such as the amount or rate of transport/delivery across the blood brain barrier, the rate and/or levels of distribution to central nervous system tissue, and/or the C max for plasma, central nervous system tissues, or any other systemic or peripheral non-central nervous system tissues, or the a bility to attenuate expression of a target gene (e.g., in CNS tissues), of a p97-siRNA conjugate relative to the siRNA molecule alone.
• a target gene e.g., in CNS tissues
• Other examples of comparisons and "statistically significant" amounts are described herein.
• Attenuating expression with reference to a gene or an m RNA as used herein means administering or expressing an amount of an siRNA to reduce translation of a target mRNA into protein, either through mRNA cleavage or through direct inhibition of translation.
• inhibitor means administering or expressing an amount of an siRNA to reduce translation of a target mRNA into protein, either through mRNA cleavage or through direct inhibition of translation.
• inhibitor means administering or expressing an amount of an siRNA to reduce translation of a target mRNA into protein, either through mRNA cleavage or through direct inhibition of translation.
• the terms “inhibit,” “silencing,” and “attenuating” as used herein refer to a measura ble reduction in expression of a target m RNA or the correspond ing protein as compared with the expression of the target m RNA or the corresponding protein in the a bsence of an interfering RNA of the invention.
• the red uction in expression of the target m RNA or the correspond ing protein can be evaluated relative to levels present following administration or expression of a non-targeting control siRNA and/or an unconjugated siRNA.
• a p97-siRNA conjugate has "increased" attenuation of target gene expression in CNS tissues (e.g., upon peripheral administration in vivo) relative to a correspond ing, u nconjugated siRNA.
• nucleotide refers to a ribonucleotide or a deoxyribonucleotide or modified form thereof, as well as an analog thereof.
• Nucleotides include species that comprise pu rines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs. In some embodiments, all nucleotides are selected from the group of modified or unmodified A, C, G or U.
• nucleotide is also meant to include modified bases and universal bases.
• universal bases include but are not limited to 3-nitropyrrole, 5-nitroindole, or nebularine.
• nucleotide is also meant to include the N3' to P5' phosphoramidate, resulting from the su bstitution of a ribosyl 3' oxygen with an amine group.
• nucleotide also includes those species that have a detecta ble la bel, such as for example a radioactive or fluorescent moiety, or mass la bel attached to the nucleotide.
• compositions may comprise a n agent that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure, including all decimals in between, as measured, for example and by no means limiting, by high pressure liquid chromatography (H PLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.
• H PLC high pressure liquid chromatography
• polypeptide and protein are used interchangea bly herein to refer to a polymer of a mino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid resid ues are synthetic non- naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
• the polypeptides described herein are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangea bly herein unless specifically indicated otherwise.
• polypeptides described herein may also comprise post- expression modifications, such as glycosylations, acetylations, phosphorylations and the like, as well as other mod ifications known in the art, both naturally occurring and non-naturally occurring.
• a polypeptide may be an entire protein, or a su bsequence, fragment, variant, or derivative thereof.
• a “physiologically cleavable” or “hydrolyzable” or “degradable” linker or bond is a bond that reacts with water (i.e., is hydrolyzed ) under physiological cond itions.
• the tendency of a bond to hydrolyze in water will depend not only on the general type of linkage connecting two central atoms but also on the su bstituents attached to these central atoms.
• Appropriate hydrolytically unsta ble or weak linkages include, but are not limited to: carboxylate ester, phosphate ester, anhydride, acetal, ketal, acyloxyalkyl ether, imine, orthoester, thio ester, thiol ester, carbonate, and hydrazone, peptides and oligonucleotides.
• a “releasable linker” includes, but is not limited to, a physiologically cleava ble linker a nd an enzymatically degrada ble linker.
• a “releasa ble linker” is a linker that may undergo either spontaneous hydrolysis, or cleavage by some other mechanism (e.g., enzyme-catalyzed, acid- catalyzed, base-catalyzed, and so forth) under physiological conditions.
• a “releasa ble linker” can involve an elimination reaction that has a base a bstraction of a proton, (e.g., an ioniza ble hydrogen atom, Ha), as the driving force.
• a "releasa ble linker” is synonymous with a “degrada ble linker.”
• An “enzymatically degradable linkage” includes a linkage, e.g., amino acid sequence that is su bject to degradation by one or more enzymes, e.g., peptidases or proteases.
• a releasa ble or otherwise degradable linker has a half-life at pH 7.4, 25°C, e.g., a physiological pH, human body temperature [e.g., in vivo), of a bout 30 minutes, a bout 1 hour, a bout 2 hour, a bout 3 hours, a bout 4 hours, a bout 5 hours, a bout 6 hours, a bout 12 hours, a bout 18 hours, a bout 24 hours, a bout 36 hours, a bout 48 hou rs, a bout 72 hou rs, or a bout 96 hours or less.
• pH 7.4, 25°C e.g., a physiological pH, human body temperature [e.g., in vivo), of a bout 30 minutes, a bout 1 hour, a bout 2 hour, a bout 3 hours, a bout 4 hours, a bout 5 hours, a bout 6 hours, a bout 12 hours, a bout 18 hours, a bout 24 hours, a bout 36 hours,
• reference sequence refers generally to a nucleic acid cod ing sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide a nd polynucleotide sequences described herein are included as references sequences, including those described by name a nd those described in the Sequence Listing.
• sequence identity or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
• a "percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base [e.g., A, T, C, G, I) or the identical amino acid residue [e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the num ber of matched positions, dividing the nu mber of matched positions by the total num ber of positions in the window of comparison [i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• the identical nucleic acid base e.g., A, T, C, G, I
• the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr,
• nucleotides and polypeptides having at least a bout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing), typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.
• sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window,” “sequence identity,”
• a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides a nd amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
• a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually a bout 50 to a bout 100, more usually a bout 100 to a bout 150 in which a sequence is compared to a reference sequence of the same nu mber of contiguous positions after the two sequences are optimally aligned.
• the comparison window may comprise additions or deletions (i.e., gaps) of a bout 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wl, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the va rious methods selected.
• GAP Garnier et al.
• FASTA Altschul et al.
• TFASTA TFASTA
• Statistical significance it is meant that the result was unlikely to have occurred by chance.
• Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or proba bility with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significa nce level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.
• solubility refers to the property of a protein or conjugate to dissolve in a liquid solvent and form a homogeneous solution.
• Solu bility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per d L (100 mL), mg/ml, etc.), molarity, molality, mole fraction or other similar descriptions of concentration.
• the maximum equilibrium amount of solute that can dissolve per amount of solvent is the solu bility of that solute in that solvent under the specified conditions, including temperature, pressure, pH, and the nature of the solvent.
• solu bility is measured at physiological pH, or other pH, for example, at pH 5.0, pH 6.0, pH 7.0, or pH 7.4.
• solu bility is measured in water or a physiological buffer such as PBS or NaCI (with or without Na P).
• solu bility is measured at relatively lower pH (e.g., pH 6.0) and relatively higher salt (e.g., 500mM NaCI and lOmM Na P).
• solu bility is measured in a biological fluid (solvent) such as blood or serum.
• the temperature can be a bout room temperature (e.g., a bout 20, 21, 22, 23, 24, 25°C) or a bout body temperature ( ⁇ 37°C).
• a p97 conjugate has a solu bility of at least a bout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mg/ml at room temperature or at a bout 37°C.
• a "subject,” as used herein, includes any animal that exhibits a symptom, or is at risk for exhibiting a symptom, which can be treated or diagnosed with a p97 conjugate of the invention.
• Suita ble su bjects include la boratory animals (such as mouse, rat, ra bbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog).
• la boratory animals such as mouse, rat, ra bbit, or guinea pig
• farm animals such as a cat or dog
• domestic animals or pets such as a cat or dog
• Non-human primates and, preferably, human patients, are included.
• substantially or “essentially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.
• compositions refers to the nearly complete or complete a bsence of a given quantity for instance, less than a bout 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of some given quantity.
• certain compositions may be "su bstantially free" of cell proteins, mem branes, nucleic acids, endotoxins, or other conta minants.
• target is used in a variety of different forms throughout this document and is defined by the context in which it is used.
• target m RNA refers to a messenger RNA to which a given siRNA can be directed against.
• target sequence and “target site” refer to a sequence within the m RNA to which the sense strand of an siRNA shows varying degrees of identity and the antisense strand exhibits varying degrees of complementarity.
• siRNA target can refer to the gene, m RNA, or protein against which an siRNA is directed.
• target silencing can refer to the state of a gene, or the corresponding m RNA or protein.
• Treatment includes any desira ble effect on the symptoms or pathology of a disease or condition, and may include even minimal changes or improvements in one or more measura ble markers of the disease or condition being treated. "Treatment” or “treating” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. The su bject receiving this treatment is any su bject in need thereof. Exemplary markers of clinical improvement will be apparent to persons skilled in the art.
• wild-type refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally-occurring source.
• a wild type gene or gene product e.g., a polypeptide
• a wild type gene or gene product is that which is most frequently observed in a population and is thus arbitrarily designed the "normal” or “wild-type” form of the gene.
• Em bodiments of the present invention relate generally to conjugates that comprise a p97 (melanotransferrin; MTf) polypeptide that is covalently or operatively linked to at least one polynucleotide, for example, at least one strand of a dou ble-stranded small interfering RNA (siRNA) molecule, and related compositions and methods of use thereof.
• a p97 polypeptide sequences e.g., Table 1
• siRNA target genes e.g., Table 2
• the p97 conjugate comprises one or more linkers (L) between the p97 polypeptide and the nucleic acid molecule, examples of which are provided herein. Variants and fragments of any of the foregoing are also included and described herein.
• the p97 conjugate comprises, consists, or consists essentially of at least one of the exemplary, non-limiting configurations illustrated in Figures 1A-1H or 2A.
• a polynucleotide strand of the siRNA molecule is conjugated to the N-terminus of the p97 polypeptide.
• a polynucleotide strand of the siRNA molecule is conjugated to the C-terminus of the p97 polypeptide.
• a polynucleotide strand of the siRNA molecule is conjugated to an internal amino acid of the p97 polypeptide, for example, an S-containing amino acid such as a methionine or cysteine.
• the polynucleotide strand of the siRNA molecule is conjugated to the p97 polypeptide via a linker.
• the p97 polypeptide is conjugated to the 3'-end of a polynucleotide strand of the siRNA molecule. In certain embodiments, the p97 polypeptide is conjugated to the 5'- end of a polynucleotide strand of the siRNA molecule. In particular embodiments, the p97 polypeptide is conjugated to an internal nucleobase of a polynucleotide strand of the siRNA molecule. In some of these and related embodiments, the p97 polypeptide is conjugated to the polynucleotide of the siRNA molecule via a linker.
• the p97 polypeptide is conjugated to the sense polynucleotide strand of the siRNA molecule, as described herein. In certain embodiments, the p97 polypeptide is conjugated to the antisense polynucleotide strand of the siRNA molecule, as described herein.
• Certain conjugates can employ more than one p97 polypeptide. For instance, in some embodiments, a first p97 polypeptide is conjugated to the sense strand and a second p97 polypeptide (being the same or different) is conjugated to the antisense strand. In some embodiments, a first p97 polypeptide is conjugated to the sense strand and a second p97 polypeptide (being the same or different) is conjugated to the antisense strand. In some embodiments, a first p97 polypeptide is conjugated to the sense strand and a second p97 polypeptide (being the same or different) is conjugated to the antisense strand. In some embodiments, a first p97 polypeptide is conjugated to the sense strand and a second p97 polypeptide (being the same or different) is conjugated to the antisense strand. In some embodiments, a first p97 polypeptide is conjugated to the sense strand and a second p97 polypeptide (being the same
• 1, 2, 3, 4, or 5 p97 polypeptides are conjugated to a polynucleotide strand of an siRNA molecule.
• a p97 polypeptide is conjugated to 1, 2, 3, 4, or 5 polynucleotide strands of an siRNA molecule.
• a p97 polypeptide is conjugated to 1, 2, 3, 4, or 5 sense strands of an siRNA molecule.
• a p97 polypeptide is conjugated to 1, 2, 3, 4, or 5 antisense strands of an siRNA molecule.
• a p97 polypeptide is conjugated to mixture of sense and antisense strands, such as 1, 2, 3, 4, or 5 sense strands and 1, 2, 3, 4, or 5 antisense strands of an siRNA molecule. Also included are combinations of any of the foregoing.
• Conjugates between p97 polypeptides and polynucleotide strands of siRNA molecules can be prepared according to a variety of techniques in the art. For example, certain embodiments may utilize thiol-containing siRNAs (see Muratovska and Eccles, FEBS Lett. 558:63-68, 2004) or other approaches (see Jeong et al., Bioconjugate Chem. 20:5-14, 2009).
• the 5'- terminus, internal nucleobase(s), or 3'-terminus of an siRNA strand can be covalently linked to the N- terminus, internal residue(s), or C-terminus of a peptide using one or more preactivated small molecules with functional groups that reside in the polypeptide and polynucleotide such as amine, thiol, carboxylate, hydroxyl, aldehyde and ketone, active hydrogen, photo-chemical and
• polymer conjugates can be prepared, for example, by conjugating an aminooxy-derivatized cationic block copolymer to the p97 polypeptide by means of genetically encoded p-acetyl phenylalanine (pAcF) (see Lu et al., J Am Chem Soc. 135:13885-91, 2013), and then conjugating a polynucleotide strand of the siRNA molecule to the copolymer.
• pAcF p-acetyl phenylalanine
• Conjugates can be constructed using any of the p97, L, or siRNA molecules described herein, including functional or active variants and fragments thereof.
• the conjugates have a purity of at least a bout 90%, as measured according to routine techniques in the art. In certain embodiments, such as diagnostic compositions or certain therapeutic compositions, the conjugates have a purity of at least about 95%. In specific embodiments, such as therapeutic or pharmaceutical compositions, the conjugates have a purity of at least about 97% or 98% or 99%. In other embodiments, such as when being used as reference or research reagents, conjugates can be of lesser purity, and may have a purity of at least about 50%, 60%, 70%, or 80%. Purity can be measured overall or in relation to selected components, such as other proteins, e.g., purity on a protein basis.
• the conjugates and/or compositions described herein are about substantially endotoxin free, including, for example, about 95% endotoxin free, preferably about 99% endotoxin free, and more preferably about 99.99% endotoxin free.
• the presence of endotoxins can be detected according to routine techniques in the art, as described herein.
• p97 Polypeptides In certain embodiments, a p97 polypeptide sequence used in a conjugate comprises, consists essentially of, or consists of a human p97 reference sequence provided in Table
• a p97 polypeptide sequence comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or homology, along its length, to a human p97 sequence in Table 1, or a fragment thereof.
• a p97 polypeptide sequence comprises a fragment of a human p97 sequence in Table 1.
• a p97 polypeptide fragment is about, at least about, or up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 100, 105, 110,
• a p97 polypeptide fragment is about 5-700, 5-600, 5-500, 5-400, 5- 300, 5-200, 5-100, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-40, 10-30, 10-25, 10-20, 10-15, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 20-40, 20-30, 20-25, 30-700, 30-600, 30-500, 30-400, 30-300, 30-200, 30-100, 30-50, 30-40, 40-700, 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-50, 50-700, 50-600, 50- 500, 50-400, 50-300, 50-200, 50-100, 60-700, 60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60- 70, 70
• Certain embodiments comprise one or more p97 polypeptides, for example, 2, 3, 4, or 5 polypeptides, as illustrated by the formula [X] n , where X is a p97 polypeptide described herein and n is an integer from 1-5.
• X is DSSHAFTLDEL (SEQ ID NO:14).
• the p97 polypeptide has one or more terminal (e.g., N-terminal, C- terminal) cysteines and/or tyrosines, which can be added for conjugation and iodination, respectively.
• the cysteine residue provides a free sulfhydryl group to allow conjugation of the p97 polypeptide to the polynucleotide strand of the siRNA molecule, either directly or via a linker such as PEG-based linker.
• p97 polypeptide sequences of interest include p97 amino acid sequences, subsequences, and/or variants of p97 that are effective for transporting an agent of interest across the blood brain barrier and into the central nervous system (CNS).
• the variant or fragment comprises the N-lobe of human p97 (residues 20-361 of SEQ ID NO:l).
• the variant or fragment comprises an intact and functional Fe 3+ -binding site.
• a p97 polypeptide sequence is a soluble form of a p97 polypeptide (see Yang et al., Prot Exp Purif. 34:28-48, 2004), or a fragment or variant thereof.
• the soluble p97 polypeptide has a deletion of the all or a portion of the hydrophobic domain (residues 710-738 of SEQ ID NO:l), alone or in combination with a deletion of all or a portion of the signal peptide (residues 1-19 of SEQ ID NO:l).
• the soluble p97 polypeptide comprises or consists of SEQ ID NO:2 (residues 20-711 of SEQ ID NO:l), including variants and fragments thereof.
• the p97 polypeptide sequence is a lipid soluble form of a p97 polypeptide.
• certain of these and related embodiments include a p97 polypeptide that comprises all or a portion of the hydrophobic domain, optionally with or without the signal peptide.
• the p97 fragment or variant is capable of specifically binding to a p97 receptor, an LRP1 receptor and/or an LRP1B receptor.
• the p97 polypeptide is a recombinant polypeptide made, for example, from a bacterial cell, a yeast cell, an insect cell, or a eukaryotic cell such as a mammalian or human cell.
• the recombinant p97 polypeptides are produced in substantially or completely serum free media.
• the p97 polypeptide is a synthetic polypeptide.
• siRNA Molecules and other Polynucleotides include p97 polypeptides that are covalently or otherwise attached to at least one polynucleotide.
• the polynucleotide is at least one strand of an siRNA molecule.
• siRNA molecule or “siRNA” refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway. As used herein, these molecules can vary in length (generally 15-30 base pairs plus optionally overhangs) and contain varying degrees of
• siRNA have unpaired overhanging bases on the 5' or 3' end of the sense strand and/or the antisense strand.
• RNA includes duplexes of two separate strands, and unless otherwise specified also includes single strands that can form hairpin structures comprising a duplex region, such as short-hairpin RNAs ("shRNA").
• shRNA short-hairpin RNAs
• the polynucleotide is a shRNA molecule, and the p97 polypeptide is covalently or otherwise attached to a single-stranded polynucleotide that comprises a duplex region (see, e.g., Figures 11-lL).
• a single-stranded hairpin shRNA molecule comprises, consists, or consists essentially of a stem of about, at least about, or no more than about 19-29 base pairs (i.e., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 bp), a loop of about, at least about, or no more than about 4, 5, 6, 7, or 8 nucleotides, and optionally a dinucleotide overhang at the 3' end.
• CRISPR clustered regularly interspaced short palindromic repeats
• TALEN transcription activator-like effector nuclease
• RNA oligonucleotides including RNAs that form part of CRISPR RNA-guided surveillance complexes such as Cas9-crRNA complexes and others (see, e.g., WO 2013/142578; and U.S. Application Nos. 2010/0076057; 2011/018977; 2013/0011828; and 20130330778, which are incorporated by reference).
• sense strand refers to a polynucleotide that comprises a sequence that is in whole or in part, the same as a target nucleic acid sequence such as messenger RNA or a sequence of DNA.
• antisense strand refers to a polynucleotide that comprises a sequence that is in whole or in part, the complement of a target nucleic acid sequence such as messenger RNA or a sequence of DNA.
• first and/or second single-stranded polynucleotides of an siRNA-conjugate are about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 bases or nucleotides in length including all ranges in between.
• These strand lengths include possible overhang regions.
• the first and second polynucleotides of an siRNA molecule form 1, 2, or 3 nucleotide overhang(s) at the 3' end of the antisense strand, the 5' end of the antisense strand, or both.
• siRNA When a sequence of an siRNA is provided, by convention, unless otherwise indicated it is of the sense strand, and the complementary antisense strand is implicit. In a duplex siRNA (formed from two separate strands) one strand may be the sense strand, and the other strand may be the antisense strand. If overhangs are present, the phrase “sense region” may refer to the nucleotide sequence portion of the sense strand other than overhang regions. Similarly, the phrase “antisense region” may refer to the nucleotide sequence portion of the antisense strand other than overhang regions.
• siRNA is a shRNA
• the "sense region” is the portion of the duplex region that has a sequence that is in whole or in part the same as the target sequence
• the "antisense region” is the sequence of nucleotides that is in whole or in part complementary to the target sequence and to the sense region.
• duplex region refers to the region in two complementary or substantially complementary polynucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a stabilized duplex between polynucleotide strands that are complementary or substantially complementary.
• sizes of duplex regions include but are not limited to about 17-30 base pairs, 17-25 base pairs, 17-23 base pairs, 18-30 base pairs, 18-25 base pairs, 18-23 base pairs, 19-30 base pairs, 19-25 base pairs and 19-23 base pairs.
• a duplex region may be defined by the length of base pairs, as well as the degree of complementarity over that range.
• each strand may contain nucleotides that are part of the duplex and nucleotides that are not part of the duplex at either the 5' end or the 3' end.
• An siRNA may be designed such that on the antisense strand, all nucleotides that are complementary to a target are part of the duplex region, and thus have complementary nucleotides on the sense strand.
• the siRNA may be also be designed such that the antisense strand also contains nucleotides at either its 3' end and/or its 5' end that although not having complementary nucleotides on the sense strand, are part of a continuous stretch of nucleotides within the antisense strand that have complementary nucleotides on the target.
• a sense strand may contain 19 nucleotides and an antisense strand may contain 21 nucleotides. All but the two 3' most nucleotides of the antisense strand may be complementary to the 19 nucleotides on the sense strand, while the entire stretch of 21 nucleotides of the antisense strand may be complementary to a stretch of 21 nucleotides of the target.
• the two 3' most nucleotides of the antisense strand may be selected so as not to be complementary to a portion of the target, or selected randomly or to facilitate processing such that one or both might or might not be complementary to the two nucleotides of the target that are adjacent to the nucleotides to which the other 19 nucleotides of the antisense strand are complementary.
• mismatch includes a situation in which Watson-Crick base pairing does not take place between a nucleotide of a sense strand and a nucleotide of an antisense strand.
• mismatches include but are not limited to an A across from a G, a C across from an A, a U across from a C, a U across from a G, an A across from an A, a G across from a G, a C across from C, and a U across from a U.
• first and second polynucleotide strands of an siRNA molecule are fully or substantially complementary to each other.
• complementary refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands.
• polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes.
• Watson-Crick manner e.g., A to T, A to U, C to G
• uracil rather than thymine is the base that is considered to be complementary to adenosine.
• a U is denoted in the context of the present invention, the ability to substitute a T is implied, unless otherwise stated.
• Perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand can hydrogen bond with a nucleotide unit of a second polynucleotide strand.
• nucleotide units of two strands can hydrogen bond with each other.
• the polynucleotide strands exhibit 10%
• the polynucleotide strands exhibit 90% complementarity.
• the antisense strand of the siRNA molecule is fully or substantially complementary to a target mRNA sequence in a target gene.
• the target mRNA sequence is pre-messenger RNA (pre-processed or pre-mRNA) sequence including introns and exons, or the processed mRNA sequence, typically including only the exonic sequences.
• the antisense strand is at least 75% complementary, at least 80% complementary, at least 90% complementary, at least 95% complementary, or 100% complementary to a target mRNA sequence in a target gene.
• the antisense strand is fully or substantially complementary to a target mRNA sequence of a target gene selected from Table 2 below.
• the sense strand of the siRNA molecule is fully or substantially identical to a target mRNA sequence in a target gene.
• the sense strand has at least 75% identity, at least 80% identity, at least 90% identity, at least 95% identity, or 100% identity to a target mRNA sequence in a target gene.
• the antisense strand is fully or substantially identical to a target mRNA sequence of a target gene selected from Table 2 below.
• NOX NADPH Oxidase
• IKK-gamma Activator of the NF-kappaB pathway NF-kb
• mediator of diseases such as asthma, arthritis, cancer, chronic inflammation, neurodegenerative diseases and heart disease
• IL-4 or IL-4 receptors Inflammatory diseases such as asthma and other respiratory diseases, cardiovascular diseases
• IL-13 or IL-13 receptors Inflammatory diseases such as asthma and other respiratory diseases, cardiovascular diseases
• influenza virus genes such as Influenza virus infection
• PTP-1B Cardiovascular, diabetes, obesity
• virus targets such as P gene,
• nucleocapsid (N), NS1 nucleocapsid (N), NS1
• NGF NGF, BDGF, PDGF and their Cancers
• H BV Hepatitis B virus targets H BV infection
• HCV Hepatitis C virus
• HIV Human Immunodeficiency HIV infection, e.g., having a neurological component
• Virus targets such as nef, vif,
• a target gene such as a target gene in Table 2
• the skilled artisan can select or design the specific polynucleotide sequences of an siRNA molecule according to a variety of techniques in the art. As one example, the skilled artisan can scan for AA dinucleotide sequences and select each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. This strategy for choosing siRNA target sites is based on the observation by Elbashir et al. (E BO J 20: 6877-6888, 2001) that siRNA molecules with 3' overhanging UU dinucleotides are highly effective.
• Particular target sequences can then be selected, for example, by identifying siRNAs with 30-50% GC content, and/or by comparing the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminating from consideration any target sequences with more than 16-17 contiguous base pairs of homology to other coding sequences.
• appropriate genome database human, mouse, rat, etc.
• a variety of software tools are also available for generating specific siRNA target sites in any given gene. See also Birmingham et al., Nat Protoc. 2:2068-78, 2007; and U.S. Patent Nos. 7,056,704; 7,078,196; 8,101,348; 8,394,628; 8,420,391;
• the target gene is a NOX (NADPH Oxidase) gene such as NOX1 (NADPH oxidase 1), NOX2 (NADPH oxidase 2; Cytochrome b-245 heavy chain) and/or NOX4 (NADPH oxidase 4).
• NOX enzymes are superoxide producing transmembrane proteins. NOX enzymes are expressed in many cell types and tissues. However, there is increasing evidence that overactivation of NOX enzymes may contribute to CNS-associated and other diseases.
• certain conjugates comprise an siRNA molecule, where the antisense strand is fully or substantially complementary to a NOX1 sequence (see, e.g., Accession: AJ438989.1; Accession: NM_007052.4; Accession:
• NM_013955.2 Accession: NM_001271815.1
• NOX2 sequence see, e.g., Accession: NM_000397.3
• NOX4 target sequence see, e.g., Accession: NM_001143837.1; Accession:
• the siRNA molecule comprises or consists of 5'-A UGU UCA CAA AGU CAG GUC TT-3' (SEQ ID NO:31) and/or 5'-GAC CUG ACU UUG UGA ACA UTT-3' (SEQ ID NO:32) (see Example 1).
• the target is a microRNA (miRNA).
• miRNA targets include miRNA-132, the inhibition of which prevented angiogenesis in an orthotopic mouse model of ovarian and breast carcinoma, and miRNA-21, the inhibition of which led to regression of malignant pre-B-lymphoid tumors.
• the antisense strand is at least 75% complementary, at least 80% complementary, at least 90% complementary, at least 95% complementary, or 100% complementary to a target miRNA sequence.
• the antisense strand is fully or substantially complementary to a target miRNA sequence.
• the sense strand of the siRNA molecule is fully or substantially identical to a target miRNA sequence.
• the sense strand has at least 75% identity, at least 80% identity, at least 90% identity, at least 95% identity, or 100% identity to a target miRNA sequence.
• the siRNA molecule or one or both of the polynucleotide strands of the siRNA are synthetic oligonucleotides.
• Certain embodiments employ modified siRNA molecules. Examples include the
• nucleotide analogs having modifications in the chemical structure of the base, sugar and/or phosphate (see, e.g., Figure 5), including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, and substitution of 5-bromo-uracil; and 2'-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 , or CN, wherein R is an alkyl moiety.
• Nucleotide analogs are also meant to include nucleotides with bases such as inosine, queuosine, xanthine, sugars such as 2'-methyl ribose, non-natural phosphodiester linkages such as methylphosphonates, phosphorothioates and peptides.
• 2'-fluoro, 2'-amino and/or 2'-thio modifications include 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino- cytidine, 2'-amino-uridine, 2'-amino-adenosine, 2'-amino-guanosine, 2,6-diaminopurine, 4-thio- uridine; and/or 5-amino-allyl-uridine.
• Additional exemplary modifications include 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and 5-fluoro-uridine.
• 2'-deoxy-nucleotides can be used within modified siRNAs of this invention, but are preferably included within the sense strand of the siRNA duplex.
• Modified bases refer to nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups.
• nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups.
• Some examples of types of modifications that can comprise nucleotides that are modified with respect to the base moieties include but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, individually or in combination.
• More specific examples include, for example, 5-propynyluridine, 5-propynylcytidine, 6- methyladenine, 6-methylguanine, ⁇ , ⁇ ,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2- aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino)propyl uridine, 5-halocytidine, 5- halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6- methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5- methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deaza-adenosine, 6- azouridine, 6-azocyt
• Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl.
• the sugar moieties may be, or be based on, mannoses, ara binoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles.
• Modifications include su bstitutions with alkyl, alkoxy, amino, deaza, halo, hydroxyl, thiol groups, or a combination thereof, for example.
• Nucleotides may be su bstituted with analogs with greater sta bility such as replacing a ribonucleotide with a deoxyribonucleotide, or having sugar modifications such as 2' OH groups replaced by 2' a mino groups, 2' O-methyl groups, 2'
• a purine or pyrimidine analog of nucleotides include a xanthine, a hypoxanthine, an azapurine, a
• Modification of the linkage between nucleotides or nucleotide ana logs is also preferred, e.g., su bstitution of phosphorothioate linkages for phosphodiester linkages.
• the phosphate group of the nucleotide may be modified by su bstituting one or more of the oxygens of the phosphate group with nitrogen or with sulfur (phosphorothioates). Modifications are useful, for example, to enhance function, to improve sta bility or permea bility, or to direct localization or targeting.
• Linkers As noted a bove, certain conjugates may employ one or more linker groups.
• linkage is used herein to refer to a linker that can be used to separate a p97 polypeptide from an agent (e.g., a strand of an siRNA molecule), or to separate a first agent from another agent or la bel (fluorescence la bel), for instance where two or more agents are linked to form a p97 conjugate.
• the linker may be physiologically sta ble or may include a releasa ble linker such as a la bile linker or an enzymatically degrada ble linker (e.g., proteolytically cleava ble linkers).
• the linker may be a peptide linker.
• the linker may be a non-peptide linker or non-proteinaceous linker.
• the linker may be particle, such as a nanoparticle.
• the linker may be charge neutral or may bear a positive or negative charge.
• a reversible or la bile linker contains a reversible or la bile bond.
• a linker may optionally include a spacer that increases the distance between the two joined atoms.
• a spacer may further add flexibility and/or length to the linker. Spacers may include, but are not be limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides.
• a la bile bond is a covalent bond other than a covalent bond to a hydrogen atom that is capa ble of being selectively broken or cleaved under conditions that will not break or cleave other covalent bonds in the same molecule. More specifically, a la bile bond is a covalent bond that is less sta ble (thermodynamically) or more rapidly broken (kinetically) under appropriate conditions than other non-la bile covalent bonds in the same molecule. Cleavage of a labile bond within a molecule may result in the formation of two molecules.
• cleavage or lability of a bond is generally discussed in terms of half-life (ti/ 2) of bond cleavage (the time required for half of the bonds to cleave).
• ti/ 2 the time required for half of the bonds to cleave.
• labile bonds encompass bonds that can be selectively cleaved more rapidly than other bonds a molecule.
• Appropriate conditions are determined by the type of labile bond and are well known in organic chemistry.
• a la bile bond can be sensitive to pH, oxidative or reductive conditions or agents, temperature, salt concentration, the presence of an enzyme (such as esterases, including nucleases, and proteases), or the presence of an added agent. For example, increased or decreased pH is the appropriate conditions for a pH-labile bond.
• the linker is an organic moiety constructed to contain an alkyl, aryl and/or amino acid backbone, and containing an amide, ether, ester, hydrazone, disulphide linkage or any combination thereof.
• Linkages containing amino acid, ether and amide bound components are stable under conditions of physiological pH, normally 7.4 in serum.
• Disulphide linkages are also included, at least in part because they are sensitive to reductive cleavage.
• amino acid linkers may be designed to be sensitive to cleavage by specific enzymes in the desired target organ or, for example, in the lysosome.
• Exemplary linkers are described in Blattler et al. (19S5) Biochem. 24:1517-1524; King et al (1986) Biochem. 25:5774-5779; Srinivasachar and Nevill (1989) Biochem. 28:2501-2509, and elsewhere (see also Figure 2).
• the linker is about 1 to about 30 atoms in length, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 atoms in length, including all ranges in between.
• the linker is about 1 to 30 atoms in length with carbon chain atoms which may be substituted by heteroatoms independently selected from the group consisting of O, N. or S.
• from 1- 4 or from 5 to 15 of the C atoms are substituted with a heteroatom independently selected from O, N, S.
• the linker comprises or consists of a structure selected from the following:— O— ,— NH— ,— S— ,— C(O)— , C(O)— NH, NH— C(O)— NH, O— C(O)— NH,— C(S)— , — CH2— , — CH2— CH2— , — CH2— CH2— CH2— , — CH2— CH2— CH2— CH2— , — O— CH2— , — CH2— O— , — O— CH 2 — CH 2 — CH 2 — , — CH 2 — O— CH 2 — , — CH 2 — CH 2 — O— , — O— CH 2 — CH 2 — CH 2 — , — CH 2 — CH 2 — O— , — O— CH 2 — CH 2 — CH 2 — , — CH 2 — O— CH 2 — CH 2 —
• the linker comprises a releasable linker.
• the releasable linker is selected from the group consisting of: carboxylate ester, phosphate ester, anhydride, acetal, ketal, acyloxyalkyl ether, imine, orthoester, thio ester, thiol ester, carbonate, and hydrazone.
• the linker contains a moiety subject to hydrolysis upon delivery to the lysosomal environment (e.g., susceptible to hydrolysis at the lysosomal pH or upon contact to a lysosomal enzyme).
• the linker comprises a stable linker.
• the stable linkage is selected from the group consisting of: succinimide, propionic acid, carboxymethylate linkages, ethers, carbamates, amides, amines, carbamides, imides, aliphatic C- C bonds, and thio ethers.
• the linker comprises or consists of polymer such as a polyethylene glycol or polypropylene glycol.
• polymer such as a polyethylene glycol or polypropylene glycol.
• PEG polyethylene glycol
• poly(ethylene glycol) as used herein, are interchangeable and meant to encompass any water-soluble poly(ethylene oxide) derivative.
• PEG is a well-known polymer with good solubility in many aqueous and organic solvents, which exhibits low toxicity, lack of immunogenicity, and is clear, colorless, odorless, and stable.
• water-soluble polymers as described herein, including without limitation; polyvinyl alcohol, other poly(alkylene oxides) such as poly(propylene glycol) and the like, poly(oxyethylated polyols) such as poly(oxyethylated glycerol) and the like,
• carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl purrolidone, poly-l,3-dioxolane, poly- 1,3,6-trioxane, ethylene/maleic anhydride, and polyaminoacids One skilled in the art will be able to select the desired polymer based on the desired dosage, circulation time, resistance to proteolysis, and other considerations.
• PEGs for use in accordance with the conjugates described herein comprise the following structure "-(OCH2CH2)n-" where (n) is about 1 to 4000, about 20 to 1400, or about 20-800.
• PEG also includes "-0-(CH2CH20)n-CH2CH2-" and “-(OCH2CH2)n-0-” depending upon whether or not the terminal oxygens have been displaced.
• the term “PEG” includes structures having various terminal or “end capping” groups.
• the term “PEG” also includes a polymer that contains a majority, that is to say, greater than 50%, of -OCH2CH2- repeating subunits. With respect to specific forms, the PEG can take any number of a variety of molecular weights, as well as structures or geometries such as “branched,” “linear,” “forked,” “multifunctional” PEG molecules.
• PEG derivatives are both commercially available and suitable for use in the preparation of the PEG-conjugates of the invention.
• NOF Corp.'s SUNBRIGHT ® Series provides numerous PEG derivatives, including methoxypolyethylene glycols and activated PEG derivatives such as succinimidyl ester, methoxy-PEG amines, maleimides, and carboxylic acids, for coupling by various methods to polypeptides and polynucleotides and Nektar Therapeutics' Advanced PEGylation also offers diverse PEG-coupling technologies to improve the safety and efficacy of therapeutics.
• PEGs for use in forming a p97-siRNA conjugate include those available from Polypure (Norway), from QuantaBioDesign LTD (Ohio) JenKem Technology, Nanocs Corporation, and Sunbio, Inc (South Korea). Further PEG reagents suitable for use in forming a conjugate, and methods of conjugation are described, for example, in Pasut et al., Expert Opin. Ther. Patents. 14(6) 859-893, 2004.
• the linker group is hydrophilic, for instance, to enhance the solubility of the conjugate in body fluids.
• the p97 polypeptide and si NA molecule are joined by a linker comprising amino acids or peptides, lipids, or sugar residues.
• the p97 polypeptide and siRNA molecule are joined at groups introduced
• Variant Sequences Certain embodiments include variants of the reference polypeptide and polynucleotide sequences described herein, whether described by name or by reference to a sequence identifier, including p97 sequences, IDS sequences, linker sequences, signal peptide sequences, purification tags, and protease sites (see, e.g., Tables 1-6 and the Sequence Listing).
• sequence identifier including p97 sequences, IDS sequences, linker sequences, signal peptide sequences, purification tags, and protease sites.
• the wild-type or most prevalent sequences of these polypeptides are known in the art, and can be used as a comparison for the variants and fragments described herein.
• variant sequence refers to a polypeptide or polynucleotide sequence that differs from a reference sequence disclosed herein by one or more substitutions, deletions (e.g., truncations), additions, and/or insertions. Certain variants thus include fragments of a reference sequence described herein. Variant polypeptides are biologically active, that is, they continue to possess the enzymatic or binding activity of a reference polypeptide. Such variants may result from, for example, genetic polymorphism and/or from human manipulation.
• a biologically active variant will contain one or more conservative substitutions.
• a “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
• modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.
• amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their utility.
• the hydropathic index of amino acids may be considered.
• the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
• Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
• threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (- 4.5).
• certain amino acids may be su bstituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein.
• substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
• Patent 4,554,101 the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
• an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
• substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
• amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity,
• hydrophilicity, charge, size, and the like are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
• Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
• negatively charged amino acids include aspartic acid and glutamic acid
• positively charged amino acids include lysine and arginine
• amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
• amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
• variant polypeptides may also, or alternatively, contain non-conservative changes.
• variant polypeptides differ from a native or reference sequence by substitution, deletion or addition of fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids, or even 1 amino acid.
• Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure, enzymatic activity, and/or hydropathic nature of the polypeptide.
• variants of the DSSHAFTLDEL (SEQ ID NO:14) p97 polypeptide can be based on the sequence of p97 sequences from other organisms, as shown in Table B below. Variant amino acids relative to the human sequence are underlined.
• the p97 peptide comprises, consists, or consists essentially of a sequence in Ta ble B.
• the p97 peptide retains the short alpha-helix (LDEL) at the C-terminus of the DSSHAFTLDEL (SEQ I D NO: 14) peptide.
• a polypeptide sequence is a bout, at least a bout, or up to a bout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610,
• a polypeptide sequence consists of a bout or no more than a bout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630
• a polypeptide sequence is a bout 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-40, 10-30, 10-20, 20-1000, 20-900, 20- 800, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 20-40, 20-30, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 100-1000, 100-900, 100-800, 100- 700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, or 200-300 contiguous amino acids, including all ranges in between, and comprises all or a portion of a reference sequence.
• the C-terminal or N- terminal region of any reference polypeptide may be truncated by a bout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 or more amino acids, or by a bout 10- 50, 20-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500- 550, 550-600, 600-650, 650-700, 700-750, 750-800 or more amino acids, including all integers and ranges in between (e.g., 101, 102, 103, 104, 105), so long as the truncated polypeptide retains the binding properties and/or activity of the reference
• variants will display at least a bout 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or sequence identity or sequence homology to a reference polypeptide sequence.
• sequences differing from the native or parent sequences by the addition e.g., C-terminal addition, N-terminal addition, both
• deletion, truncation, insertion, or su bstitution e.g., conservative su bstitution
• su bstitution e.g., conservative su bstitution
• variant polypeptides differ from reference sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In other embodiments, variant polypeptides differ from a reference sequence by at least 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) Calculations of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.
• the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
• the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
• the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
• the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
• the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
• the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (J. Mol. Biol. 48: 444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
• the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
• a particularly preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
• the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (Cabios. 4:11-17, 1989) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
• nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases, for example, to identify other family members or related sequences.
• Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (1990, 7. Mol. Biol, 215: 403-10).
• Gapped BLAST can be utilized as described in Altschul et al., (Nucleic Acids Res. 25 : 3389-3402, 1997).
• the default parameters of the respective programs e.g., XBLAST and NBLAST
• XBLAST and NBLAST can be used.
• polynucleotides and/or polypeptides can be evaluated using a BLAST alignment tool.
• a local alignment consists simply of a pair of sequence segments, one from each of the sequences being compared.
• a modification of Smith-Waterman or Sellers algorithms will find all segment pairs whose scores cannot be improved by extension or trimming, called high-scoring segment pairs (HSPs).
• HSPs high-scoring segment pairs
• the results of the BLAST alignments include statistical measures to indicate the likelihood that the BLAST score can be expected from chance alone.
• the raw score, S is calculated from the num ber of gaps and su bstitutions associated with each aligned sequence wherein higher similarity scores indicate a more significant alignment.
• Su bstitution scores are given by a look-up ta ble (see PAM, BLOSUM ).
• the gap cost would be G+Ln.
• the choice of gap costs, G and L is empirical, but it is customary to choose a high value for G (10-15), e.g., 11, and a low value for L (1-2) e.g., 1.
• bit score is derived from the raw alignment score S in which the statistical properties of the scoring system used have been taken into account. Bit scores are normalized with respect to the scoring system, therefore they ca n be used to compare alignment scores from different searches.
• bit score and “similarity score” are used interchangea bly. The bit score gives an indication of how good the alignment is; the higher the score, the better the alignment.
• the E-Value or expected value, describes the likelihood that a sequence with a similar score will occur in the data base by chance. It is a prediction of the num ber of different alignments with scores equiva lent to or better than S that are expected to occur in a data base search by chance. The smaller the E-Value, the more significant the alignment. For exa mple, an alignment having an E value of e "117 means that a sequence with a similar score is very unlikely to occur simply by chance.
• the expected score for aligning a random pair of amino acids is required to be negative, otherwise long alignments would tend to have high score independently of whether the segments aligned were related.
• the BLAST algorithm uses an appropriate su bstitution matrix, nucleotide or amino acid and for gapped alignments uses gap creation and extension penalties. For example, BLAST alignment and comparison of polypeptide sequences are typically done using the BLOSUM62 matrix, a gap existence penalty of 11 and a gap extension penalty of 1.
• sequence similarity scores are reported from BLAST analyses done using the BLOSUM62 matrix, a gap existence penalty of 11 and a gap extension penalty of 1.
• sequence identity/similarity scores provided herein refer to the value obtained using GAP Version 10 (GCG, Accelrys, San Diego, Calif.) using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, PNAS USA. 89:10915-10919, 1992).
• GAP uses the algorithm of Needleman and Wunsch (J Mol Biol. 48:443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
• the variant polypeptide comprises an amino acid sequence that can be optimally aligned with a reference polypeptide sequence (see, e.g., Sequence Listing) to generate a BLAST bit scores or sequence similarity scores of at least about 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750,
• a reference polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, additions, and insertions. Methods for such manipulations are generally known in the art.
• amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (PNAS USA. 82: 488-492, 1985); Kunkel et al., (Methods in Enzymol. 154: 367-382, 1987), U.S. Pat. No. 4,873,192, Watson, J. D.
• REM recursive ensemble mutagenesis
• the p97 conjugate is operatively linked to a "detectable entity.”
• detectable entities include, without limitation, iodine-based labels, radioisotopes, fluorophores/fluorescent dyes, and nanoparticles.
• Exemplary iodine-based labels include diatrizoic acid (Hypaque ® , GE Healthcare) and its anionic form, diatrizoate.
• Diatrizoic acid is a radio-contrast agent used in advanced X-ray techniques such as CT scanning. Also included are iodine radioisotopes, described below.
• radioisotopes that can be used as detectable entities include 32 P, 33 P, 35 S, 3 H, 18 F, C, 13 N, 15 0, m ln, 169 Yb, 99m TC, 55 Fe, and isotopes of iodine such as 123 l, 124 l, 125 l, and 131 l. These radioisotopes have different half-lives, types of decay, and levels of energy which can be tailored to match the needs of a particular protocol. Certain of these radioisotopes can be selectively targeted or better targeted to CNS tissues by conjugation to p97 conjugates, for instance, to improve the medical imaging of such tissues.
• fluorophores or fluorochromes that can be used as directly detectable entities include fluorescein, tetramethylrhodamine, Texas Red, Oregon Green ® , and a number of others (e.g., Haugland, Handbook of Fluorescent Probes - 9th Ed., 2002, Molec. Probes, Inc., Eugene OR; Haugland, The Handbook: A Guide to Fluorescent Probes and Labeling Technologies-lOth Ed., 2005, Invitrogen, Carlsbad, CA). Also included are light-emitting or otherwise detectable dyes. The light emitted by the dyes can be visible light or invisible light, such as ultraviolet or infrared light.
• the dye may be a fluorescence resonance energy transfer (FRET) dye; a xanthene dye, such as fluorescein and rhodamine; a dye that has an amino group in the alpha or beta position (such as a naphthylamine dye, l-dimethylaminonaphthyl-5-sulfonate, l-anilino-8- naphthalende sulfonate and 2-p-touidinyl-6-naphthalene sulfonate); a dye that has 3-phenyl-7- isocyanatocoumarin; an acridine, such as 9-isothiocyanatoacridine and acridine orange; a pyrene, a bensoxadiazole and a stilbene; a dye that has 3-(s-carboxypentyl)-3'-ethyl-5,5'- dimethyloxacarbocyanine (CYA);
• FRET flu
• Certain em bodiments include conjugation to chemotherapeutic agents (e.g., paclitaxel, adriamycin) that are la beled with a detecta ble entity, such as a fluorophore (e.g., Oregon Green ® , Alexa Fluor 488).
• chemotherapeutic agents e.g., paclitaxel, adriamycin
• a detecta ble entity such as a fluorophore (e.g., Oregon Green ® , Alexa Fluor 488).
• Nanoparticles usually range from a bout 1-1000 nm in size a nd include diverse chemical structures such as gold and silver particles and quantum dots.
• silver or gold nanoparticles When irradiated with angled incident white light, silver or gold nanoparticles ranging from a bout 40-120 nm will scatter monochromatic light with high intensity. The wavelength of the scattered light is dependent on the size of the particle. Four to five different particles in close proximity will each scatter monochromatic light, which when superimposed will give a specific, unique color.
• Derivatized nanoparticles such as silver or gold particles can be attached to a broad array of molecules including, proteins, antibodies, small molecules, receptor ligands, and nucleic acids.
• nanoparticles include metallic nanoparticles and metallic nanoshells such as gold pa rticles, silver particles, copper particles, platinum particles, cadmium particles, composite particles, gold hollow spheres, gold-coated silica nanoshells, and silica-coated gold shells. Also included are silica, latex, polystyrene, polycarbonate, polyacrylate, PVDF nanoparticles, a nd colored particles of any of these materials.
• Quantum dots are fluorescing crystals a bout 1-5 nm in diameter that are excita ble by light over a large range of wavelengths. Upon excitation by light having an appropriate wavelength, these crystals emit light, such as monochromatic light, with a wavelength dependent on their chemical composition and size. Quantum dots such as CdSe, ZnSe, InP, or InAs possess unique optical properties; these and similar quantum dots are availa ble from a number of commercial sou rces (e.g., N N-La bs, Fayetteville, AR; Ocean Nanotech, Fayetteville, AR; Nanoco Technologies, Manchester, U K; Sigma-Aldrich, St. Louis, MO).
• Certain em bodiments of the present invention relate to methods of using the p97 conjugates described herein. Examples of such methods include methods of treatment and methods of d iagnosis, including for instance, the use of p97 conjugates for medical imaging of certain organs/tissues, such as those of the nervous system.
• Some em bodiments include methods of diagnosing a nd/or treating disorders or conditions of the central nervous system (CNS), or d isorders or conditions having a CNS component.
• Particular aspects include methods of treating a lysosomal storage disorder (LSD), including those having a CNS component.
• certain em bodiments include methods of treating a su bject in need thereof, comprising administering a p97 conjugate described herein.
• kits for delivering an I DS enzyme to the nervous system (e.g., central nervous system tissues) of a su bject comprising administering a composition that comprises a p97 conjugate described herein.
• the methods increase the rate of delivery of the agent to the central nervous system tissues, relative, for exa mple, to delivery by a composition that comprises an unconjugated siRNA molecule.
• the su bject has a disease or condition in Ta ble 2.
• the siRNA molecule is targeted against a NOX gene such as NOX1, NOX2 and/or NOX4, and the su bject has a d isease or condition such as a disease associated with cell death, chronic kidney disease, dia betes or dia betic complications such as dia betic nephropathy (see, e.g., Jha et al., J Am Soc Nephrol.
• a neurocardiovascular d isease such as myocardial infarction- induced cardiac dysfunction, or a disorder of the CNS (see, e.g., Nayernia et al., Antioxidants & Redox Signaling, doi: 10.1089/ars.2013.5703, 2014; Sorce and Kra use, Antioxidants & Redox
• Neurodegenerative diseases for example, where activation of NOX enzymes in microglia generate a pathological cycle of cell damage leading to increased microglia activation, which leads to further cell damage and so on.
• Parkinson's disease for example, where NOX (e.g., NOX1) expression in dopaminergic neurons could contribute to neurodegeneration.
• NOX2 is increasingly implicated in animal models of psychotic d isease, in particular in ketamine-induced psychosis.
• disease-relevant NOX2 activity might be in neurons, and not exclusively be restricted to microglia.
• Particu lar examples include Acute Stress Disorder; Adjustment Disorder Unspecified; Adjustment Disorder with Anxiety; Adjustment Disorder with Depressed Mood; Adjustment Disorder with Disturbance of Conduct; Adjustment Disorder with Mixed Anxiety and Depressed Mood; Adjustment Disorder with Mixed Disturbance of Emotions and Conduct; Agoraphobia without History of Panic Disorder; Anorexia Nervosa; Antisocial Personality Disorder; Anxiety Disorder Due to Medical Condition; Anxiety Disorder, NOS; Avoidant Personality Disorder; Bipolar Disorder NOS; Bipolar I Disorder, Most Recent Episode Depressed, In Full Remission; Bipolar I Disorder, Most Recent Episode Depressed, In Partial Remission; Bipolar I Disorder, Most Recent Episode Depressed, Mild; Bipolar I Disorder, Most Recent Episode Depressed, Moderate; Bipolar I Disorder, Most Recent Episode Depressed, Severe With Psychotic Features; Bipolar I Disorder, Most Recent Episode Depressed, Severe Without Psychotic Features; Bipolar I Disorder, Most Recent Episode Depressed, Unspecified; Bipolar I Disorder, Most
• Certain embodiments include the treatment of neurodegenerative disorders, such as nerve inflammation, certain types of cancers, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Friedreich's ataxia, spinal muscular atrophy, Huntington's disease, Dementia with Lewy bodies, spinal muscular atrophy, neuromyelitis optica, major depressive disorder, schizophrenia, glaucoma or peripheral neuropathies (diabetic or AIDS neuropathy).
• neurodegenerative disorders such as nerve inflammation, certain types of cancers, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Friedreich's ataxia, spinal muscular atrophy, Huntington's disease, Dementia with Lewy bodies, spinal muscular atrophy, neuromyelitis optica, major depressive disorder, schizophrenia, glaucoma or peripheral neuropathies (diabetic or AIDS neuropathy).
• diseases of the central and peripheral nervous system include Absence of the Septum Pellucidum, Acid Lipase Disease, Acid Maltase Deficiency, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Adie's Pupil, Adie's Syndrome, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS - Neurological Complications, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation, Asperger Syndrome, Ataxia
• ADHD Attention Deficit-Hyperactivity Disorder
• Autism Autism, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Becker's Myotonia, Behcet's Disease, Bell's Palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy, Benign Intracranial Hypertension, Bernhardt- oth Syndrome, Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injuries, Bradbury- Eggleston Syndrome, Brain and Spinal Tumors, Brain Aneurysm, Brain infarction, Brain ischemia, Brain Injury, Brown-Sequard Syndrome, Bulbospinal Muscular Atrophy, , CADASIL, Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma, Cavernous Malformation, Central
• CIDP Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, COFS, Colpocephaly, Coma, Complex Regional Pain Syndrome, Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Malformations, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Creutzfeldt-Jakob Disease, Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic Inclusion Body Disease, Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome, Deep Brain Stimulation for Parkinson's Disease, Dejerine-Klumpke Palsy, Dementia, Dementia - Multi-lnfarct, Dementia - Semantic, Dementia - Su bcortical, Dementia
• Encephalopathy Empty Sella Syndrome, Encephalitis, Encephalitis Lethargica, Encephaloceles, Encephalopathy, Encephalopathy, familial infantile, with intracranial calcification and chronic cerebrospinal fluid lymphocytosis; Cree encephalitis; Pseudo-Torch syndrome; Pseudotoxoplasmosis syndrome, Encephalotrigeminal Angiomatosis, Epilepsy, Epileptic Hemiplegia, Erb-Duchenne and Dejerine-Klumpke Palsies, Erb's Palsy, Essential Tremor, Extrapontine Myelinolysis, Fabry Disease, Fahr's Syndrome, Fainting, Familial Dysautonomia, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Periodic Paralyses, Familial Spastic Paralysis, Farber's Disease, Febrile Seizures, Fibromuscular
• Neuromyelitis Optica Neuromyotonia, Neuronal Ceroid Lipofuscinosis, Neuronal Migration
• Neuropathy - Hereditary Neurosarcoidosis, Neurotoxicity, Nevus Cavernosus, Niemann- Pick Disease, Normal Pressure Hydrocephalus, Occipital Neuralgia, Ohtahara Syndrome,
• Olivopontocerebellar Atrophy Opsoclonus Myoclonus, Orthostatic Hypotension, O'Sullivan-McLeod Syndrome, Overuse Syndrome, Pain - Chronic, Pantothenate Kinase-Associated Neurodegeneration, Paraneoplastic Syndromes, Paresthesia, Parkinson's Disease, Paroxysmal Choreoathetosis,
• p97-siRNA conjugates directed to NOX genes e.g., NOX1, NOX2, NOX4
• NOX genes e.g., NOX1, NOX2, NOX4
• p97-siRNA conjugates directed to NOX genes could provide therapeutic utility in the treatment of these and other NOX-associated d iseases or conditions of the CNS.
• the p97 conjugates described herein are generally incorporated into a pharmaceutical composition prior to administration.
• a pharmaceutical composition comprises one or more of the p97 conjugates described herein in combination with a physiologically accepta ble carrier or excipient.
• an effective or desired amount of one or more conjugates is mixed with a ny pharmaceutical carrier(s) or excipient known to those skilled in the a rt to be suita ble for the particular mode of administration.
• a pharmaceutical carrier may be liquid, semi-liquid or solid.
• Solutions or suspensions used for parenteral, intradermal, su bcutaneous or topical application may include, for example, a sterile diluent (such as water), saline solution (e.g., phosphate buffered saline; PBS), fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; a ntimicrobial agents (such as benzyl alcohol a nd methyl para bens); antioxidants (such as ascorbic acid and sod ium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates and phosphates).
• a sterile diluent such as water
• saline solution e.g., phosphate buffered saline; PBS
• PBS phosphate buffered saline
• fixed oil polyethylene glycol, glycerin, propylene glyco
• suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof.
• PBS physiological saline or phosphate buffered saline
• compositions can be carried out via any of the accepted modes of administration of agents for serving similar utilities.
• the pharmaceutical compositions can be prepared by combining a conjugate-containing composition with an appropriate physiologically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
• suitable excipients such as salts, buffers and stabilizers may, but need not, be present within the composition.
• Administration may be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, subcutaneous or topical. Preferred modes of administration depend upon the nature of the condition to be treated or prevented. Particular embodiments include administration by IV infusion.
• Carriers can include, for example, pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
• physiologically acceptable carrier is an aqueous pH buffered solution.
• physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as polysorbate 20 (TWEENTM) polyethylene glycol (PEG), and poloxamers (PLURONICSTM), and the like.
• buffers such as phosphate, citrate, and other organic acids
• antioxidants including ascorbic acid
• the p97 polypeptide sequence and the siRNA molecule(s) are each, individually or as a pre-existing conjugate, bound to or encapsulated within a particle, e.g., a nanoparticle, bead, lipid formulation, lipid particle, or liposome, e.g., immunoliposome.
• a particle e.g., a nanoparticle, bead, lipid formulation, lipid particle, or liposome, e.g., immunoliposome.
• the p97 polypeptide sequence is bound to the surface of a particle
• the siRNA molecule of interest is bound to the surface of the particle and/or encapsulated within the particle.
• the p97 polypeptide and the siRNA molecule(s) are covalently or operatively linked to each other only via the particle itself (e.g., nanoparticle, liposome), and are not covalently linked to each other in any other way; that is, they are bound individually to the same particle.
• the p97 polypeptide a nd the si NA molecule(s) are first covalently or non-covalently conjugated to each other, as described herein (e.g., via a linker molecule), and are then bound to or encapsulated within a particle (e.g., liposome, nanoparticle).
• the particle is a liposome
• the composition comprises one or more p97 polypeptides, one or more siRNA molecule(s) of interest, and a mixture of lipids to form a liposome (e.g., phospholipids, mixed lipid chains with surfactant properties).
• the p97 polypeptide and the siRNA molecule(s) are individually mixed with the lipid/liposome mixture, such that the formation of liposome structures operatively links the p97 polypeptide and the siRNA molecule(s) without the need for covalent conjugation.
• the p97 polypeptide and the siRNA molecule(s) are first covalently or non-covalently conjugated to each other, as described herein, and then mixed with lipids to form a liposome.
• the p97 polypeptide, the siRNA molecule(s), or the p97 conjugate may be entrapped in microcapsules prepared, for exa mple, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate)microcapsules, respectively), in colloida l drug delivery systems (for example, liposomes, al bumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions.
• Such techniques are disclosed in Remington's
• the particle(s) or liposomes may further comprise other therapeutic or diagnostic agents, such as cytotoxic agents.
• the precise dosage and d uration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosages may also vary with the severity of the condition to be alleviated.
• composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesira ble side effects.
• the composition may be administered one time, or may be divided into a num ber of smaller doses to be administered at intervals of time. For any particular su bject, specific dosage regimens may be adjusted over time according to the individual need.
• Typical routes of administering these and related pharmaceutical compositions thus include, without limitation, oral, topical, transdermal, inhalation, parenteral, su blingual, buccal, rectal, vaginal, and intranasal.
• parenteral as used herein includes su bcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
• Pharmaceutical compositions according to certain embodiments of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.
• compositions that will be administered to a subject or patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein described conjugate in aerosol form may hold a plurality of dosage units.
• dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition
• composition to be administered will typically contain a therapeutically effective amount of a conjugate described herein, for treatment of a disease or condition of interest.
• a pharmaceutical composition may be in the form of a solid or liquid.
• the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form.
• the carrier(s) may be liquid, with the compositions being, for example, an oral oil, injecta ble liquid or an aerosol, which is useful in, for example, inhalatory administration.
• the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
• the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like.
• a solid composition will typically contain one or more inert diluents or edible carriers.
• binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
• a liquid carrier such as polyethylene glycol or oil.
• the pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension.
• the liquid may be for oral administration or for delivery by injection, as two examples.
• preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
• a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
• the liquid pharmaceutical compositions may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl para ben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• Physiological saline is a preferred adjuvant.
• a liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of a conjugate such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the agent of interest in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of the agent of interest. In certain embodiments, pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of the agent of interest prior to dilution.
• the pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
• the base for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
• Thickening agents may be present in a pharmaceutical composition for topical administration.
• the composition may include a transdermal patch or iontophoresis device.
• the pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug.
• the composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient.
• bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.
• the pharmaceutical composition may include various materials, which modify the physical form of a solid or liquid dosage unit.
• the composition may include materials that form a coating shell around the active ingredients.
• the materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
• the active ingredients may be encased in a gelatin capsule.
• the pharmaceutical composition in solid or liquid form may include an agent that binds to the conjugate or agent and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome.
• the pharmaceutical composition may consist essentially of dosage units that can be administered as an aerosol.
• aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation may determine preferred aerosols.
• compositions described herein may be prepared with carriers that protect the conjugates against rapid elimination from the body, such as time release formulations or coatings.
• carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others known to those of ordinary skill in the art.
• compositions may be prepared by methodology well known in the pharmaceutical art.
• a pharmaceutical composition intended to be administered by injection may comprise one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution.
• a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
• Surfactants are compounds that non-covalently interact with the conjugate so as to facilitate dissolution or homogeneous suspension of the conjugate in the aqueous delivery system.
• compositions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
• a therapeutically effective daily dose is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., ⁇ 0.07 mg) to about 100 mg/kg (i.e., ⁇ 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., ⁇ 0.7 mg) to about 50 mg/kg (i.e., ⁇ 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., ⁇ 70 mg) to about 25 mg/kg (i.e., ⁇ 1.75 g).
• compositions described herein may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents, as described herein.
• the conjugate is administered with an anti-inflammatory agent.
• Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including
• betamethasone betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone,
• hydrocortisone methylprednisolone, prednisolone, prednisone, triamcinolone
• nonsteroidal antiinflammatory drugs NSAIDS
• aspirin ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.
• Such combination therapy may include administration of a single pharmaceutical dosage formulation, which contains a compound of the invention (i.e., conjugate) and one or more additional active agents, as well as administration of compositions comprising conjugates of the invention and each active agent in its own separate pharmaceutical dosage formulation.
• a conjugate as described herein and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations.
• a conjugate as described herein and the other active agent can be administered to the patient together in a single parenteral dosage composition such as in a saline solution or other physiologically accepta ble solution, or each agent administered in separate parenteral dosage formulations.
• compositions comprising conjugates and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.
• p97-siRNA Conjugates To test the ability of p97 to enhance delivery of si NA molecules across the blood brain barrier (BBB), the C-terminus of a p97 polypeptide DSSHAFTLDERYC (SEQ ID NO:29) was conjugated via a linker moiety to the 3' end of the antisense strand of an siRNA molecule targeted against the NOX4 gene (5'-A UGU UCA CAA AGU CAG GUC TT-3') (SEQ ID NO:31).
• the sense strand of the siRNA molecule (5'-GAC CUG ACU UUG UGA ACA UTT-3') (SEQ ID NO:32) was conjugated via a linker to Alexa Fluor ® 680 (AF680).
• AF680 Alexa Fluor ® 680
• the positive control the p97-conjugated antisense and AF680-labeled sense strands were annealed to form the p97-siRNA conjugate (see Figure 2A).
• an unconjugated antisense strand and the AF680-labeled sense strands were annealed (see Figure 2B).
• Test molecules were injected into mice, as outlined in the study design shown in Table El below.
• injection volume 5 ml/g ( ⁇ 0.1ml per mouse)
• Vascular Perfusion 10mins@lml/min with PBS pH 7.4 with 2.7% BSA, 100 U/ml heparin
• Test molecules were provided in powder form and re-constituted in Nuclease-free PBS, pH 7.4 at
• mice were injected (i.v.) with tomato Lectin- Texas Red (80mg) for 10 min to stain the brain vasculature. Blood was cleared by intracardiac perfusion of 10 ml heparinized saline (lml/min). Brains were excised and frozen on dry ice and stored at -80 ° C. Brains were mounted in Tissue Tek and sectioned (25 mm, 3 sections/ brain) with a cryostat at -20 ° C.
• AF680 fluorescent signal was not detected in the brain parenchyma of any mice (see Figure 4B) and no AF680 signal was observed in the vasculature.
• siRNA AF6S0 perinuclear AF680 signal was not detected in the brain parenchyma of any mice (see Figure 4B) and no AF680 signal was observed in the vasculature.
• p97-siRNA AF6S0 perinuclear AF680 signal was detected in the brain parenchyma of all mice (see Figure 4D), and no AF680 signal was observed in the vasculature.
• Figure 7 shows the calculated volume fractions of the siRNA AF6S0 and the p97-siRNA AF6S0 conjugate in brain tissues relative to the PBS control.
• mice were injected (as above) with a low dose of test molecules (10 mg/kg), and tissue samples were collected for analysis one hour after injection.
• Total RNA was extracted from frozen brain tissues (see TRIzol ® reagents and methods) and real-time PCR was performed on the RNA samples using the primers in Table E2 below.
• RNA sample was reverse transcribed to cDNA with qScript cDMA SuperMix agent (QuantaBio) using random hexamers and oligo dT.
• Real-time RT-PCR was then performed using SYBR Green I technology on ABI 7500 FAST REAL TIME PCR System (Applied Biosystems, Foster city, USA).
• a master mix for each PCR run was prepared with SYBR Green PCR Core Reagents (Applied Biosystems, Foster City, USA) containing: lx SYBR Green PCR Buffer, 3 mM MgCI2, 1 mM dNTP, 0.625U Taq polymerase, and 0.25 U Amperase UNG. 10 ng of cDNA were added, and 300 nM each for specific sense and anti-sense primers were used.
• Amplification program for Real-time PCR was as follows: 50°C 2 min, 95°C 10 min, 40 cycles at 95°C for 15 s followed by 60°C for 1 min. All samples were amplified in triplicate from the same RNA preparation and the mean value was considered. The real-time PCR efficiency was determined for each gene and each stress with the slope of a linear regression model. For this, each cDNA sample was bulked and then used as the PCR template in a range of 50, 25, 10, 5, and 2 ng. For each gene, PCR efficiency was determined by measuring the CT to a specific threshold (Walker, 2002) for a serial dilution of bulked cDNA. All PCR reactions displayed efficiencies between 84% and 96%.
• Figure 8 shows the RT-PCR results for the seven different mice tested (one PBS control and three for each siRNA test molecule). Levels of NOX4 mRNA are shown relative to GAPDH control.
• Figure 9A shows that NOX4 mRNA is significantly down-regulated in brain tissues from mice treated with p97-siRNA conjugate relative to controls (PBS and unconjugated si NA).
• Figure 9B shows the percentage reduction in endogenous NOX4 expression in brain tissues from mice treated with p97-siRNA conjugate relative to unconjugated siRNA.
• Figure 9C shows the fold-reduction ( ⁇ 1.8-fold) in NOX4 expression in brain tissues from mice treated with p97-siRNA conjugate relative to unconjugated siRNA.
• NOX4 gene-targeted p97-siRNA conjugates from Example 1 were tested for their ability to provide neuroprotection in a mouse model of middle cerebral artery occlusion (MCAO) (see, e.g., Chang et al., J Vis Exp. 2761:2011; Althenhofer et al., Cell. Mol. Life Sci. 69:2327-2343, 2012; and Kleinschnitz et al., PLoS Biol. 8(9):el000479, 2010).
• MCAO middle cerebral artery occlusion
• C57/BI6 mice were pretreated 3 times (1 hour interval between each injection) with either PBS or 30 mg/kg of siRNA alone or p97-siRNA via intravenous (IV) injection. Animals were subjected to stroke via MCAO about 1 hour following the last injection, as follows.
• the external and internal carotid arteries (ECA and ICA, respectively) were dissected and isolated and the common carotid artery (CCA) ligated.
• An 11cm 8-0 nylon thread coated in dental silicone was inserted into a small hole in the ECA towards the bifurcation of the CCA. The thread was gently introduced into the ICA and fed until only 1mm is protruding from the hole.
• the thread passes into the Circle of Willis and occludes the opening of the middle cerebral artery allowing no blood to pass.
• the thread was left in place for 1 hour and then removed and the ligature on the CCA removed allowing reperfusion of the affected brain area. Mice were sacrificed at 24 hours post reperfusion, and the brains dissected and removed for sample analysis.
• Infarct Volume Analysis To measure ischemic damage at 24 hours after 1 hour MCA occlusion, the brains were removed from euthanized animals, the cerebrum was cut into five 2-mm- thick coronal slices, which were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC). The brain slices were then cut in half to separate the ischemic hemisphere and the contralateral hemisphere. The ischemic and contralateral brain slices were transferred to separate vials containing 2% of TTC solution and continue to stain at 37°C for 60min. Images of the brain slices were obtained with a HP scanner after 10-15 min staining at RT.
• TTC 2,3,5-triphenyltetrazolium chloride
• infarct size was measured by a colorimetric staining method. Briefly, the tissue was rinsed with saline and subsequently exposed to a mixture of ethanol/dimethyl sulfoxide (1:1) to solubilize the formazan product. After 24-hour incubation in the dark, the red solvent extracts were diluted 1:20 with fresh ethanol/dimethyl sulfoxide (1:1) solvent in three glass tubes and then transferred into cuvettes.
• % loss lOOx (1 - absorbance of ischemic hemisphere/absorbance of contralateral hemisphere)
• the neurological deficits were scored as follows:
• pretreatment with MTfp-siRNA conjugates improved the neurological deficit in the animals at 0.5 hours post-stroke induction relative to pretreatment with PBS or siRNA- only.
• NOX4 mRNA Expression Real-time PCR was performed to analyze mRNA levels of NOX4 following pretreatment and stroke induction by MCAO.
• Figure 12A shows that MTfp-siRNA pretreatment led to lower Nox4 induction in the stroke-induced portion of the brain relative to PBS or siRNA-only.
• Figure 12B shows that MTfp-siRNA pretreatment reduced NOX4 mRNA expression in the stroke-induced brain relative to PBS or siRNA-only.
• MTfp mediated the transport of siRNA (targeting NOX4 gene) across the BBB into the brain.
• pretreatment with MTfp-siRNA significantly reduced the infarct volume 24 hours after ischemic induction of stroke via MCAO.
• the smaller infract areas correlated with better neurologic scores in mice injected with MTfp-siRNA prior to induction of stroke.
• pretreatment with MTfp-siRNA improved the neurological deficit in mice at both 0.5 hours and 24 hours post stroke induction relative to pretreatment with PBS or siRNA-only.
• MTfp-siRNA pretreatment also reduced Nox4 expression at the mRNA level relative to PBS or siRNA-only controls in the stroke-induced brain (left brain).
• siRNA can be delivered to the brain at a therapeutic dose after conjugation to p97 polypeptides such as MTfp.

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