WO2015025217A2 - Structure, manufacturing and uses of human-derived cell-permeable peptides conjugated with specific biologically active cargo peptides - Google Patents

Structure, manufacturing and uses of human-derived cell-permeable peptides conjugated with specific biologically active cargo peptides Download PDF

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Publication number
WO2015025217A2
WO2015025217A2 PCT/IB2014/002029 IB2014002029W WO2015025217A2 WO 2015025217 A2 WO2015025217 A2 WO 2015025217A2 IB 2014002029 W IB2014002029 W IB 2014002029W WO 2015025217 A2 WO2015025217 A2 WO 2015025217A2
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WIPO (PCT)
Prior art keywords
conjugate
region
subject
seq
formulation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/IB2014/002029
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English (en)
French (fr)
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WO2015025217A3 (en
WO2015025217A4 (en
Inventor
Bruce H. Littman
John F. Thompson
Frank W. MARCOUX
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
PPL BVI Ltd
Original Assignee
Portage Pharmaceuticals Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EP19175082.7A priority Critical patent/EP3581207B1/en
Priority to ES14838596T priority patent/ES2745566T3/es
Priority to DK14838596.6T priority patent/DK3007729T3/da
Priority to JP2016518595A priority patent/JP6479777B2/ja
Priority to CN201480033651.9A priority patent/CN105555317A/zh
Priority to MX2015016588A priority patent/MX2015016588A/es
Priority to AU2014310360A priority patent/AU2014310360B2/en
Priority to EP14838596.6A priority patent/EP3007729B1/en
Priority to CA2915296A priority patent/CA2915296A1/en
Priority to US14/897,450 priority patent/US10301629B2/en
Priority to HK16112193.2A priority patent/HK1223838A1/zh
Application filed by Portage Pharmaceuticals Ltd filed Critical Portage Pharmaceuticals Ltd
Publication of WO2015025217A2 publication Critical patent/WO2015025217A2/en
Publication of WO2015025217A3 publication Critical patent/WO2015025217A3/en
Anticipated expiration legal-status Critical
Publication of WO2015025217A4 publication Critical patent/WO2015025217A4/en
Priority to ZA2016/00162A priority patent/ZA201600162B/en
Priority to US16/268,814 priority patent/US10947539B2/en
Priority to AU2020200454A priority patent/AU2020200454A1/en
Priority to US17/179,079 priority patent/US20210180069A1/en
Priority to AU2022202225A priority patent/AU2022202225A1/en
Ceased legal-status Critical Current

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    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/1101—IkappaB kinase (2.7.11.10)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/06—Sulfuric ester hydrolases (3.1.6)
    • C12Y301/06013—Iduronate-2-sulfatase (3.1.6.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01045—Glucosylceramidase (3.2.1.45), i.e. beta-glucocerebrosidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01076—L-Iduronidase (3.2.1.76)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y406/00—Phosphorus-oxygen lyases (4.6)
    • C12Y406/01—Phosphorus-oxygen lyases (4.6.1)
    • C12Y406/01002—Guanylate cyclase (4.6.1.2)

Definitions

  • the embodiments disclosed herein relate generally to compositions and methods for treating a variety of conditions or diseases through administration of cell permeable peptide conjugates, particularly, but not exclusively, in the form of a fusion protein or protein/nucleic acid complex utilizing peptides derived from the amino acid homeodomain sequences coded for by human homeobox (HOX) genes and other human genes containing a homeodomain with cell permeabilizing properties. More particularly, the embodiments relate to a delivery system comprising a human homeodomain or variant or portion thereof, linked to one or more functional or regulatory peptides or proteins. These peptides facilitate entry into tissues, cells and the nucleus of cells thus allowing the fusion protein or conjugate containing a biologically active second region or "cargo" to reach its site of action for therapeutic purposes.
  • HOX human homeobox
  • genes may be regulated through the direct delivery of biologically active molecules, such as nucleic acids, peptides and proteins, to their intracellular and intranuclear sites of action to influence gene expression either directly or indirectly through interference with transcription, translation or transcription factor production and action and also missing or defective protein products may be replaced to provide these types of molecules in individuals with germ-line or somatic mutations.
  • biologically active molecules such as nucleic acids, peptides and proteins
  • compositions for conjugates including fusion proteins, and methods of using them to treat a variety of conditions.
  • the conjugates or fusion proteins incorporate a human HOX gene-derived 60 amino acid sequence or variant, or other human homeodomain sequence or variant or portion thereof to translocate functional and regulatory molecules, which are not naturally associated with the human homeodomain or variant or portion thereof, across cell and nuclear membranes to their intended sites of action without provoking an unwanted immune response that may reduce exposure and/or effectiveness of the conjugate, or which may produce an adverse clinical event.
  • conjugates and fusion proteins also allow entry into the CNS by facilitating passage through the blood-brain barrier and then entry into cells in the CNS, such as microglia and neurons; they also allow for engagement of these functional or regulatory molecules to their intra-cytoplasmic and intranuclear targets.
  • the ability to deliver directly into a cell (i) the expression product of a gene of interest or (ii) novel molecules able to regulate gene function has wide applicability in the medical field.
  • peptide sequences from human HOX genes also facilitate transport across the blood-brain barrier thus providing for CNS intracellular exposure to molecules containing these HOX gene sequences.
  • systemic administration can target cancers of the central nervous system by delivering drugs across the blood brain barrier.
  • Her2 e.g., trastuzumab
  • antibacterial and anti-viral agents that otherwise do not achieve therapeutic levels in the CNS are able to penetrate the blood brain barrier and successfully treat intracellular and extracellular bacterial and viral CNS infections in animal models.
  • the conjugate comprises a first region, having a sequence described in any of SEQ ID Nos. 1 -19 or variant or portion thereof, conjugated to a second region not naturally associated with the first region, wherein the second region is a polypeptide having a sequence set forth in any one of SEQ ID Nos. 20, 23, 26, 30, 33, 36, 39, 42, 45, 48, 51 , 54, 57, 60, 63, 66, 69, and 72 or a variant or variant or portion thereof.
  • the first region is derived from the human gene.
  • the conjugate comprises:
  • the second region may comprise a nucleic acid.
  • This may be associated via the specific 1 ,3 dipolar Huisgen cycloaddition reaction known as 'click-reaction' between azide and alkyne groups and is employed for the synthesis of peptide-oligonucleotide conjugates.
  • this embodiment may be seen as a protein/nucleic acid complex where the second region may comprise a nucleic acid binding domain, capable of binding nucleic acids as part of the complex.
  • the first and second regions are associated via the specific 1 ,3 dipolar Huisgen cycloaddition reaction (i.e., 'click- reaction'), wherein the oligonucleotides act as miRNAs or antagonists to miRNAs in order to affect gene expression.
  • These oligonucleotides may comprise DNA, RNA, LNA (locked nucleic acid), PNA (peptide nucleic acid) or other nucleic acids.
  • the first and second regions are connected via standard peptide bonds wherein nucleobases are attached to a peptide backbone rather than to a phosphodiester backbone. Some or all of these nucleobases are capable of hybridizing to other nucleic acids in the cell and affecting gene expression. These nucleobases may be PNA, ⁇ or contain other side chains with similar properties.
  • peptide or peptide-nucleic acid conjugates can be used to affect the activity of miRNAs including, but not limited to, antagonizing miRNA-132 for inhibition of the Ras pathway and cancer, antagonizing miR-21 for inhibiting cancer or the NF- ⁇ pathway and inflammation, or affecting the function of other miRNAs and pathways that may have detrimental and/or undesirable effects on health.
  • the conjugate is in the form of a fusion protein.
  • the second region is a functional or regulatory protein.
  • a conjugate can comprise:
  • a second region not naturally associated with the first region comprising a protein such as a functional enzyme.
  • a composition comprises a conjugate or fusion protein that may take any of the following forms: an inhalable composition; an eye drop or other ophthalmic composition for local or injectable use; an enema; a topical composition; or an injectable composition, including an injectable implant for sustained release.
  • Another embodiment disclosed is a method comprising steps that permit a conjugate or fusion protein containing one or more biologically active peptides or proteins to be translocated to cells of the CNS.
  • Other embodiments comprise methods to prevent immunogenicity of otherwise antigenic proteins delivered as conjugates or fusion proteins to their intended cytosolic or nuclear sites of action.
  • FIG. 1 shows an exemplary 60-amino acid human HOX C12 homeodomain (first region) or variant or portion thereof at the N-terminus of a Cargo peptide.
  • FIG. 2 shows an exemplary cargo peptide (second region) at the N- terminus of 60-amino acid human HOX D12 homeodomain or variant or portion thereof.
  • FIG. 3a shows an exemplary 60-amino acid human HOX D12 homeodomain (first region) or variant or portion thereof at its C-terminus with Cargo peptides (second and third regions), where the third region may be used to target the entire structure to specific tissues or cell types.
  • FIG. 3b shows an exemplary 60-amino acid human HOX D12 homeodomain (first region) or variant or portion thereof at its N-terminus with Cargo peptides (second and third regions), where the third region may be used to target the entire structure to specific tissues or cell type.
  • FIG. 4 shows an exemplary 60-amino acid human HOX C12 homeodomain (first region) or variant or portion thereof at the N-terminus of a Cargo peptide (second region), and His-tail conjugated to C-terminus through GS linker.
  • FIG. 5 shows an exemplary 60-amino acid human HOX D12 homeodomain (first region) or variant or portion thereof at N-terminus of NBD Cargo peptide (second region), and His-tail conjugated to C-terminus through GS linker.
  • FIG. 6 shows an exemplary sequence for plasmid pJ41 1 :129925, showing a 6 HIS leader, with a transition sequence of SAA followed by the TEV cleavage site (ENLYFQS) to produce SEQ. ID No. 21 (also referred to as PPL-002) with the appropriate N-terminus.
  • ENLYFQS TEV cleavage site
  • FIG. 7 shows an exemplary sequence for plasmid pJ41 1 :129926, showing a 6 HIS leader, with a transition sequence of SAA followed by the TEV cleavage site (ENLYFQS) to produce SEQ ID No. 22 (also referred to as PPL-003) with the appropriate N-terminus.
  • ENLYFQS TEV cleavage site
  • FIG. 8a shows inhibition of NF- ⁇ activation in murine RAW 264.7 cells (LPS 4-hour stimulation). Error bars represent SEM.
  • FIG. 8b shows inhibition of NF- ⁇ activation in Human Embryonic Kidney (HEK) 293 cells (TNF-cc 4-hour stimulation). Error bars represent SEM.
  • FIG. 9 shows inhibition of TN F-cc-stimulated N F- ⁇ activation in HEK 293 N F-KB Luciferase Reporter Cells by NBD cargo containing cell permeable peptides
  • FIG. 10 Timeline for determining in vivo activity of NBD cargo-containing cell permeable peptides: Mice were pretreated with peptides, challenged by injection of lipopolysaccharide (LPS) and blood samples were taken for cytokine assays at the indicated times
  • FIG. 1 1 a shows TNF-cc response following LPS injection and peptide or dexamethasone (Dex) pretreatment. Each datapoint represents the value for a single mouse.
  • FIG. 1 1 b shows IP-10 response following LPS injection and peptide or dexamethasone (Dex) pretreatment. Each datapoint represents the value for a single mouse.
  • FIG. 12 shows IL-10 response following LPS injection and peptide or dexamethasone (Dex) pretreatment. Each datapoint represents the value for a single mouse.
  • FIG. 13 shows PPL-003 concentration in plasma following dosing at 500, 1000, and 2000 microgram.
  • FIG. 14 shows PPL-003 C ma x in plasma by dose, showing average and median at C ma x (30 min.).
  • FIGS. 15a and 15b show amyloid A protein (SAA) response at 2 hours following LPS challenge with pretreatment of2000 microgram of PPL-003 at 0, 30- and 60-minutes prior to LPS stimulation. Error bars represent SEM in FIG 15a and data points in FIG 15b are individual mice, horizontal lines are median values.
  • SAA amyloid A protein
  • FIGS. 16a and 16b show amyloid A protein (SAA) response to LPS following PPL-003 pretreatment at doses of 500, 1000, and 2000 micrograms. Error bars represent SEM in FIG 16a, and data points in FIG 16b are individual mice, horizontal lines are median values. Percentages are percent inhibition relative to vehicle/LPS.
  • SAA amyloid A protein
  • FIGS. 17a and 17b show IL-10 and Mouse Kc responses, respectively, following 2000 microgram PPL-003 dose at 0, 30- and 60-minutes prior to LPS stimulation. Error bars represent SEM.
  • FIGs. 18a and 18b show inhibition of GM-CSF response following 2000 microgram PPL-003 or PPL-004 dose, respectively, at 0, 30- and 60-minutes prior to LPS stimulation. Error bars represent SEM. DETAILED DESCRIPTION
  • the human HOX genes and other homeodomain coding genes are important in embryonic development and include small regions homologous to the gene antennapedia (Antp) but where the majority of amino acids are included in uniquely human sequences that facilitate their function.
  • the protein sequence of antennapedia is characterized by the presence of a 60-amino acid motif (homeodomain) that binds to specific DNA target elements.
  • the Antp homeodomain see US Pat. No. 7,968,512
  • much smaller sequences from the Antp gene have been shown to facilitate the entry of biologically active (“cargo”) peptides into tissues and cells to reach their site of action for therapeutic purposes.
  • peptide sequences from human HOX genes and certain other human homeodomain sequences can also facilitate transport of biologically active molecules such as peptides, proteins and nucleotides to their intracellular and intranuclear sites of action (i) without provoking an unwanted immune response that may reduce exposure and/or effectiveness of the conjugate or may produce an adverse event, and (ii) with greater efficiency than Antp conjugates and fusion proteins.
  • biologically active molecules including the expression product of a gene of interest directly into a cell has wide applicability, particularly in the medical field.
  • HOX and other human homeodomain peptides are also able to translocate nucleic acids. This is especially advantageous for applications utilizing a mechanism of gene regulation.
  • novel conjugates which may take the form of fusion proteins, each comprising a 60- amino acid peptide or variant or portion thereof (1 e.g. as shown in FIG. 1 ) derived from human HOX genes or from other human genes containing a homeodomain with cell permeabilizing properties, linked (e.g. 12 or 13 as shown in FIG. 1 ) to a second region (2): at least one of the biologically active "cargo" peptides disclosed herein.
  • FIG. 1 fusion proteins, each comprising a 60- amino acid peptide or variant or portion thereof (1 e.g. as shown in FIG. 1 ) derived from human HOX genes or from other human genes containing a homeodomain with cell permeabilizing properties, linked (e.g. 12 or 13 as shown in FIG. 1 ) to a second region (2): at least one of the biologically active "cargo" peptides disclosed herein.
  • FIG. 1 shows one embodiment comprising the 60-amino acid human HOX C12 homeodomain ("first region") (1 ) linked on its C-terminus (4) to the N-terminus (5) of a biologically-active cargo peptide ("second region”) (2).
  • FIG. 2 shows an alternative embodiment comprising the 60-amino acid human HOX D12 homeodomain (“first region”) (1 ) is linked on its N-terminus (3) to the C-terminus (6) of a biologically- active cargo peptide ("second region”) (2).
  • the links in both embodiments may be a simple peptide bond (13) or a linker sequence known in the art (12) provided that function of the fusion protein or conjugate is not compromised by its addition.
  • the term "compromised” refers to reduced function of the conjugate embodiments as reflected in reduced efficacy of treatment for any of the outcomes discussed herein, e.g., compromised function could be reflected in an attenuated reduction in immunogenicity as compared to the reduction in immunogenicity observed with (1 ) the conjugate in the form of a fusion protein, or (2) the conjugate comprising an alternative linker.
  • Cargo peptides include both small synthetic peptides and larger proteins such as antibodies or the binding regions of antibodies into cells for therapeutic purposes.
  • human homeodomain refers to (1 ) human HOX-derived homeodomains such as the HOX C12 and HOX D12 sequences shown in Table 3 as SEQ ID No. 1 and SEQ ID No. 2, respectively, or variants or portions thereof; and (2) any other human homeodomain that has cell permeabilizing activity, such as SEQ ID Nos. 3 through 19, or variants or portions thereof.
  • the phrase "not naturally associated with” means that entire sequence of the conjugate or fusion protein is not found in nature, and that the entire sequence is not encoded for by a single gene found in nature.
  • the phrase "operably linked” means that the first and second region are linked such that the second region is able to translocate a cell membrane. Such linkage may be produced via application of "click” chemistry methods or other methods known in the art or may be incorporated as a fusion protein with a peptide bond between regions.
  • homology refers to identity or near identity of nucleotide or amino acid sequences.
  • nucleotide mismatches can occur at the third or wobble base in the codon without causing amino acid substitutions in the translated polypeptide sequence.
  • minor nucleotide modifications e.g., substitutions, insertions or deletions
  • substitutions, insertions or deletions in certain regions of the gene sequence can be tolerated whenever such modifications result in changes in amino acid sequence that do not alter functionality of the final gene product.
  • Homologs of specific DNA sequences may be identified by those skilled in the art using the test of cross-hybridization of nucleic acids under conditions of stringency as is well understood m the art (as described in Hames et al.. Nucleic Acid Hybridisation, (1985) IRL Press, Oxford, UK). Extent of homology is often measured in terms of percentage of identity between the sequences compared.
  • variant refers to a polypeptide or protein that differs from a reference polypeptide or protein, but retains essential properties.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall (homologous) and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions).
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of skill in the art and include, but are not limited to (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lie: Leu, Val), (Leu: lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: lie, Leu).
  • Embodiments of this disclosure therefore, consider functional or biological equivalents of a polypeptide or protein as set forth above.
  • embodiments of the polypeptides and proteins can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide and protein of interest.
  • Identity is a relationship between two or more polypeptide or protein sequences, as determined by comparing the sequences.
  • identity also refers to the degree of sequence relatedness between polypeptides or proteins, as determined by the match between strings of such sequences. “Identity” can be readily calculated by known bioinformational methods.
  • the first region of the conjugate or fusion protein embodiments disclosed may comprise a natural or synthetic 60-amino acid peptide or variant or portion thereof derived from the HOX C12 or HOX D12 gene or any other human homeodomain that has cell permeabilizing activity, such as SEQ ID Nos. 3 through 19, or variants or portions thereof.
  • the HOX C12 (SEQ ID No. 1 ) amino acid sequence is:
  • HOX D12 (SEQ ID No. 2) amino acid sequence is:
  • embodiments will include any one of 17 additional human 60-amino acid homeodomain sequences or variants or portions thereof with permeabilizing properties to allow for intracellular and intranuclear delivery of conjugates and fusion proteins. Further, these additional sequences each provide for CNS penetration of molecules that otherwise would not penetrate the blood brain barrier. Examples of amino acid sequences of these conjugates and fusion proteins with HOX C12 and HOX D12 homeodomains in the first region are shown in Table 3 (SEQ ID Nos.
  • each of the human homeodomain sequences is less than 50% identical to the 60-amino acid Antp sequence and thus is structurally distinct and has unique human properties.
  • synthetic variants may be used provided that they retain the ability to translocate the membrane.
  • Synthetic variants will generally differ from the naturally-occurring proteins by substitution, particularly conservative substitution.
  • conservative amino acid changes herein means replacing an amino acid from one of the amino acid groups, namely hydrophobic, polar, acidic or basic, with an amino acid from within the same group.
  • An example of such a change is the replacement of valine by methionine and vice versa.
  • Other examples of conservative substitutions may be seen by reference to Table 1 below:
  • Such variants may be synthesized directly or prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed to make the encoded protein.
  • These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.
  • Variants that retain at least 50% sequence identity with the claimed 60-amino acid sequences or variants or portions thereof derived from HOX-C12 and HOX-D12 will likely maintain their cell permeability characteristics and retain their human characteristics resulting in low immunogenicity potential.
  • the ability of a naturally occurring or synthetic HOX sequence to translocate the membrane may be tested by routine methods known in the art. Any polynucleotide which encodes the amino acid of SEQ ID Nos. 1 -19 (Table 3) or variant or portion thereof can be used in a fusion protein or conjugate herein.
  • the second region of the conjugate or fusion protein embodiments disclosed may comprise any peptide or protein sequence not naturally associated with the first region.
  • the gene encoding the first region may or may not also encode the second region.
  • the second region also may or may not be from the same species as the first region, but the first and second regions will be present in the conjugate or fusion protein embodiments in a manner different from the natural situation.
  • the second region of the fusion protein or conjugate embodiments may be a peptide or protein of any length as long as it is biologically active on its target when included in the fusion protein or conjugate.
  • the second region may include any nucleic acid that may be therapeutically active.
  • the nucleic acid can be DNA or RNA.
  • the nucleic acid is an oligonucleotide or a PNA.
  • the second region may include any peptide nucleic acid such as PNAs in which nucleobases replace standard amino acids and the second region may be therapeutically active, including acting via antagonizing or activating miRNAs, in order to affect gene expression or other biological process.
  • PNAs peptide nucleic acid
  • the second region may be therapeutically active, including acting via antagonizing or activating miRNAs, in order to affect gene expression or other biological process.
  • the second region may include any peptide nucleic acid such as PNAs and wherein the second region may be therapeutically active, including acting via antagonizing or activating mRNAs, by activating or repressing splicing or translation in order to affect gene expression or other biological process.
  • PNAs peptide nucleic acid
  • the second region may be therapeutically active, including acting via antagonizing or activating mRNAs, by activating or repressing splicing or translation in order to affect gene expression or other biological process.
  • the complex can further comprise a nucleic acid or PNA as part of the second region that, when inside a cell, binds to a RISC complex or an miRNA.
  • a nucleic acid or PNA as part of the second region that, when inside a cell, binds to a RISC complex or an miRNA.
  • binding to miR132 miRNA may inhibit cancer or NF- ⁇ .
  • binding to an miRNA molecule can form a stable complex, wherein the miRNA molecule can become incapable of affecting the expression of genes as would normally be the case for an unbound miRNA.
  • the complex can further comprise a nucleic acid or PNA as part of the second region that, when inside a cell, binds to genomic DNA.
  • a nucleic acid or PNA as part of the second region that, when inside a cell, binds to genomic DNA.
  • binding to DNA at transcription factor binding sites may prevent transcription factor binding and/or activity and thus affect gene transcription.
  • the complex can further comprise a nucleic acid, LNA or PNA as part of the second region that, when inside a cell, binds to an mRNA. For example, binding to DYRKI b mRNA can down-regulate DYRKI b expression and/or translation.
  • the complex can further comprise a nucleic acid (or DNA) binding domain as part of the second region. In one embodiment, the nucleic acid binding domain may serve to mediate the specific, high affinity and non-covalent interaction of the protein component with the nucleic acid or PNA component.
  • the nucleic acid binding domain may be an RNA or DNA binding domain, e.g., the DNA binding domain of a transcription factor, particularly a yeast or human transcription factor.
  • a GAL4-derivable domain mediates the selective binding of the protein to the DNA sequence CGGAGGACAGTCCTCCG (Cavey et al J Mol Biol 209:423, 1989).
  • the DNA binding domain consists of GAL4 amino acids 2 to 147.
  • a DNA binding domain may bind to single-stranded or to a double-stranded DNA on the second domain.
  • conjugate or fusion protein embodiments disclosed include development of antibacterial and antiviral measures.
  • HOX gene-derived or other human homeodomain-derived cell permeable peptides may be used to transport in the cytoplasm of infected cells recombinant antibodies, naturally occurring innate immunity effecter molecules or DNA binding molecules and which destroy bacteria, parasites or interfere with a crucial step of bacterial or viral replication.
  • Suitable peptides and proteins include those that are of therapeutic and/or diagnostic application such as, but are not limited to: cytokines, chemokines, hormones, antibodies, engineered immunoglobulin-like molecules, a single chain antibody, conjugates, enzymes, immune co-stimulatory molecules, immunomodulatory molecules, anti-sense RNA, a transdominant negative mutant of a target protein, a toxin, such as endotoxin A, Colicin A, d-endotoxin, diphtheria toxin, Bacillus anthrox toxin, Cholera toxin, Pertussis toxin, E.
  • One or more embodiments may provide a pharmaceutical composition for treating an individual by gene therapy in animals or humans, wherein the composition comprises a therapeutically effective amount of the conjugate or fusion protein. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular individual.
  • Fusion protein or conjugate embodiments delivering one or more therapeutic molecules such as genes or proteins may be used alone or in combination with other treatments or components of the treatment.
  • Diseases which may be treated include, but are not limited to: cancer, neurological diseases, inherited diseases, heart disease, stroke, arthritis, viral and bacterial infections, and diseases of the immune system.
  • Suitable therapeutic genes include those coding for tumor suppressor proteins, enzymes, pro-drug activating enzymes, immunomodulatory molecules, antibodies, engineered immunoglobulin-like molecules, conjugates, hormones, membrane proteins, vasoactive proteins or peptides, cytokines, chemokines, anti-viral proteins, antisense RNA and ribozymes.
  • the conjugate may also contain one or more cytokine-encoding nucleic acids or cytokines.
  • Suitable cytokines and growth factors include but are not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1 , EGF, ENA-78, Eotaxin, Eotaxin-2. Exodus-2, FGF-acidic, FGF-basic, fibroblast growth factor-10 (Marshall 1998 Nature Biotechnology 16: 129).FLT3 ligand (Kimura et al.
  • the fusion protein or conjugate embodiments disclosed may comprise further suitable domains known to those skilled in the art.
  • an endoplasmic reticulum retention signal functions to affect the intracellular routing of the internalized conjugate or protein/nucleic acid complex.
  • a suitable endoplasmic retention signal may be a mammalian endoplasmic reticulum retention signal.
  • translocation domain which serves to enhance nucleic acid or protein escape from the cellular vesicle system and thus to augment nucleic acid transfer by this route.
  • This domain may serve to reduce or avoid lysosomal degradation after internalization of the protein/nucleic acid into the target cell.
  • Suitable translocation domains are derivable from toxins, particularly bacterial toxins, such as exotoxin A, Colicin A, d-endotoxin, diphtheria toxin, Bacillus anthrax toxin, Cholera toxin, Perussis toxin, E. coli toxin toxins, Shigatoxin or Shiga-like toxin.
  • the first binding domain may be modified to target cell sites other than the nucleus.
  • a target cell-specific binding domain recognizing a cell surface structure may be present, such as a receptor protein or surface antigen on the target cell.
  • conjugate comprises a category of structures, including fusion proteins, in which the first region, a 60-amino acid human homeodomain sequence or variant or portion thereof, is conjugated directly via a peptide bond or other type of bond including both covalent and non-covalent bonds.
  • Conjugates may include a linker region that connects the homeodomain sequence to a second region, a functional or regulatory peptide or protein ("cargo" peptide) that is not naturally associated with the first region. Any of a wide variety of linkers (short, connecting sequences) known in the art may be utilized to form the conjugate provided that function of the conjugate is not compromised by its addition.
  • translocation of the second region is enabled through a cellular or nuclear membrane.
  • linkers known in the art in Chen et al. "Fusion protein linkers: property, design and functionality.” Advanced Drug Delivery Reviews. http://dx.doi.Org/10.1016/J.addr.2012.09.039.
  • fusion protein is used to refer to a particular subcategory of conjugate that exists when no such linkers are used to form the conjugate and the domains are linked entirely by peptide bonds.
  • the first (HOX or other human homeodomain) region and second (cargo) regions may be linked by a cleavable linker region this may be any region suitable for this purpose provided the function of the conjugate is not compromised by its addition.
  • the cleavable linker region is a protease cleavable linker, although other linkers, cleavable for example by small molecules, may be used. These include Met- X sites, cleavable by cyanogen bromide, Asn-Gly, cleavable by hydroxylamine, Asp- Pro, cleavable by weak acid and Trp-X cleavable by, inter alia, NBS-skatole.
  • protease cleavage sites require milder cleavage conditions and are found in, for example, factor Xa, thrombin and collagenase. Any of these may be used. The precise sequences are available in the art and the skilled person will have no difficulty in selecting a suitable cleavage site.
  • the protease cleavage region targeted by Factor Xa is I E G R.
  • the protease cleavage region targeted by Enterokinase is D D D D D K.
  • the protease cleavage region targeted by Thrombin is L V P R G.
  • the cleavable linker region may be one that is targeted by endocellular proteases. Linkers may not be required for function but linkers may be included between first and second regions to allow targeted release of the second region without compromising function or to enhance biological activity of the second region with linker cleavage.
  • RVLjAEA RVLjAEA
  • EDWCQSMSY GGIEGRjGS
  • TRHRQPRJ G WE
  • AGNRVRRJ SVG
  • the embodiments disclosed allow for potent therapeutic action, including efficient translocation into intracellular and intranuclear sites of action of any of a number of identified peptides for the specified therapeutic treatments, wherein (i) the CNS is a target tissue; and (ii) regardless of the target tissue, such translocation does not provoke an unwanted immune response that may reduce the exposure and/or effectiveness of the conjugate or produce an adverse or undesirable clinical event.
  • the biologically-active peptide of the "second region” is not naturally associated with the human homeodomain sequence or variant or portion thereof (the "first region”) before they are joined.
  • the conjugate or fusion protein may comprise a third region also comprising a cargo peptide.
  • FIGS. 3a and 3b show alternative embodiments comprising the 60-amino acid human homeodomain ("first region,” shown abbreviated here) and an additional cargo protein that may target the fusion protein or conjugate to its cellular site of action.
  • first region shown abbreviated here
  • additional cargo protein that may target the fusion protein or conjugate to its cellular site of action.
  • the human HOX D12 homeodomain on the left (1 ) is linked on its C-terminus (4) to the N-terminus (5) of a biologically-active cargo peptide of interest ("second region") (2), while a third cargo peptide of interest (“third region”) (7) is linked on its N-terminus (8) to the C- terminus (6) of the "second region” (2).
  • This third region may remain linked or fused via peptide bonds to the entire fusion protein or may be designed for removal after uptake or binding to specific sites in targeted tissues.
  • FIG. 3b shows an alternative embodiment, with the human HOX D12 homeodomain on the right (1 ), linked through its N-terminus (3) to the C-terminus (6) of the second region (2), while a third region cargo peptide (7) is linked through the N-terminus (5) of the second region (2).
  • the links may be simple peptide bonds (13) or a linker sequences known in the art (12), or a combination of both, provided that the function of the conjugate is not compromised by the addition of a linker sequence.
  • a second cargo peptide (“third region”) (7) may target the conjugate or fusion protein to its cellular site of action. See Example 2.
  • Conjugate and fusion protein embodiments herein may be produced in accordance with any of the standard molecular biology techniques described in the literature. See, for example, Ausubel et al. (2002) Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 5th Ed. John Wiley & Sons. Manufacturing methods including purification methods that may be used are also disclosed in U.S. Pat. No. 7,968,512, the entirety of which is incorporated herein by reference.
  • Expression vectors or “plasmids” may be used for producing conjugates or components thereof to introduce heterologous DNA into host cells, either for expression or replication. Selection by the artisan of the appropriate vector will depend on its intended use, i.e. (DNA amplification or DNA expression), the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector.
  • Each vector contains various components depending on its intended use, which comprise one or more of: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.
  • Sources of nucleic acid may be ascertained by reference to published literature or databanks provided by organizations such as NCBI or EMBL. Identification of sequences of interest may be accomplished by using BLAST, BLAT, or other homology search algorithms. Further, nucleic acid encoding the desired first or second region may be obtained from academic or commercial sources where such sources are willing to provide the material or by synthesizing or cloning the appropriate sequence where only the sequence data are available. Generally, this may be done by reference to literature sources which describe the cloning of the gene in question.
  • exemplary nucleic acids can be characterized as those nucleotide sequences which hybridize to the nucleic acid sequences known in the art.
  • stringency of hybridization refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skill in the art. Also as understood by persons skilled in the art, the stability of hybrids is reflected in the melting temperature (T m ) of the hybrid which decreases approximately 1 to 1 .5[deg.] C. with every 1 % decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. The hybridization reaction typically is performed under conditions of higher stringency, followed by washes of varying stringency.
  • high stringency refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 1 M Na+ at 65-68[deg.] C.
  • High stringency conditions can be provided, for example, by hybridization in an aqueous solution containing 6*SSC, 5* Denhardt's, 1 % SDS (sodium dodecyl sulphate), 0.1 Na+ pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as nonspecific competitor.
  • high stringency washing may be done in several steps, with a final wash (about 30 minutes) at the hybridization temperature in 0.2-0.1 *SSC, 0.1 % SDS.
  • Moderate stringency refers to conditions equivalent to hybridization in the above described solution, except that the temperature is at about 60-62[deg.] C. In that case the final wash is performed at the hybridization temperature in 1 *SSC, 0.1 % SDS.
  • Low stringency refers to conditions equivalent to hybridization in the above described solution at about 50-52[deg.] C. In that case, the final wash is performed at the hybridization temperature in 2*SSC, 0.1 % SDS.
  • the conjugate or fusion protein may be produced through use of an expression vector comprising the nucleic acid sequence and a promoter for recombinant synthesis in, for example, plant cells (including algae), in bacteria such as E. coli, or in eukaryotic cells such as Chinese Hamster Ovary (CHO) cells or yeast cells.
  • plant cells including algae
  • bacteria such as E. coli
  • eukaryotic cells such as Chinese Hamster Ovary (CHO) cells or yeast cells.
  • a host cell is transformed with the expression vector.
  • nucleic acid sequence encodes the conjugate or fusion protein for the purposes of synthesis and manufacture by recombinant technology.
  • the conjugate or fusion protein is non-denatured, meaning it may exist in its native state, the form in which the protein occurs in the intact cell in its three-dimensional structure.
  • non-denatured may also, but need not, imply a specific non- denaturing step. Denaturing alters the three-dimensional shape of the protein molecule without rupture of its peptide bonds; disulfide bonds may be ruptured, or certain groups in the protein may be chemically modified if such processes are also accompanied by changes in its overall three-dimensional structure.
  • the conjugate or fusion protein is renatured, a process by which the denatured protein is returned to its original conformation prior to denaturation.
  • reversible denaturation is generally brought about by disulfide reducing agents and urea, and for nucleic acid, by heat and salts.
  • the first region is at the N-terminus of the second region. In another embodiment the first region is at the C-terminus of the second region.
  • conjugates and fusion proteins Purification methods known in the art may be used in the process of preparing conjugates and fusion proteins according to the embodiments disclosed, for example, as described in Zachariou, M. (2010) Affinity Chromatography: Methods and Protocols, 2nd Ed. Totoua, NJ:Humana Press.
  • the conjugate or fusion protein can be obtained from bacterial or eukaryotic cell lysates , as denaturing reagents, small changes in pH, and differences in osmolarity may have an effect on the translocation properties of the peptides.
  • Conditions for obtaining and purifying the human homeodomain peptides, including HOX-derived peptides are disclosed herein.
  • HOX peptide-conjugates to translocate across the cell surface membrane may be dependent on the conformation of the recombinant proteins. For example, translocation of the polypeptide by using either bacteria cell extracts or purified proteins exposed to small amounts of detergent (ionic and non- ionic) or denaturating agents (urea or guanidinuim) may prevent or inhibit translocation.
  • This conformation-dependent property may be preserved by purifying the HOX peptide-conjugate or other human homeodomain conjugate under native conditions.
  • both the first (the human homeodomain or variant or portion thereof) and second (biologically active peptide or protein) regions are purified from a bacterial lysate.
  • the conjugate or fusion protein is purified from a plant cell lysate.
  • the conjugate or fusion protein is purified from an eukaryotic cell lysate , culture medium or fermentation broth.
  • One embodiment is a method for preparing a conjugate or fusion protein, comprising:
  • Another embodiment is a method for preparing a conjugate or fusion protein comprising:
  • a first region comprises the HOX peptide or other human homeodomain peptide
  • a second region not naturally associated with the first region comprises a peptide or protein, under conditions which provide for expression of the fusion protein from the expression vector within the host cell;
  • (ii) recovering the fusion protein which method comprises fusing an amino acid tail or other ligand to the conjugate, which tail is capable of binding to at least one substrate and not to another substrate, and wherein the conjugate is caused to bind via the tail to at least one substrate such that components of the host cell do not bind to this substrate; and the conjugate is contacted with the other substrate such that the conjugate is not bound and remaining components of the host cell are bound to the other substrate.
  • the embodiments include the use of a tail or ligand that is attached to the conjugate or fusion protein; this allows for both positive and negative purification steps.
  • additional amino acid sequences can be added to either the amino or carboxy termini in order to facilitate purification.
  • sequences may include FLAG-tags (DYKDDDDK), myc- tags (EQKLISEEDL), His-tags (such as HHHHHH, HHHHHHGS, the latter utilizing a GS linker, see FIGs. 4 and 5), and other similar tags known to those in the art.
  • ligands such as the biotin-acceptor protein (GLNDIFEAQKIEWHE) together with the active BirA protein may be used.
  • sequences that include an N-terminal initiating methionine if a N-terminal purification domain is added the methionine will be on the N-terminal of the purification domain instead of at the N-terminal of the human homeodomain peptide first region.
  • a peptide linker known in the art may be used and removed after purification with a specific protease.
  • the amino acid tail or ligand is fused to the C- terminus of the conjugate. For example, FIG.
  • FIG. 4 shows an embodiment comprising a biologically-active cargo peptide (2), linked on its C-terminus (6) through either a peptide bond (13) or a linker sequence known in the art (12) to the N-terminus (3) of the human homeodomain "first region” shown abbreviated here (1 ).
  • a GS linker (14) is shown attaching a His-tag ("tail") to the C-terminus (6) of the cargo peptide (2). Any linker may be used provided it does not compromise the function of the conjugate.
  • the immobilized substrate is a nickel or cobalt column, avidin column, or an antibody column with affinity for the amino acid tail.
  • the conjugate's amino acid tail is serially brought into contact with at least two immobilized substrates with which the tail has affinity, in which case the nickel or cobalt column and/or avidin and/or antibody may be used in any order.
  • the method of purifying a conjugate comprises fusing an amino acid tail or ligand to the conjugate or fusion protein, which tail is capable of binding to at least one substrate while impurities bind only to a second substrate; the conjugate is contacted with the other substrate such that the conjugate is not bound and remaining impurities are bound to the other substrate.
  • a complex with nucleic acid may be formed by mixing the conjugate formed with the nucleic acid.
  • a conjugate or fusion protein comprises a biologically active peptide or protein that is a functional enzyme, linked to the human homeodomain or variant or portion thereof.
  • a bacterial expression construct was generated for two homeobox candidate proteins PPL-002 (SEQ ID No. 21 , Table 3) and PPL-003 (SEQ ID No. 22, Table 3) capable of regulated expression of the target protein with a TEV cleavable N-terminal His tag in the T7 vector system.
  • the T7 expression system is suitable for producing material for animal efficacy studies; however, other expression vectors/system are also contemplated and are known by one of ordinary skill in the molecular biology/biotechnology art.
  • composition embodiments disclosed herein may comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • pharmaceutically acceptable carrier diluent or excipient refers to any substance, not itself a therapeutic agent, used as a carrier or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a unit dose of the composition, and that does not produce unacceptable toxicity or interaction with other components in the composition.
  • compositions may comprise any agents that may aid, regulate, release or increase entry into the body compartment, tissue, intracellular or intranuclear target site, such as binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), or other agents.
  • An injectable implant for the sustained release of the protein may also be used to obtain prolonged exposure and action.
  • sustained release refers to formulations from which the conjugate is released at a slow rate allowing for a longer period of exposure at active concentrations.
  • compositions comprising one or more conjugates or fusion proteins disclosed herein can be administered, depending on condition to be treated or other considerations, in any number of ways, for example without limitation, by any one or more of the following: (1 ) inhalation; (2) in the form of a suppository or pessary; (3) in the form of a topical lotion, solution, cream, ointment or dusting powder; (4) by use of a skin patch; (5) orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavoring or coloring agents; (6) injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously; (7) for ophthalmic diseases, they may be formulated as eye drops or for intraocular injection; (8) for parenteral administration, they may be in the
  • the conjugate comprises: a first region comprising a structure derived from (1 ) the human HOX genes, or (2) other human genes having a sequence described in any of SEQ ID Nos. 1 - 19, wherein 1 - 20, 1 - 15, or 1 - 10 amino acid residues are substituted, deleted, added, and/or inserted; and
  • a second region is a functional or regulatory polypeptide or protein not naturally associated with the first region.
  • the second region is selected from the group consisting of NBD peptide, PC1 CTT-derived p200, PC1 CTT-derived p21 , glucocerebrosidase, alpha-L-iduronidase, iduronidate-2-sulfatase, retinal membrane guanylyl cyclase, the zinc finger peptide construct, ZF6xHunt-Kox- 1 , the zinc finger peptide construct, ZF12xHunt-Kox-1 , and the zinc finger peptide construct, ZF18xHunt-Kox-1 .
  • the conjugate comprises:
  • a first region comprising a structure derived from (1 ) the human HOX genes, or (2) other human genes having a sequence described in any of SEQ ID Nos. 1 - 19, wherein 1 - 20, 1 - 15, or 1 - 10 amino acid residues are substituted, deleted, added, and/or inserted and wherein said amino acid has (i) has human homeodomain activity; and (ii) maintains the cell permeabilizing activity of the helical regions of the human HOX genes or other human genes containing a homeodomain with cell permeabilizing properties; and
  • a second region is a functional or regulatory polypeptide or protein not naturally associated with the first region.
  • the second region is selected from the group consisting of NBD peptide, PC1 CTT-derived p200, PC1 CTT-derived p21 , glucocerebrosidase, alpha-L-iduronidase, iduronidate-2-sulfatase, retinal membrane guanylyl cyclase, the zinc finger peptide construct, ZF6xHunt-Kox- 1 , the zinc finger peptide construct, ZF12xHunt-Kox-1 , and the zinc finger peptide construct, ZF18xHunt-Kox-1 .
  • the active concentration of fusion protein or conjugate in cell culture is less than about 1 15 ⁇ , less than about 100 ⁇ , less than about 90 ⁇ , less than about 80 ⁇ , less than about 70 ⁇ , less than about 65 ⁇ , less than about 60 ⁇ , less than about 55 ⁇ , less than about 50 ⁇ , less than about 45 ⁇ , less than about 40 ⁇ , less than about 35 ⁇ , less than about 30 ⁇ , less than about 25 ⁇ , less than about 20 ⁇ , less than about 15 ⁇ , less than about 10 ⁇ , less than about 5 ⁇ , or less than about 1 ⁇ for example, less than about 1 ⁇ to about 3 ⁇ , less than about 1 ⁇ to about 6 ⁇ , less than about 1 ⁇ to about 8 ⁇ , less than about 1 ⁇ to about 15 ⁇ , less than about 1 ⁇ to about 25 ⁇ , less than about 1 ⁇ to about 50 ⁇ , less than about 1 ⁇ to about 70 ⁇ , less than about 1 ⁇ to about 85 ⁇ ,
  • the dosage delivered (daily or as required) in mouse models (per 20 g mouse) through (1 ) an intravenous (i.v.) or intraperitoneal (i.p.) injection, (2) a topical formulation, or (3) an inhaled formulation is at least 500 ⁇ g, at least 450 ⁇ g, at least 400 ⁇ g, at least 350 ⁇ g, at least 300 ⁇ g, at least 250 ⁇ g, at least 200 ⁇ g, at least 150 ⁇ g, at least 100 ⁇ g, at least 80 ⁇ g, at least 70 ⁇ g, at least 60 ⁇ g, at least 50 ⁇ g, at least 40 ⁇ g, at least 30 ⁇ g, at least 20 ⁇ g, at least 10 ⁇ g, or at least 1 ⁇ g, for example, about 1 ⁇ g to about 500 ⁇ g, about 10 ⁇ g to about 450 ⁇ g, about 20 ⁇ g to about 400 ⁇ g about 30 ⁇ g to about 350 ⁇ g, about 30 ⁇
  • the dosage delivered (daily or as required) through an intravenous (i.v.) or intraperitoneal (i.p.) injection in a mouse model is at least 100 mg/kg, less than about 80 mg/kg, less than about 45 mg/kg, less than about 40 mg/kg, less than about 30 mg/kg, less than about 25 mg/kg, less than about 20 mg/kg, less than about 15 mg/kg, less than about 12 mg/kg, less than about 10 mg/kg, less than about 8 mg/kg, less than about 4 mg/kg, less than about 2 mg/kg, or less than about 1 mg/kg, for example, less than about 1 mg/kg to about 50 mg/kg, about 5 mg/kg to about 40 mg/kg, about 8 mg/kg to about 30 mg/kg, about 10 mg/kg to about 20 mg/kg, or about 12 mg/kg to about 15 mg/kg, about 8 mg/kg to about 12 mg/kg, about 5 mg/kg to about 9 mg/kg, about 3 mg/kg, less than about 1 mg/
  • the dosage delivered (daily or as required) through an inhaled formulation in humans is at least about 600 mg, at least about 500 mg, at least about 450 mg, at least about 400 mg, at least about 350 mg, at least about 300 mg, at least about 250 mg, at least about 200 mg, at least about 150 mg, at least about 125 mg, at least about 100 mg, at least about 75 mg, at least about 50 mg, at least about 25 mg, at least about 20 mg, at least about 15 mg, at least about 10 mg, at least about 5 mg, at least about 1 mg, at least about 500 ⁇ g, at least about 450 ⁇ g, at least about 400 ⁇ g, at least about 350 ⁇ g, at least about 300 ⁇ g, at least about 250 ⁇ g, at least about 200 ⁇ g, at least about 150 ⁇ g, at least about 100 ⁇ g, at least about 80 ⁇ g, at least about 70 ⁇ g, at least about 60 ⁇ g, at least about 50 ⁇ g,
  • the dosage delivered (daily or as required) through topical formulation in humans and in mouse models is less than about 5% wt/vol, less than about 4.5% wt/vol, less than about 3.5% wt/vol, less than about 2.5% wt/vol, less than about 1 .5% wt/vol, less than about 0.5% wt/vol, less than about 0.4% wt/vol, less than about 0.3% wt/vol, less than about 0.2%, less than about 0.1 % wt/vol, less than about 0.09% wt/vol, less than about 0.08% wt/vol, less than about 0.07% wt/vol, less than about 0.06% wt/vol, less than about 0.05% wt/vol, less than about 0.04% wt/vol, less than about 0.03% wt/vol, less than about 0.02% wt/vol, less than about 0.01 % wt/vol, less than about 0.008%
  • the dosage delivered (daily or as required) through a topical formulation in humans is less than about 70 ⁇ g, less than about 50 ⁇ g, less than about 45 ⁇ g, less than about 40 ⁇ g, less than about 30 ⁇ g, less than about 25 ⁇ g, less than about 20 ⁇ g, less than about 15 ⁇ g, less than about 12 ⁇ g, less than about 10 ⁇ g, less than about 8 ⁇ g, less than about 4 ⁇ g, less than about 2 ⁇ g, or less than about 1 ⁇ g, for example, about 1 ⁇ g to about 50 ⁇ g, about 5 ⁇ g to about 40 ⁇ g, about 8 ⁇ g to about 30 ⁇ g, about 10 ⁇ g to about 20 ⁇ g, or about 12 ⁇ g to about 15 ⁇ g, about 8 ⁇ g to about 12 ⁇ g, about 5 ⁇ g to about 9, about 3 ⁇ g to about 6 ⁇ g, about 2 ⁇ g to about 5 ⁇ g, or
  • the systemic dosage delivered (daily or as required) through an intravenous, subcutaneous or intramuscular injection or an injectable implant for sustained release formulations in humans is less than about 100 mg/kg, less than about 80 mg/kg, less than about 45 mg/kg, less than about 40 mg/kg, less than about 30 mg/kg, less than about 25 mg/kg, less than about 20 mg/kg, less than about 15 mg/kg, less than about 12 mg/kg, less than about 10 mg/kg, less than about 8 mg/kg, less than about 4 mg/kg, less than about 2 mg/kg, less than about 1 mg/kg, less than about 0.1 mg/kg, or less than about 0.01 mg/kg, for example, less than about 0.01 mg/kg to about 50 mg/kg, less than about 5 mg/kg to about 40 mg/kg, less than about 8 mg/kg to about 30 mg/kg, less than about 10 mg/kg to about 20 mg/kg, or less than about 12 mg/kg to about 15 mg/kg, less than
  • the dosage delivered (daily or as required) to humans (based on 70 kg weight) through any formulation other than an intravenous, subcutaneous, or intramuscular injection or injectable implant for the sustained release, inhaled or topical formulation is at least about 600 mg, at least about 500 mg, at least about 450 mg, at least about 400 mg, at least about 350 mg, at least about 300 mg, at least about 250 mg, at least about 200 mg, at least about 150 mg, at least about 125 mg, at least about 100 mg, at least about 75 mg, at least about 50 mg, at least about 25 mg, at least about 20 mg, at least about 15 mg, at least about 10 mg, at least about 5 mg, at least about 1 mg, at least about 500 ⁇ g, at least about 450 ⁇ g, at least about 400 ⁇ g, at least about 350 ⁇ g, at least about 300 ⁇ g, at least about 250 ⁇ g, at least about 200 ⁇ g, at least about 150 ⁇ g, at least about 100 ⁇ g, at least about 600 mg, at least about 500
  • the formulation is administered systemically or locally (e.g. intraocular injection, enema formulation, inhalation) at intervals of 6 hours, 12 hours, daily or every other day or on a weekly or monthly basis to elicit the desired benefit or otherwise provide a therapeutic effect.
  • the formulation is administered as required to elicit the desired benefit or otherwise provide a therapeutic effect.
  • the improvement in post-myocardial infarction (Ml) cardiac output will be assessed by ultrasound.
  • the improvement in post-stroke neurological function will be assessed by determining the percent of patients with a NIHSS of less than 17 one month after presentation.
  • immunogenicity of the human homeodomain or variant or portion thereof conjugates or fusion proteins will be assessed by determining the incorporation of 3H-thymidine by lymphocytes.
  • immunogenicity of the human homeodomain or variant or portion thereof conjugates or fusion proteins will be assessed by determining the level of specific antibodies produced by the recipient animal or human.
  • the extent of ischemia-reperfusion injury will be assessed using known histopathology techniques to examine the area of necrotic tissue in a stroke model.
  • the GAG accumulation will be assessed using known histopathology techniques.
  • behavioral and motor deficits in a mouse model of Huntington's Disease will be assessed using published methods evaluating grip strength in the forepaws and balance ability on a rotorod balance beam. In other embodiments each outcome will be assessed by known methods.
  • the subject(s) upon treatment of one or more human or animal subjects with any of the fusion protein or conjugate embodiments disclosed, the subject(s) will exhibit one or more of the following outcomes:
  • BALF cytokine levels e.g. TNF- a, IL-1 , IL-6, IL-8
  • IL-10 interleukin-10
  • the patient will be treated over a period, for example, of about 1 day through the lifetime of the patient, over a period of about 1 day to about 200 weeks, about 1 day to about 100 weeks, about 1 day to about 80 weeks, about 1 day to about 50 weeks, about 1 day to about 40 weeks, about 1 day to about 20 weeks, about 1 day to about 15 weeks, about 1 day to about 12 weeks, about 1 day to about 10 weeks, about 1 day to about 5 weeks, about 1 week to about 4 weeks, about 2 weeks to about 3 weeks, about 1 day to about 2 weeks, about 1 week, about 1 to 5 days, about 1 to 3 days, or about 1 to 2 days.
  • the treatment group members, or the treatment group(s) will exhibit one or more of the following outcomes, each compared to baseline or control, unless otherwise indicated:
  • cytokine levels e.g. TNF-cc, IL-1 , IL-6, IL-8;
  • the treatment with a formulation comprising a fusion protein and/or conjugate embodiment disclosed in clinical studies will extend over a period, for example, of about 1 day to about 52 weeks, about 1 day to about 26 weeks, about 1 day to about 16 weeks, about 1 day to about 12 weeks, about 1 day to about 10 weeks, about 1 day to about 5 weeks, about 1 week to about 4 weeks, about 2 weeks to about 3 weeks, about 1 day to about 2 weeks, about 1 week, about 1 to 6 days, about 1 to 4 days, or about 1 to 2 days.
  • BALF bronchoalveolar lavage fluid
  • inflammatory cell numbers of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);
  • a reduction in bronchoalveolar lavage fluid (BALF) cytokines e.g., TNF-cc, IL-1 , IL-6, IL-8 of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);
  • BALF bronchoalveolar lavage fluid
  • a reduction in acute mortality i.e., at one month
  • a reduction in acute mortality i.e., at one month
  • at least about 99% at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);
  • (dd) a reduction or repression of the mutant Huntingtin gene expression in the HD murine model of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);
  • (gg) a reduction in neuronal loss and/or reduced striatal cell volume as assessed by molecular methods and histological analysis in the HD murine model of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);
  • (hh) an increase in IL-10 in plasma (blood) and/or BALF of at least about 3,000%, at least about 2,000%, at least about 1 ,000%, at least about 500%, at least about 380%, at least about 360%, at least about 340%, at least about 320%, at least about 300%, at least about 280%, at least about 260%, at least about 240%, at least about 220%, at least about 200%, at least about 180%, at least about 160%, at least about 140%, at least about 120%, at least about 1 10%, at least about 100%, at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 400% to about 500%, about 450% to about 500%, about 420% to about 480%, about 300% to about 45
  • an increase in IL-10 in blood or BALF to least about 300 pg/mL, to at least about 400 pg/mL, to least about 500 pg/mL, to least about 600 pg/mL, to least about 800 pg/mL, to least about 1000 pg/mL, to least about 1200 pg/mL, to least about 1400 pg/mL, to least about 1600 pg/mL, to least about 1800 pg/mL, to least about 2000 pg/mL, to least about 2200 pg/mL, to least about 2400 pg/mL, to least about 2600 pg/mL, to least about 2800 pg/mL, to least about 3000 pg/mL, to at least about 4000 pg/ml, for example, about 300 mg/mL to about 3900 pg/mL, about 380 pg/mL to about 2900 pg/mL, about 400 pg/
  • (jj) a reduction in the level of IP-10 in blood and BALF to at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control),
  • mm a reduction in GM-CSF in blood or BALF of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control); (nn) prevention of a clinically significant increase in graded vitreous haze and/or graded anterior chamber cells based on commonly accepted clinical criteria known to those expert in the field.
  • (ss) reduced activation of NF- ⁇ regulated genes of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control).
  • the conjugates or fusion proteins may be used alone or in combination with other treatments or components of other treatments.
  • Diseases and disorders or conditions that may be treated include, but are not limited to: cancer, inflammation or inflammatory disease, dermatological disorders, fever, cardiovascular effects, hemorrhage, coagulation and acute phase response, cachexia, anorexia, acute infection, HIV infection, shock states, graft-versus-host reactions, autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-dependent anti- thrombosis; tumor growth, invasion and spread, angiogenesis, metastases, malignant, ascites and malignant pleural effusion; cerebral ischemia, ischemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis; ps
  • follicle-stimulating hormone modulation of fertility
  • haemostatic and thrombolytic activity e.g.
  • antiinflammatory activity for treating hemophilia and stroke
  • antiinflammatory activity for treating e.g. septic shock or Crohn's disease
  • antimicrobials modulators of e.g. metabolism or behavior; as analgesics; treating specific deficiency disorders; in treatment of e.g. psoriasis, in human or veterinary medicine; macrophage inhibitory and/or T cell inhibitory activity and thus, anti-inflammatory activity; anti-immune activity, i.e.
  • inhibitory effects against a cellular and/or humoral immune response including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as up-regulated fas receptor expression in T cells; inhibit unwanted immune reaction and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto- rhino-lary
  • retinitis or cystoid macular oedema retinitis or cystoid macular oedema, sympathetic ophthalmia, scieritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g.
  • monocyte or leukocyte proliferative diseases e.g. leukemia
  • monocytes or lymphocytes by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.
  • GCase Entire DRLNLSDQQVKIWFQNRRMKKKRLLARPCIPKSFG sequence: YSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSG combined SEQ RRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGF ID Nos.
  • GCase Entire SNRLNLSDQQVKIWFQNRRMKKKRVVARPCIPKSF sequence: GYS S VVCVCNATYCD SFDPPTFP ALGTF SRYESTRS combined SEQ GRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKG ID Nos.
  • PAADALYALLRAFGWARVALVTAPQDLWVEAGRS 33 also LSTALRARGLPVASVTSMEPLDLSGAREALRKVRD showing GPRVTAVIMVMHSVLLGGEEQRYLLEAAEELGLTD initiating GSLVFLPFDTIHYALSPGPEALAALANSSQLRRAHD methionine A VLTLTRHCP SEGS VLD SLRRAQERRELP SDLNLQQ residue) VSPLFGTIYDAVFLLARGVAEARAAAGGRWVSGA
  • VFEWKDLVSSLARRYIGRYGLAHVSKWNFETWNE 45 also PDHHDFDNVSMTMQGFLNYYDACSEGLRAASPAL showing RLGGPGDSFHTPPRSPLSWGLLRHCHDGTNFFTGEA initiating GVRLDYISLHRKGARSSISILEQEKVVAQQIRQLFPK methionine FADTPIYNDEADPLVGWSLPQPWRADVTYAAMVV residue KVIAQHQNLLLANTTSAFPYALLSNDNAFLSYHPHPHPHP
  • EPDHHDFDNVSMTMQGFLNYYDACSEGLRAASPA showing LRLGGPGDSFHTPPRSPLSWGLLRHCHDGTNFFTGE initiating AGVRLDYISLHRKGARSSISILEQEKVVAQQIRQLFP methionine KFADTPIYNDEADPLVGWSLPQPWRADVTYAAMV residue VKVIAQHQNLLLANTTSAFPYALLSNDNAFLSYHP
  • SWSFPPYHPSSEKYENTKTCRGPDGELHANLLCPV 48 also DVLDVPEGTLPDKQSTEQAIQLLEKMKTSASPFFLA showing VGYHKPHIPFRYPKEFQKLYPLENITLAPDPEVPDG initiating LPPVAY PWMDIRQREDVQALNISVPYGPIPVDFQR methionine) KIRQSYFASVSYLDTQVGRLLSALDDLQLANSTIIAF
  • Ag85A Entire RLNLSDQQVKIWFQNRRMKKKRLL AFSRPGLP sequence, VEYLQVPSPS MGRDIKVQFQ SGGANSPALY combined SEQ LLDGLRAQDD FSGWDINTPA FEWYDQSGLS ID nos. 1 and VVMPVGGQSS FYSDWYQPAC GKAGCQTYKW 72, TB ETFLTSELPG WLQANRHVKP TGSAVVGLSM Vaccine) AASSALTLAI YHPQQFVYAG AMSGLLDPSQ

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DK14838596.6T DK3007729T3 (da) 2013-06-11 2014-06-11 Struktur, fremstilling og anvendelser af humanderiverede cellegennemtrængelige peptider, der er konjugeret med specifikke biologisk aktive cargo-peptider
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ES14838596T ES2745566T3 (es) 2013-06-11 2014-06-11 Estructura, fabricación y usos de péptidos que pueden penetrar en las células derivados de seres humanos conjugados con péptidos de carga biológicamente activos específicos
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US12454706B2 (en) * 2020-03-04 2025-10-28 Flagship Pioneering Innovations Vi, Llc Methods and compositions for modulating a genome
US20240316217A1 (en) * 2021-02-05 2024-09-26 Regents Of The University Of Minnesota Methods for preventing cardiac or skeletal defects in diseases including mucopolysaccharidoses
EP4308234A4 (en) * 2021-03-16 2025-01-22 Rush University Medical Center METHODS OF TREATING NEURODEGENERATIVE DISEASES WITH PEPTIDE WITH INTRANASALE NF-KAPPAB ETHER TARGET MODIFIER (NEMO) BINDING DOMAIN (NBD)
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