WO2012016162A2 - Compositions, procédés et kits pour modéliser, diagnostiquer et traiter des troubles du complément - Google Patents

Compositions, procédés et kits pour modéliser, diagnostiquer et traiter des troubles du complément Download PDF

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Publication number
WO2012016162A2
WO2012016162A2 PCT/US2011/045933 US2011045933W WO2012016162A2 WO 2012016162 A2 WO2012016162 A2 WO 2012016162A2 US 2011045933 W US2011045933 W US 2011045933W WO 2012016162 A2 WO2012016162 A2 WO 2012016162A2
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Prior art keywords
protein
cells
complement
sample
vector
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WO2012016162A3 (fr
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Rajendra Kumar-Singh
Siobhan M. Cashman
John Harry Sweigard
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Tufts University
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Tufts University
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Priority to US13/692,734 priority Critical patent/US8877896B2/en
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    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4716—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology

Definitions

  • Systems, compositions, methods and kits are provided related to the role of complement in ocular pathology, for diagnosing a complement-based ocular disease, and for identifying potential therapeutic agents using C3 protein.
  • Methods, composition and kits are provided for preventing or treating a complement-related condition using CD46 protein, CDS 5 protein, or a recombinant soluble terminator of activated complement (STAC) protein.
  • Activation of complement results in generation of anaphylatoxins that are pleiotropic effector molecules that mediate inflammatory processes such as chemoattraction, vasodilation and vasopermeability (Markiewski et al. 2007 Am J Pathol 171: 715-727), and non-inflammatory processes such as tissue regeneration, lipid metabolism, and synapse formation (Klos et al. 2009 Mol Immunol 46: 2753-2766).
  • Activation of complement terminates in formation of a pore on the surface of target cells referred to as the membrane attack complex (MAC), resulting in cell lysis and cell death.
  • MAC membrane attack complex
  • Age-related macular degeneration is a disease associated with aging that gradually destroys sharp, central vision, and is the leading cause of blindness in the elderly (Klein et al. 2007 Ophthalmology 114: 253-262).
  • the macula is a specific tissue located in the center of the retina, the light-sensitive tissue at the back of the eye that converts light or an image into electrical impulses.
  • AMD is classified as either wet age-related macular degeneration or diy age-related macular degeneration.
  • Wet AMD is characterized by growth of abnormal blood vessels behind the retina under the macula. These new blood vessels are fragile and often leak blood and fluid. The blood and fluid raise the macula from its normal place at the back of the eye, causing loss of central vision.
  • Wet AMD is treated with laser surgery, photodynamic therapy, and injections into the eye. None of these treatments, however, cures wet AMD, rather the treatments slow progression of the disease.
  • Dry AMD is characterized by slow breakdown of light-sensitive cells in the macula, gradually blurring central vision in the affected eye. Over time, less of the macula functions and central vision is gradually lost. There is no known form of treatment for advanced stage dry AMD, and vision loss is inevitable.
  • a specific high-dose formulation of antioxidants and zinc has been shown to prevent intermediate stage AMD from progressing to advanced AMD.
  • Complement proteins are deposited on the choriocapillaris of patients with diabetic retinopathy, as well as in the retinal vessels of diabetic subjects (Gerl et al. 2002 Invest Ophthalmol Vis Sci 43: 1104-1108). These vessels also exhibited significant reduction in expression of the complement regulatory proteins decay accelerating factor (CD55) and CD59. Hyperacute rejection of organ transplantation, mainly the liver and kidney, has shown evidence of complement activity on the endothelium, and is considered a key reason for transplant rejection (Satoh et al. 1997 Transplantation 64: 11 17-1123).
  • An aspect of the invention provides a method for treating AMD in a ocular tissue of a subject, including contacting the ocular tissue of the subject with a composition comprising a CD46 protein or a CD55 protein, such that the ocular tissue is treated for AMD.
  • the ocular tissue is treated for any complement-related disease or condition.
  • the composition includes at least one selected from the group of: a nucleic acid vector with a gene encoding the protein; the protein; or the protein expressed directly from naked nucleic acid.
  • the vector is a viral vector or a plasmid for example, the viral vector is derived from a genetically engineered genome of at least one virus selected from the group consisting of adenovirus, adeno-associated virus, a herpesvirus, and a lentivirus.
  • the lentivirus is a retrovirus.
  • the ocular tissue is selected from the group of: retinal pigment epithelium, retina, choroid, sclera, lens, cornea, Bruch's membrane, and an ocular blood vessel.
  • the ocular blood vessel includes a choroidal blood vessel.
  • delivery of the protein or the vector is by at least one injection route selected from the group consisting of intravenous, intra-ocular, intramuscular, subcutaneous, and intraperitoneal.
  • the macular degeneration is dry AMD.
  • the macular degeneration is wet AMD.
  • the CD46 protein includes an amino acid sequence as shown in SEQ ID NO: 4 or a portion or homologue thereof. In related embodiments, the CD46 protein includes an amino acid sequence at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NO: 4.
  • the nucleic acid with a gene encoding the CD46 protein includes an nucleotide sequence as shown in SEQ ID NO: 3 or a portion or homologue thereof.
  • the CD46 nucleotide sequence is at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NO: 3.
  • the CD55 protein includes an amino acid sequence as shown in SEQ ID NO: 6 or a portion or homologue thereof. In related embodiments, the CD55 protein includes an amino acid sequence at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NO: 6.
  • the nucleic acid with a gene encoding the CD55 protein includes an nucleotide sequence as shown in SEQ ID NO: 5 or a portion or homologue thereof.
  • the CD55 protein includes the nucleotide sequence having at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% identity to SEQ ID NO: 5.
  • the CD46 protein or the CD55 protein is non-membrane bound, for example the protein lacks or has a non-fuctional (mutated) hydrophobic
  • the CD46 protein or the CD55 protein is derived from a mammal, for example the CD46 protein or the CD55 protein is derived from a human, a mouse, a cow, and a sheep.
  • An aspect of the invention provides a pharmaceutical composition for treating a complement-related condition in a subject including a chimeric soluble terminator of activated complement (STAC) protein having amino acid sequences from at least two of a CD46 protein, a CD55 protein, and a CD59 protein, or a nucleic acid expressing or encoding the recombinant STAC protein, such that the STAC protein negatively modulates classical and alternative complement pathways.
  • STAC activated complement
  • the subject is a mammal for example a human, a dog, a cat, a cow, a pig, and a horse.
  • the STAC protein comprises two component proteins selected from the group of: the CD46 protein and the CD55 protein, the CD46 protein and the CD59 protein, and the CD55 protein and the CD59 protein.
  • the composition comprises the STAC protein including each of the CD46 protein, the CD55 protein, and the CD59 protein.
  • a related embodiment provides the STAC protein having amino acid sequence as shown in SEQ ID NO: 1 or a portion or homologue thereof.
  • the STAC protein includes an amino acid sequence at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NO: 1.
  • An alternative embodiment provides the nucleic acid encoding an amino acid sequence of the CD59 protein having at least one mutation conferring loss of function of a glycosyl phosphatidyl inositol (GPI) anchoring domain, the mutation including at least one of a substitution, deletion, and addition.
  • GPI glycosyl phosphatidyl inositol
  • a related embodiment provides the composition formulated in a dose effective to treat the subject for the complement-related condition.
  • a related embodiment provides the CD59 protein having an amino acid sequence that includes at least one of a secretory signal peptide and a short consensus repeat (SCR) domain.
  • SCR short consensus repeat
  • the composition in various embodiments further includes a linker connecting amino acid sequences of the CD59 protein and the CD46 protein.
  • the composition further includes a linker connecting amino acid sequences of the CD46 protein and the CD55 protein.
  • the composition further having a linker connecting amino acid sequences of the CD55 protein and the CD59 protein.
  • the linker includes at least one amino acid, for example glycine.
  • the amino acid includes at least one of: aspartate, threonine, alanine, tyrosine, serine, or proline.
  • the linker for example covalently connects each binding region of the STAC protein.
  • the linker is a single amino acid or a plurality of amino acids that does not reduce the stability, orientation, binding, neutralization, and/or clearance characteristics of the STAC protein.
  • a related embodiment provides the amino acid sequences of the CD46, CD55 and
  • CD59 proteins are encoded by nucleic acid as a protein fusion in the same reading frame as a transcription fusion in which expression of the protein is operably linked and expressed.
  • a related embodiment provides the CD46 protein having an amino acid sequence that includes at least one of: a short consensus repeat domain and a serine/threonine/proline (STP) rich domain.
  • the nucleic acid encoding the CD46 protein amino acid sequence includes at least one mutation, for example a substitution, a deletion or an addition.
  • the nucleic acid encoding CD55 protein amino acid sequence in a related embodiment includes at least one mutation resulting in loss of function of a glycosyl phosphatidyl inositol (GPI) anchoring domain of the CD55 protein, the mutation including for example a substitution, a deletion, or an addition.
  • the CD55 protein amino acid sequence includes at least one of: a short consensus repeat domain and a
  • a related embodiment provides the nucleotide sequence encoding the STAC protein as a plasmid.
  • the nucleotide sequence is provided as a viral vector.
  • the vector encodes amino acid sequence SEQ ID NO: 1 or a portion thereof, for example at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of SEQ ID NO: 1.
  • the vector is at least one selected from the group of: an adenovirus, an adeno- associated virus, a herpesvirus, a poxvirus, and a lentiviras.
  • the composition is formulated to include a dose of the vector selected from: about 10 6 to about 10 7 ; 10 7 to about 10 8 ; about 10 s to about 10 9 ; about 10 9 to about 10 10 ; and about 10 10 to about 10 11 .
  • the nucleotide sequence in various embodiments includes that the promoter is from a gene selected from: a beta-actin for example a chicken beta actin, a peripherin/RDS, cGMP phosphodiesterase, and a rhodopsin.
  • a beta-actin for example a chicken beta actin, a peripherin/RDS, cGMP phosphodiesterase, and a rhodopsin.
  • the nucleotide sequence in related embodiments further encodes a linker including at least one amino acid for example a glycine, a serine, or an alanine.
  • the linker is located between the CD59 protein and the CD46 protein amino acid sequences.
  • the linker is located between the CD46 protein and the CD55 protein amino acid sequences.
  • the linker in certain embodiments includes a molecule that joins two other amino acids, proteins, or domains either covalently or noncovalently, e.g., through ionic or hydrogen bonds or van der Waals interactions. It is envisioned that the CD46, CD55, and CD 59 amino acid sequences can be arranged in any order without limitation, and that linkers are located between adjacent amino acid sequences.
  • the composition further includes a delivery vehicle engineered to target a cell or tissue selected from the group of: a liposome, a lipid, a polycation, a peptide, a nanoparticle, a gold particle, and a polymer.
  • the composition further includes at least one agent selected from: anti-tumor, anti-coagulant, anti-viral, antibacterial, anti-mycobacterial, anti-fungal, anti-proliferative and anti-apoptotic.
  • An aspect of the invention provides a method of treating a complement-related condition in a subject including: contacting a cell of the subject with a composition including a vector carrying a nucleotide sequence encoding a recombinant chimeric soluble terminator of activated complement (STAC) protein operably linked to a promoter sequence causing expression of the STAC protein in a cell, such that the nucleotide sequence encodes amino acid sequences of each of a CD59 protein, a CD46 protein, and a CD55 protein; and, observing a decrease in a symptom of the complement-related condition in the subject in comparison to prior to contacting, thereby treating the complement-related condition.
  • STAC activated complement
  • the method in a related embodiment further includes observing as measuring an amount of a protein of a complement pathway.
  • the protein is MAC and measuring includes identifying or determining MAC deposition on a cell or in the subject.
  • determining extent of MAC deposition is analyzing by
  • the cell is selected from: muscular, epithelial, endothelial, and vascular.
  • the cell is selected from a tissue, for example the tissue is in at least one of: eye, heart, kidney, thyroid, brain, stomach, lung, liver, pancreas, and vascular system.
  • the condition in related embodiments is selected from the group of: macular degeneration, age-related macular degeneration, inflammatory bowel disease, thyroiditis, cryoglobulinaemia, foetal loss, organ graft rejection, sepsis, viral infection, fungal infection, bacterial infection, toxic shock syndrome (TSS), membranoproliferative glomerulonephritis, dense deposit disease, peroximal nocturnal hemoglobinurea, lupus nephritis, membranous nephritis, immunoglobulin A nephropathy, goodpasture syndrome, post-streptococcal glomerulonephritis, systemic lupus erythematosus, atypical hemolytic uremic syndrome, systemic lupus erythromatosis, lupus arthritis, rheumatoid arthritis, Sjogren's syndrome, Behcet's syndrome, systemic sclerosis, Alzheimer's disease, multiple sclerosis,
  • Alternative embodiments of the method include contacting the cell in vitro, or contacting the cell ex vivo, in vivo or in situ.
  • the method in a related embodiment further includes engineering the vector carrying the nucleotide encoding the recombinant STAC protein, such that the STAC protein is a chimeric protein.
  • engineering includes mutating nucleic acid encoding the CD55 protein amino acid sequence such that at least one mutation results in loss of function of glycosyl phosphatidyl inositol (GPI) anchoring domain.
  • GPI glycosyl phosphatidyl inositol
  • the engineering step includes in a related embodiment mutating nucleic acid encoding the CD46 protein amino acid sequence such that at least one mutation results in removal of a secretoiy signal.
  • engineering includes mutating nucleic acid sequence encoding CD55 protein amino acid sequence such that at least one mutation results in loss of function of GPI anchoring domain.
  • the mutation includes for example at least one of: a substitution, a deletion, and an addition.
  • engineering includes recombinantly joining nucleic acid encoding the CD59 protein C-terminus with nucleic acid encoding amino acids of CD46 protein N-terminus.
  • engineering includes recombinantly joining nucleic acid sequence encoding the CD46 protein C-terminus with nucleic acid encoding the CD55 protein N-terminus.
  • the STAC protein further in various embodiments includes a purification tag and/or a protease cleavage site for removal of the tag.
  • the purification tag is at least one selected from: Arg-tag, calmodulin-binding peptide, cellulose-binding domain, DsbA, c-myc-tag, glutathione S-transferase, FLAG-tag, HAT-tag, His-tag, maltose-binding protein, NusA, S-tag, SBP-tag, Strep-tag, and thioredoxin.
  • the subject is in various embodiments a mammal, for example a human, a research animal, a high value zoo animal, and an agricultural animal.
  • the method includes in various embodiments contacting the cell by administering the composition by at least one route selected from: parenteral, intravenous, intramuscular, intraperitoneal, intradermal, intracistemal, mucosal, subcutaneous, sublingual, intranasal, oral, ocular, bucal, intra-ocular, intravitreal, topical, transdermal, vaginal, rectal, and infusion.
  • parenteral intravenous, intramuscular, intraperitoneal, intradermal, intracistemal, mucosal, subcutaneous, sublingual, intranasal, oral, ocular, bucal, intra-ocular, intravitreal, topical, transdermal, vaginal, rectal, and infusion.
  • the method includes in related embodiments observing by analyzing amount or presence of at least one of: an antibody, a peptide, a carbohydrate, an enzyme, a sugar, and a surface receptor.
  • observing includes analyzing an amount of membrane attack complex (MAC).
  • MAC membrane attack complex
  • An aspect of the invention provides, a kit for regulating or of treating a complement- related condition in a subject, the method including: a composition including a chimeric soluble terminator of activated complement (STAC) recombinant protein including amino acid sequences from each of a CD46 protein, a CD55 protein, and a CD59 protein, or a nucleotide sequence encoding the recombinant STAC protein, such that the composition negatively modulates classical and alternative complement pathways and is formulated in a dose effective to treat the subject for the complement-related condition; instructions for treating the subject; and, a container.
  • STAC activated complement
  • the STAC protein in various embodiments of the kit has an amino acid sequence as shown in SEQ ID NO: 1 or a portion thereof.
  • the STAC protein amino acid sequence is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NO: 1.
  • the nucleotide sequence in related embodiments of the kit includes a vector or a plasmid.
  • the vector is in various embodiments an engineered viral vector selected from: an adenovirus, an adeno-associated virus, a herpesvirus, a poxvirus, and a lentivirus.
  • the STAC protein in various embodiments of the kit is a genetic fusion of the CD46,
  • composition includes any of the pharmaceutical compositions described herein.
  • the kit further includes in various embodiments a therapeutic agent.
  • the therapeutic agent is selected from: anti-coagulant, anti-tumor, anti-viral, anti-bacterial, anti- mycobacterial, anti-fungal, anti-proliferative, and anti-apoptotic.
  • An aspect of the invention provides, a pharmaceutical composition for treating a complement-related condition in a subject including: an adenovirus viral vector including a nucleotide sequence encoding a recombinant chimeric soluble terminator of activated complement (STAC) protein including: an amino acid sequence as shown in SEQ ID NO: 1 or portion thereof, such that the composition negatively modulates classical and alternative complement pathways and is formulated in a dose effective to treat the subject for the complement-related condition.
  • STAC activated complement
  • An aspect of the invention provides a method of assaying extent of human MD in a model cell system or a method in a model cell system of assaying a serum complement component for prognosis or diagnosis of macular degeneration (MD), the method including: exposing a first sample of cells to serum and measuring resulting lysis, and comparing extent of lysis to that in a second sample of control cells not so exposed to serum, such that the extent of lysis in the first sample compared to that in the second sample is a measure of complement- induced MD.
  • An aspect of the invention provides a method of assaying in a model cell system potential therapeutic agents for human MD, the method including: contacting a first sample of cells to serum and measuring resulting lysis, and contacting a second sample of otherwise identical control cells with serum and a source of mammalian CD46 protein or mammalian CD55 protein and measuring resulting lysis; and contacting at least a third sample of cells to a candidate therapeutic composition and otherwise identically to serum, such that the extent of lysis of the third sample compared to that in the first and second sample is a measure of protection by the candidate composition, thereby providing the method of assaying for potential therapeutic agents.
  • the mammalian protein is a human protein.
  • a related embodiment of the above methods further includes contacting cells or tissues with a recombinant vector having a gene capable of expressing the protein such as the CD46 protein or CD55 protein. Lysis is measured for example by propidium iodide uptake and cell sorting.
  • the cells are hepatocytes.
  • the cells are of murine origin.
  • the source of the protein is human.
  • the serum is normal human serum. Alternatively, the serum is from a diseased subject, for example, the diseased subject has MD.
  • An aspect of the invention provides a method of diagnosing or prognosing presence or progression of macular degeneration, the method including determining extent of MAC deposition on retina.
  • determining extent of MAC deposition is analyzing by immunohistochemistry with antibodies that are specific for human MAC.
  • An aspect of the invention provides a pharmaceutical composition for treating macular degeneration including CD46 protein or CD55 protein or a source of expression of CD46 protein or CD55 protein in vivo, in which the composition is formulated for ocular delivery, in a dose effective to treat macular degeneration.
  • the protein or source of expression of protein is at least one selected from the group consisting of: a nucleic acid vector with a gene encoding the protein; a viral vector with a gene encoding the protein; and the protein.
  • the gene includes a nucleotide sequence selected from SEQ ID NO: 3 or SEQ ID NO: 5.
  • the composition includes at least one selected from the group of: a nucleic acid vector with a gene encoding the protein; the protein; or protein expressed directly from naked nucleic acid.
  • the composition includes a nucleic acid vector with a gene encoding CD55 protein; CD55 protein; or CD55 expressed directly from naked nucleic acid.
  • the CD55 protein includes an amino acid sequence shown in SEQ ID NO: 6 or a portion or homologue thereof.
  • the composition includes a nucleic acid vector with a gene encoding CD46 protein; CD46 protein; or CD46 expressed directly from naked nucleic acid.
  • the CD46 protein includes an amino acid sequence shown in SEQ ID NO: 4 or a portion or homologue thereof.
  • the composition formulated for ocular delivery is at least one selected from the group consisting of: injection, eye drop, and ointment.
  • injection is at least one selected from the group consisting of: intra-ocular injection, subconjunctival injection, and subtenon injection.
  • the composition further includes at least one drug selected from the group consisting of: anti-tumor, antiviral, antibacterial, anti-mycobacterial, anti-fungal, anti- proliferative and anti-apoptotic.
  • the protein is expressed as a soluble protein.
  • the protein has a deletion encoding a GPI anchoring domain.
  • the CD55 protein has a deleted GPI anchoring domain and thus is membrane independent.
  • kits for assaying MAC deposition on ocular tissue or cells and for screening agents that inhibit deposition includes anti-MAC antibody, a container, and instructions for use with normal human serum and the ocular tissue or cells.
  • the kit further includes anti-emmprin antibody and/or normal human serum.
  • the kit further includes at least one of CD46 protein, CD55 protein, and STAC protein as a positive control and the protein is a soluble form or a membrane-bound form, the latter for example embedded in a liposome preparation.
  • at least one of the antibody, the serum, and the protein is a lyophil.
  • An aspect of the invention provides a method in a model cell system of assaying a serum complement component for prognosis or diagnosis of macular degeneration (MD), the method including: contacting detectably labeled cells with serum from a subject and measuring amount of extracellular and/or intracellular detectable agent for contacted cells; and comparing extracellular and/or intracellular agent in the cells to that in detectably labeled control cells not exposed to the serum and otherwise identical, such that amount of extracellular and/or intracellular agent in the contacted cells is compared to that in the control cells, such that a greater amount of extracellular detectably labeled agent in cells contacted with serum compared to the control cells is an indication of prognosis or diagnosis of MD.
  • An aspect of the invention provides a method of assaying in a model cell system a potential therapeutic agent for efficacy in treatment of human macular degeneration (MD), the method including: contacting a first sample of detectably labeled cells with serum from a subject and measuring amount of extracellular and/or intracellular detectable agent, and contacting a second sample of otherwise identical detectably labeled control cells with serum and a source of human CD46 protein or human CD55 protein and measuring amount of extracellular and/or intracellular detectable agent; and contacting at least a third sample of detectably labeled cells to at least one candidate therapeutic composition and otherwise identically to serum and measuring amount of extracellular and/or intracellular detectable agent, such that the amount of extracellular and/or intracellular detectable agent of the third sample compared to that in the first sample and the second sample is a measure of protection by the candidate composition, such that a greater amount of extracellular detectably labeled agent is an indication of MD, thereby assaying for a potential therapeutic agent for efficacy in treatment of human MD.
  • the detectable agent is at least one composition selected from the group consisting of a recombinant vector having a gene capable of expressing a detectable protein, a fluorescent agent, a colorimetric agent, an enzymatic agent, and a radioactive agent.
  • the detectable protein is at least one fluorescent protein selected from the group consisting: green fluorescent protein, aequorin, cyan fluorescent protein, DsRed fluorescent protein, enhanced green fluorescent protein, and yellow fluorescent protein.
  • the detectable agent is not a protein, for example, the detectable agent is at least one fluorescent agent selected from the group consisting of: Indocyanine Green, Doxorubicin, Riboflavin, Chlorophyll, and Porphyrin.
  • the detectable protein is enzyme, for example, ⁇ -galactosidase or alkaline phosphatase.
  • the cells are hepatocytes; exemplary cells are of murine origin.
  • the source of protein i.e., the CD46 protein or the CD55 protein
  • the serum is normal human serum.
  • the serum is from a diseased subject. In general, the subject is in need of diagnosis or prognosis of MD.
  • the human CD46 or human CD55 protein is soluble.
  • the human CD46 or human CD55 protein is membrane-bound.
  • An aspect of the invention herein provides a model system for diagnosing or prognosing a complement-based ocular pathology or for screening potential therapeutic agents to treat the pathology, the model system including: a first sample of cells or tissue contacted with a vector including a nucleic acid encoding complement component 3 (C3) to overactivate natural complement takeover in the cells or the tissue; a second sample of cells or tissue contacted with a control nucleic acid encoding a detectable protein, such that the vector and the control vector include the same promoter sequence operably linked to nucleic acids encoding the C3 and the detectable protein, such that the detectable protein is a control that does not overactivate the natural complement takeover, such that the second sample of the cells or tissue are otherwise identical to the first sample of the cells or tissue contacted with the vector; a control sample of cells or a tissue from a normal subject, such that the normal subject is not affected by the complement-based ocular pathology; and a marker associated with complement activation for measuring in each of the first
  • the nucleic acid includes a nucleotide sequence shown in SEQ ID NO: 7.
  • the nucleotide sequence is obtained from a mammal such as a human or mouse.
  • the C3 includes an amino acid sequence including SEQ ID NO: 8.
  • the amino acid sequences includes at least about 30% identity, or at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% identity.
  • the vector includes a viral vector, for example an adenovirus, an adeno-associated virus, a retrovirus, a herpesvirus, and a lentivirus.
  • the vector includes a plasmid vector.
  • the detectable protein is at least one selected from: a fluorescent protein, an enzyme having a colorimetric assay, and a chemifluorescent protein
  • the fluorescent protein is at least one selected from the group consisting: green fluorescent protein, enhanced green fluorescent protein, aequorin, cyan fluorescent protein, DsRed fluorescent protein, and yellow fluorescent protein.
  • the marker includes at least one selected from: a membrane attack complex protein, glial fibrillary acidic protein, vascular endothelial growth factor, and Griffonia simplicifolia lectin I.
  • the marker is a complement component or complement protein such as an anaphylatoxin, such as C3a or C5a.
  • the cells or the tissues are cultured in vitro.
  • the cells or tissue are contacted in an animal model in vivo.
  • the animal model is a mouse model.
  • the cells or the tissue includes ocular cells or ocular tissues, respectively.
  • the cells or tissue are contacted by injection, for example by at least one route of administration selected from: intra-ocular, invitreal, subconjunctival, subretinal, and subtenon.
  • the cells or tissue are contacted topically.
  • the marker includes at least one selected from the group of: a membrane attack complex protein, glial fibrillary acidic protein, vascular endothelial growth factor, and Griffonia simplicifolia lectin I.
  • measuring includes observing cellular staining or tissue staining, for example staining of a cellular membrane or nucleus.
  • measuring includes observing at least one from the group of: increased vascular permeability, increased endothelial cell proliferation and migration, RPE atrophy, loss of photoreceptor outer segments, reactive gliosis, increased drusen formation, Muller cell activation, formation of membrane attack complex, retinal detachment, and reduced retinal function.
  • the first sample of the cells or the tissue is characterized as having loss of integrity of retinal vessels.
  • An aspect of the invention herein provides a method for diagnosing a complement- based ocular disease in cells or tissue from a subject, the method including: contacting a first sample of the cells or tissue from the subject with a vector including a nucleic acid encoding complement component 3 (C3), such that the C3 overactivates natural complement takeover in the cells or the tissue; measuring an amount of a marker in the first sample, such that the marker is characteristic of the disease; and comparing amounts of the marker in the first sample to amount of the marker in a second sample of cells or tissue from the subject not so contacted to the vector and otherwise identical, such that amount of the marker in the first sample of cells is compared to that in the cells or the tissue from the subject, such that a substantially similar amount or even greater amount of the marker in the second sample compared to the amount of the marker in first sample is an indication of the diagnosis of the complement-based ocular disease in the subject.
  • the cells or the tissue includes ocular cells or ocular tissues, respectively.
  • the method further includes obtaining a control sample of cells or tissue from a normal subject, such that the normal subject is not affected by the complement-based ocular disease, and measuring an amount of the marker in the control sample, such that a greater amount of the marker in the second sample and/or first sample compared to the amount of the marker in the control sample is an indication of presence of the complement-based ocular disease in the subject.
  • the method indicates the presence of age-related macular degeneration.
  • An aspect of the invention herein provides a method of identifying in a model system a potential therapeutic agent for treating or preventing a complement based-ocular disease, the method including: contacting a first sample of cells or tissue with a vector including a nucleic acid encoding complement component 3 (C3) to overactivate natural complement takeover in the cells or the tissue, contacting a second sample of the cells or tissue with a control nucleic acid encoding a detectable protein, such that the vector and the control vector include the same promoter sequence operably linked to nucleic acids encoding the C3 and the detectable protein, such that the detectable protein is a control that does not overactivate the natural complement takeover, and contacting at least a third sample of cells or tissue with the nucleic acid encoding the C3 and at least one of a plurality of potential therapeutic agents; and measuring in the first sample, the second sample and the third sample, an amount of the marker, such that the marker is characteristic of the disease, such that the amount of the marker in the third sample
  • the complement based-eye disease includes
  • AMD condition for example wet AMD or dry AMD.
  • the detectable protein is at least one selected from: a fluorescent protein, an enzyme having a colorimetric assay, and a chemifluorescent protein.
  • the fluorescent protein is at least one selected from the group of: green fluorescent protein, enhanced green fluorescent protein, aequorin, cyan fluorescent protein, DsRed fluorescent protein, and yellow fluorescent protein.
  • the promoter includes a cytomegalovirus (CMV) promoter, for example a human CMV promoter or mouse CMV promoter.
  • CMV cytomegalovirus
  • the vector includes a viral vector, for example the viral vector is an adenovirus, an adeno- associated virus, a retrovirus, a herpesvirus, or a lentivirus.
  • the marker includes a protein, an enzyme, a lipid, a carbohydrate, or a nucleic acid.
  • the vector is selected from the viral vector encoding a DNA or an RNA, a naked DNA vector, and a microencapsulated DNA or R A.
  • the marker includes at least one selected from the group of: MAC or a MAC protein, glial fibrillary acidic protein, vascular endothelial growth factor, and Griffonia simplicifolia lectin I.
  • measuring further includes observing a localization of the marker in the cells or the tissue contacted by at least one of the first sample, the second sample, and the third sample.
  • the method includes observing the localization of the marker within the cytoplasm or in a nucleus.
  • the cells or the tissues are cultured in vitro.
  • the cells or tissue are cultured in a tube, a flask, or a plate.
  • contacting is in an animal model in vivo.
  • contacting includes injecting the cells or the tissue, for example by at least one route of administration selected from: intra-ocular, invitreal, subconjunctival, subretinal, and subtenon.
  • contacting includes administering topically, for example to an ocular tissue or mucosal lining.
  • the method prior to contacting, includes engineering the vector having the nucleic acid encoding the C3 to express the C3 as a soluble protein.
  • measuring further includes observing cellular staining or tissue staining, for example staining of a cellular membrane or nucleus.
  • measuring further includes observing the marker specifically located on the cells or the tissue, for example retinal pigment epithelium or retina.
  • measuring further includes observing cellular morphology, cell viability, or tissue functionality in the samples.
  • the method includes observing decreased ocular functionality (e.g., retinal functionality) in the first sample compared to the second sample and/or third samples.
  • measuring further includes observing in the samples at least one selected from the group consisting of: retinal detachment, cellular pathology, and tissue pathology. In related embodiment of the method, measuring further includes performing electroretinography.
  • An aspect of the invention herein provides a kit for diagnosing or prognosing an ocular pathology and/or identifying a potential therapeutic agent for treating or preventing a complement based-ocular disease including: a vector including a nucleic acid encoding a component 3 (C3) that overactivates natural complement takeover in cells or tissue; a container; and, instructions for use.
  • the instructions for use include methods described herein for identifying the potential therapeutic agent or for diagnosing or prognosing an ocular pathology.
  • the kit further includes an anti- C3 antibody or an anti-MAC antibody.
  • the kit further includes a control vector including a control nucleic acid encoding a detectable protein absent the C3, such that the vector and the control vector include the same promoter sequence operably linked to nucleic acids encoding the C3 and the detectable protein, such that the detectable protein does not overactivate the natural complement takeover in the cells or the tissue.
  • the instructions for use in kits herein include any of the methods described herein.
  • the vector is at least one selected from: an adenovirus, an adeno-associated virus, a retrovirus, a herpesvirus, and a lentivirus.
  • the kit further includes a plurality of cells and/or a culture medium.
  • An aspect of the invention provides a kit for diagnosing a complement based-ocular disease in a subject including: a vector including a nucleic acid encoding a component 3 (C3) to overactivate natural complement takeover in cells or tissue, for example the vector is a viral vector; a container; and, instructions for use.
  • a vector including a nucleic acid encoding a component 3 (C3) to overactivate natural complement takeover in cells or tissue for example the vector is a viral vector
  • a container for example the vector is a viral vector
  • instructions for use for use.
  • the kit further includes a control nucleic acid encoding a detectable protein, such that the vector and the control vector include the same promoter sequence operably linked to nucleic acids encoding the C3 and the detectable protein, such that the detectable protein does not overactivate the natural complement takeover in the cells or the tissue.
  • the kit farther includes control cells or tissue from a normal subject that is not infected by any complement-based ocular disease.
  • the kit further includes in a related embodiment a culture medium.
  • the instructions for use include any of methods, compositions, and kits described herein.
  • Figure 1 is a set of photomicrographs and photographs of Western blots showing efficient secretion of C3 expressed from adenovirus in vitro.
  • Figure 1 panel A is a set of photomicrographs showing immunostaining of human embryonic retinoblasts contacted with recombinant adenovirus a gene encoding C3
  • Figure 1 panel B is a set of photographs of Western blots of lysates and media of human embryonic retinoblasts contacted with AdcmvC3 or AdcmvGFP, or control retinoblasts that were not injected with adenovirus, analyzed under non-reducing (left) or reducing (right) conditions using a monoclonal antibody specific for binding to human C3 protein. Data show correct processing of C3 intracellularly and efficient secretion into the media for retinoblasts contacted with AdcmvC3. Under non-reducing conditions, the disulfide-linked a and ⁇ chains of C3 are observed in the media and lysates of AdcmvC3 -contacted cells.
  • AdcmvC3 -contacted cells showed dark bands at the predicted molecular weights, 107 kDa and 62 kDa, of a chain and ⁇ chains of C3. Unfd, uninfected; NHS, Normal Human Serum.
  • Figure 2 is set of photomicrographs of murine subjects subretinally injected with recombinant adenovirus AdcmvC3 (right photmicrographs) or AdcmvGFP (left
  • AdcmvC3, n 4 for AdcmvGFP.
  • Figure 3 panel A is a set of photomicrographs for eyes injected with AdcmvGFP and then visualized for GFP fluorescence (left) and stained with DAPI (right). GFP fluorescence for AdcmvGFP-contacted eyes was observed in the RPE.
  • Figure 3 panel B is a set of photomicrographs showing intense FITC-GSL I staining, particularly in an area of retinal detachment, in AdcmvC3 -contacted retinas (left) compared with the control AdcmvGFP-contacted retinas (right). Representative images from two independent injections for each virus are shown (top row and bottom row respectively). GSL I staining was observed in the choroid indicated on the photomicrographs as an asterisk (*) in each of the AdcmvGFP-contacted eyes. GSL I staining was also observed in retinas of particular AdcmvGFP-contacted subjects specifically in areas of retinas close to the site of injection ( ⁇ ).
  • Figure 3 panel C is a set of photomicrographs at 10X magnification showing that injection of eyes with AdcmvGFP or AdcmVC3 resulted in strong GSL I staining at the RPE/retinal junction and throughout the retina in AdcmvC3 -contacted eyes. GSL I binding was observed mostly in the choroid within the region of injection in AdcmvGFP-contacted eyes.
  • Figure 4 is a set of photographs showing increased disruption of murine RPE and choroid in AdcmvC3 -contacted eyes compared to AdcmvGFP-contacted eyes.
  • Bright field illuminated eyes are shown in the left column and eyes stained with FITC-GSL I stain are shown in the right column.
  • BF bright-field
  • GSL I Griffonia simplicifolia lectin I
  • R RPE
  • O outer nuclear layer
  • I inner nuclear layer
  • Figure 4 panel A shows photomicrographs showing disruption to the RPE/choroid of AdcmvC3-injected eyes (top row) and no disruption to the RPE/choroid of AdcmvGFP- injected eyes (bottom row).
  • Data show that cells in the RPE/choroid of AdcmvC3 -injected eyes stained positive for GSL I. In a number of eyes (2/6) examined, it was observed that pigmented cells migrated into the retina (see inset). In AdcmvGFP -injected eyes the GSL I staining was observed to be restricted primarily to the choroid, and the RPE cell layer remaining intact. Photomicrograph magnification was 20X.
  • Figure 4 panel B is a set of magnified (40X) photomicrographs showing AdcmvC3- contacted retinas visualized with BF (left) and stained with GSL I (right).
  • the AdcmvC3- contacted retinas showed a dramatic loss of RPE cells and pigment along the RPE/choroid region.
  • Figure 5 is a set of photomicrographs and bar graphs that show increased retinal detachment in AdcmvC3 -contacted murine eyecups.
  • Figure 5 panel A is a set of photomicrographs of FITC-GSL I stained eyes subretinally injected with either AdcmvC3 (left) or AdcmvGFP (right). Increased staining was observed at the site of injection ( ⁇ ) for eyes injected with either AdcmvC3 or AdcmvGFP. Staining in
  • AdcmvC3-injected eyes extended beyond the region of injection. Discontinuous (dashed) lines delineate the border of the eyecups.
  • Figure 5 panel B is a bar graph showing quantitation of area (mm 2 ) of GSL I staining in eyecups on the ordinate as a function of the adenovirus subretinal injection (abscissa).
  • Murine subjects were injected with either AdcmvC3 or AdcmvGFP.
  • Data show a significantly greater area of staining in eyecups of AdcmvC3 -contacted eyes compared to eyecups of AdcmvGFP- contacted eyes (*p ⁇ 0.05).
  • Figure 5 panel C is a bar graph showing quantitation of retinal detachment ( ⁇ ) in eyecups on the ordinate as a function of each of the adenovirus vectors injected into the eyes, AdcmvC3 or control AdcmvGFP (abscissa). Retinal detachment was significantly increased (2.6-fold) in AdcmvC3 -injected eyes compared to AdcmvGFP-injected (*p ⁇ 0.05).
  • SH sodium hyaluronate
  • Figure 5 panel E is a photograph, 40X objective magnification, of the RPE/choroid of a representative eye injected with 0.25% SH. Data show an intact RPE cell layer. BF, bright- field; C, choroid; R, RPE; O/I, outer/inner nuclear layer.
  • Figure 6 is a set of photomicrographs taken nine days after injection with AdcmvC3, showing disturbance of the outer nuclear layer of murine retinas , outer segment loss, and Muller cell activation.
  • BF bright-field
  • O outer nuclear layer
  • I inner nuclear layer
  • Unjd uninjected.
  • n 4 for AdcmvGFP and AdcmvC3.
  • Figure 6 panel A is a set of photomicrographs of retinas contacted by injection with either AdcmvC3 (top row) or control AdcmvGFP (bottom row) and illuminated with bright field (first column) and stained with DAPI (second and third columns).
  • DAPI staining of AdcmvC3 -contacted retinas showed perturbations in the photoreceptor cell layer, with little or no disturbance to the INL. Proliferation of cells was observed also in the ganglion cell and inner plexiform layers (*). DAPI staining of AdcmvGFP-contacted retinas showed no perturbations in the photoreceptor cell layer and no disturbances to the INL. Higher magnifications of boxed regions in the second column are shown in the third column.
  • Figure 6 panel B is a set of photomicrographs of retinas contacted by injection with either AdcmvC3 (left) or AdcmvGFP (middle) or control eyes not injected (right), and stained with an antibody specific for rhodopsin.
  • Rhodopsin (RHO) staining showed a loss of outer segments in AdcmvC3 -contacted retinas, the RHO staining was observed predominantly in the outer segments of both AdcmvGFP-contacted retinas and uninjected retinas. Low levels of non-specific staining were observed in the sclera of some subjects.
  • Figure 6 panel C is a set of photomicrographs of retinas contacted by injection with either AdcmvC3 (left) or AdcmvGFP (middle) or control eyes not injected (right), and stained for glial fibrillary acidic protein (GFAP), a protein found in glial cells.
  • Data show an altered distribution of GFAP in the Muller cells of AdcmvC3 -contacted retinas, specifically, GFAP staining was observed throughout the Muller cell from inner to outer limiting membranes. It was observed that Muller cells in AdcmvGFP-injected retinas showed GFAP staining that extended towards the outer retina.
  • GFAP is typically localized to astrocytes and to the end feet of non-reactive Muller cells at the inner limiting membrane, as was observed in uninjected retinas.
  • Figure 7 is a set of bar graphs showing that electroretinogram amplitude values
  • Figure 8 is a set of bar graphs showing that expression of C3 mRNA was increased in AdcmvC3 -injected retinas compared to uninjected retinas.
  • the bar graphs show fold increase of mRNA above uninjected subjects on the ordinate as a function of the adenovirus vector injected into the eyes of the mice, AdcmvC3 (C3) or AdcmvGFP (GFP).
  • C3 AdcmvC3
  • GFP AdcmvGFP
  • VEGF vascular endothelial growth factor
  • C3 mRNA was observed to be significantly increased in AdcmvC3 -contacted retinas at both three days and eight days following injection compared to AdcmvGFP-contacted retinas.
  • a significant fold-increase in VEGF mRNA in AdcmvC3 -injected subjects was observed three days following injection, and no change in VEGF mRNA was observed at eight days following injection.
  • a significant fold-increase in VEGF mRNA was also observed in AdcmvGFP- contacted retinas compared to uninjected retinas.
  • Figure 9 is a set of photomicrographs showing that AdcmvC3-injected retinas exhibited MAC deposition on endothelial cells and outer segments.
  • Murine retinas were subretinally injected with either AdcmvC3 or AdcmvGFP and were visualized by bright field, and stained for DAPI, or for MAC using anti-human C5b-9 antibody.
  • BF bright-field
  • C choroid
  • R RPE
  • O outer nuclear layer
  • GSL I Griffonia simpUficolia Lectin I
  • MAC membrane attack complex.
  • Figure 9 panel A shows photomicrographs of AdcmvC3 -injected retinas visualized by
  • FIG. 9 panel B is a set of photomicrographs of AdcmvC3 -injected retinas visualized by BF (top row, left) and stained with DAPI (top row, right), an anti-human C5b-9 antibody for MAC staining (bottom row, left), or GSL I (bottom row, right). Intense MAC staining was observed on the remaining outer segments of the photoreceptors of the AdcmvC3 -contacted eyes.
  • Figure 9 panel C is a set of photomicrographs of AdcmvGFP-injected retinas visualized by BF (top row, left) or GFP fluorescence (bottom row, right) and stained with DAPI (top row, right), or an anti-human C5b-9 antibody for MAC staining (bottom row, left). Strong MAC staining was observed in transduced RPE cells at the site of injection for AdcmvGFP-contacted retinas, and little or no MAC staining was observed in the retina.
  • Figure 10 is a drawing and a set of photographs showing human CD46 expressed in HER cells and on the membrane of mouse Hepa lclc7 cells.
  • Figure 10 panel A is a drawing showing an adenovirus vector expressing a gene encoding either human CD46 protein (hCD46) or GFP protein under control of the chicken beta actin promoter. Also shown is a control empty vector expressing no transgene. Expression cassettes encoding hCD46, GFP, or control vector were cloned into the deleted El region ( ⁇ 1) of a first generation adenovirus vector. Symbols used: CAG, cytomegalovirus chicken ⁇ -actin ⁇ -globin promoter; pA, polyadenylation signal; LITR, left inverted terminal repeat; RITR, right inverted terminal repeat; ⁇ , Ad packaging signal; MLT, major late transcript; E, early region labels.
  • CAG cytomegalovirus chicken ⁇ -actin ⁇ -globin promoter
  • pA polyadenylation signal
  • LITR left inverted terminal repeat
  • RITR right inverted terminal repeat
  • ⁇ Ad packaging signal
  • MLT major late transcript
  • Figure 10 panel B is a set of photographs of Western blots of lysates of human embryonic retinoblasts cells contacted with AdCAGCD46 (left column), AdCAGpA having no transgene (middle column) or control cells not contacted (right column), and then analyzed using a monoclonal antibody specific for binding to either human CD46 (top photograph) or beta (P)-actin (bottom photograph).
  • Figure 10 panel C is a set of photomicrographs of mouse Hepalclc7 cells contacted with AdCAGCD46 (top row) or AdCAGpA (bottom row), visualized with BF (left column) and stained with mouse anti human CD46 (MEM258; right column) or DAPI (middle column). Hepalclc7 cells contacted with AdCAGCD46 were observed to have human CD46 localized on the cell membrane. No CD46 expression was observed for Hepalclc7 cells contacted with AdCAGpA.
  • Figure 11 is a set of bar graphs and photomicrographs showing that AdCAGCD46 protected mouse Hepalclc7 cells specifically from alternative pathway-mediated MAC deposition.
  • Hepalclc7 cells were pre-treated with adenovirus then contacted with either 25 ⁇ g ml emmprin antibody followed by 10% NHS to activate both classical and alternative pathways, or with 10 mM ethyleneglycoltetraacetic acid (MgEGTA)-treated NHS for inhibition of the classical pathway (i.e., activation of the alternative complement only).
  • MgEGTA ethyleneglycoltetraacetic acid
  • Figure 11 panel A is a set of bar graphs showing percent propidium iodide (PI) uptake on the ordinate for mouse hepa- 1 c lc7 cells as a function of contact with media from
  • AdCAGCD46-contacted cells right column or media from AdCAGpA-contacted cells (third column from left).
  • cells were further contacted for 30 minutes at 4°C with 25 ⁇ g/ml rat anti mouse emmprin (Abd Serotec Inc., MCA2283) in either NHS (left column) or HI-NHS (second column from the left).
  • NHS left column
  • HI-NHS second column from the left.
  • Reduced PI uptake was observed for cells contacted with HI-NHS compared to cells contacted with NHS.
  • Cells contacted with media from AdCAGCD46-contacted cells showed slightly decreased PI uptake and cell death compared to cells contacted with media from AdCAGpA contacted cells.
  • Figure 11 panel B shows a set of bar graphs showing fluorescence-activated cell sorter (FACS) cell lysis analysis for the alternative pathway monitored by percent PI uptake on the ordinate in contacted mouse hepa-lclc7 cells as a function of contact with media from
  • FACS fluorescence-activated cell sorter
  • AdCAGCD46-contacted cells (right column) or media from AdCAGpA-contacted cells (third column from left).
  • the alternative pathway only was activated by contacting cells for 30 minutes at 4°C with 7mM magnesium and 10 mM EGTA in either NHS (left column) or HI- NHS (second column from the left).
  • Control cells were not contacted with adenovirus vector and were contacted with NHS or HI-NHS only. In control cells, PI uptake was observed to be greater for cells contacted with NHS than for cells contacted with HI-NHS. Data show that cells contacted with AdCAGCD46 were effectively protected from cell lysis mediated by the alternative pathway.
  • FIG. 11 panel C is a set of photomicrographs of mouse Hepalclc7 cells contacted with AdCAGCD46 (top row) or AdCAGpA (bottom row), contacted with 7mM magnesium and 10 mM EGTA and NHS to activate the alternative complement pathway only, and then visualized with BF (left column) and stained for MAC (right column). Data show that Hepalclc7 cells contacted with AdCAGCD46 had healthier cell morphology and cell characteristics and had less MAC deposition on cellular membranes than cells contacted with AdCAGpA.
  • Photomicrographs are representative of three separate experiments performed in duplicate each time.
  • Figure 12 is a bar graph and set of photomicrographs showing that media conditioned by AdCAGCD46 contacted cells protected mouse primary RPE cells from alternative pathway mediated MAC deposition. Images and data are representative of three independent experiments performed in duplicate each time.
  • Figure 12 panel A is a set of photomicrographs of mouse hepa-lclc7 cells contacted with either AdCAGCD46 (top row) or AdCAGpA (bottom row) and then incubated with EGTA in NHS and visualized by BF (left column) and stained for MAC deposition (right column).
  • Media conditioned by AdCAGCD46 contacted cells reduced MAC deposition on hepa-lclc7 cells compared to media conditioned by AdCAGGFP contacted cells.
  • Cell morphology of the hepa-lclc7 cells treated with AdCAGCD46 was relatively normal and unchanged, and cells generally showed reduced MAC deposition compared to cells contacted with AdCAGpA.
  • MAC staining intensity was faint and not uniform on cells contacted with AdCAGCD46.
  • AdCAGpA infected cells displayed strong uniform MAC deposition.
  • Figure 12 panel B is a graph of quantification of mean MAC pixel (staining) intensity on the ordinate as a function of media from adenovirus-contacted cells and serum to which mouse hepa-lclc7 cells were contacted (abscissa).
  • the hepa-lclc7 cells were contacted with either AdCAGpA (left column) or AdCAGCD46 (right column) and then contacted with EGTA in NHS to produce alternative pathway MAC deposition.
  • AdCAGCD46 contacted RPE cells compared to AdCAGpA contacted RPE cells.
  • Figure 13 is a set of photomicrographs showing that hCD46 is expressed in mouse primary RPE cells following transfection (or contacting) with AdCAGCD46.
  • RPE cells were contacted withmedia conditioned by either AdCAGCD46 (top row) or AdCAGpA (bottom row) and were visualized with BF (left column) and stained with DAPI (middle column) or an antibody specific for human CD46 (right column).
  • Data show that mouse primary RPE cells efficiently expressed hCD46 on their membranes following transfection with AdCAGCD46. Little or no hCD46 was detectable in cells transfected with AdCAGpA.
  • Figure 14 is a set of photomicrographs showing that hCD46 was expressed on mouse
  • RPE cells following a sub-retinal injection of AdCAGCD46 Eight days after injection with AdCAGCD46, subjects were sacrificed and the lens, cornea and retina were removed from the eye to expose the RPE cells. Control subjects received no adenovirus vector injection. The eyecups were incubated in rate anti-mouse emmprin and NHS containing calcium and magnesium. Tissues were visualized with BF and stained with an antibody specific for human CD46.
  • Figure 14 panel A is a set of photomicrographs of a flat-mount of murine eyecups with the RPE cells exposed. Data show a patch of CD46 expression eight days after a sub-retinal injection of AdCAGCD46. Two views of the eyecups are shown including a higher magnification (right) of the boxed region of MAC staining (left) .
  • Figure 14 panel B shows BF visualizations (top row) and hCD46 expression staining (bottom) of cross sections through the injection site of subjects injected with AdCAGCD46. Data show visualizations of the un-injected side (left column) and the injected side (right column). It was observed that hCD46 expression was strongest on the basal and lateral surface of the RPE cells and that there was little or no detectable hCD46 expression in the un-injected region of the same eye.
  • Figure 15 is a bar graph and a set of photomicrographs showing that sub-retinal delivery of AdCAGCD46 reduced the amount of alternative pathway-mediated MAC deposition on RPE cells.
  • Murine eyes were injected with either a mixture of AdCAGpA and AdCAGGFP (9: 1 ratio) or a mixture of AdCAGCD46/AdCAGGFP (9: 1 ratio) and the eyes were then visualized for GFP fluorescence and stained for expression of MAC.
  • AdCAGGFP was included in each mixture to facilitate visualization of the injection site.
  • Figure 15 panel A is a set of photomicrographs showing the visualization of GFP (left column) and immunohistochemistry staining for MAC complex (right column) of eyes injected with either a mixture of AdCAGpA and AdCAGGFP (top row) or a mixture of AdCAGCD46 and AdCAGGFP (bottom row) .
  • a substantial decrease in MAC pixel intensity was observed for eyes injected with a mixture of AdCAGCD46 and AdCAGGFP compared to eyes injected with a mixture of AdCAGpA and AdCAGGFP.
  • Arrows in the inset demarcate the periphery of the injected area.
  • Figure 15 panel B is a set of photomicrographs using a higher magnification of the boxed regions of MAC staining for the eyecups injected with either a mixture of AdCAGpA and AdCAGGFP (left) or a mixture of AdCAGCD46 and AdCAGGFP (right) as shown in Figure 15 panel A.
  • Figure 15 panel C is a bar graph showing quantification of MAC pixel intensity of eyecups on the ordinate as a function of the mixture injected into the eyes (abscissa). Eyes were injected with either a mixture of AdCAGpA and AdCAGGFP or a mixture of
  • FIG. 15 panel D is a photomicrograph of a cross-section of an uninjected eye exposed to serum then stained for MAC. Data show that most of the MAC was deposited on the apical surface of RPE cells.
  • Figure 16 is a drawing and a set of photomicrographs showing that human CD55 (hCD55) expressed in vitro from an adenovirus vector was processed correctly and localized in the cell membrane.
  • Figure 16 panel A is a drawing showing an adenovirus vector expressing under control of the chicken beta actin promoter a gene encoding either hCD55 or GFP protein, and a control vector expressing no transgene.
  • Expression cassettes were cloned into the deleted El region of serotype 5 adenovirus in an anti-sense orientation with respect to the El enhancer.
  • hCD55 and GFP were expressed from a CAG promoter. Symbols used: CAG, cytomegalovirus chicken ⁇ -actin ⁇ -globin promoter; pA, polyadenylation signal; LITR, left inverted terminal repeat; RITR, right inverted terminal repeat; ⁇ , Ad packaging signal; MLT, major late transcript; E, early region labels.
  • Figure 16 panel B is a set of photographs of Western blots of human embryonic retinoblasts (HER) cell administered lysates (L) or media (M) contacted with adenovirus vector expressing a gene encoding hCD55 (AdCAGCD55) or a control adenovirus vector encoding no transgene (AdCAGpA).
  • Western blots were analyzed using a monoclonal antibody specific to hCD55 (top photograph) or a monoclonal antibody specific to ⁇ -actin (bottom photograph). Control lysates or media were not contacted with adenovirus (Uninfected). Data show detectable hCD55 expression in AdCAGCD55-contacted HER cells and not in AdCAGpA- contacted HER cells.
  • Figure 16 panel C is a set of photomicrographs of mouse Hepalclc7 cells contacted with media conditioned with AdCAGpA (top row) or AdCAGCD46 (bottom row). Cells were visualized with BF (left column) and were stained with mouse anti human CD55 (right column). Photomicrographs were visualized at 40x magnification. It was observed that AdCAGCD55-contacted mouse hepalcl c7 cells showed hCD55 expression and localization at the cell membrane.
  • Figure 17 is a bar graph and a set of print outs showing that adenovirus vectors expressing hCD55 protected mouse hepalclc7 cells from complement-mediated cell lysis.
  • Figure 17 panel A is a bar graph showing percent cell lysis on the ordinate of hepalclc7 cells as a function of adenovirus vector (abscissa): uninfected, AdCAGpA, or AdCAGCD55.
  • Cells were injected at a multiplicity of infection (MOI) of 1000 viral particles in 10% NHS (open bars) or HI-NHS (closed bars).
  • MOI multiplicity of infection
  • Data show greater cell lysis for hepalclc7 cells contacted with NHS compared to cells contacted with HI-NHS.
  • Figure 17 panel B are printouts of cell sorting data showing results of human serum cell lysis assays with extent of propidium iodide (PI) labeling of injected cells shown on the abscissa (acquired in the FL3-H channel) and the number of cells on the ordinate.
  • Hepalcl c7 were contacted with AdCAGpA. (middle) or AdCAGCD55 (right), or control cells were not injected (left), and then treated with either NHS or HI-NHS.
  • Data show that untreated cells treated with HI-NHS sorted to a location of lesser PI uptake than untreated cells treated with NHS (i.e., greater PI uptake and cell lysis).
  • Figure 18 is a set of photomicrographs and a bar graph showing that AdCAGCD55 protected mouse hepalclc7 cells from complement-mediated MAC deposition.
  • Figure 18 panel A is a set of representative photomicrographs of murine hepalclc7 cells contacted with either AdCAGpA (top row) or AdCAGCD55 (bottom row) at a MOI of 1000 viral particles, incubated with 10% NHS, and then visualized with BF (left column) and stained with a monoclonal antibody specific for MAC (right column). Photomicrographs show dramatically reduced MAC staining for cells contacted with AdCAGCD55 compared to cells contacted with AdCAGpA.
  • Figure 19 is a set of photomicrographs showing that subretinal injection of
  • AdCAGCD55 to murine eyes caused localization of hCD55 to the apical, basal and lateral membrane of RPE cells.
  • Flatmounts of eyecups were pretreated by injection with a mixture of vectors AdCAGpA and AdCAGGFP (9: 1 ratio) or a mixture of vectors AdCAGCD55 and AdCAGGFP (9: 1 ratio).
  • Cells were contacted six days post-injection with 10% NHS for five minutes, and then were visualized with BF and stained for hCD55 expression using a protein specific monoclonal antibody.
  • Figure 19 panel A is a set of photomicrographs showing GFP fluorescence (left column) and hCD55 expression (right column) for flatmounts of eyecups injected with a mixture of vectors AdCAGpA and AdCAGGFP (top row) or a mixture of vectors
  • AdCAGCD55 and AdCAGGFP bottom row. Immunohistochemistry of AdCAGCD55- injected eyes showed a section/patch of hCD55 expression coincident with the GFP expression at the site of injection. A portion of eye cup that folded onto itself is indicated by (*).
  • Figure 19 panel B is a set of photomicrographs of representative cross sections through an injection site of an eye injected with a mixture of vectors AdCAGCD55 and AdCAGGFP. It was observed that hCD55 expression was located on the apical, basal and lateral surface of the RPE cells.
  • Figure 20 is a bar graph and a set of photomicrographs showing protection against human MAC deposition as a result of adenovirus vector expressing hCD55 delivered to mouse RPE cells in vivo.
  • Flatmounts of eyecups were pretreated by injection with a mixture of vectors AdCAGpA and AdCAGGFP (9: 1 ratio) or a mixture of vectors AdCAGCD55 and AdCAGGFP (9:1).
  • Cells were contacted six days post-injection with 10% NHS for five minutes, and were then visualized for GFP fluorescence and stained for MAC.
  • Figure 20 panel A is a set of photomicrographs of flatmounts of eyecups contacted with a mixture of vectors AdCAGCD55 and AdCAGGFP (top row) or a mixture of vectors of AdCAGpA and AdCAGGFP (bottom row), and then contacted with NHS.
  • Flatmounts were visualized for GFP fluorescence (left column in each set), and then stained for MAC staining (middle column in each set) with anti-human C5b-9 antibody.
  • Merge (right column in each set) is an overlay of the GFP and the MAC dissected tissue photograph.
  • MAC staining of flat mounted eyecups injected with a mixture of AdCAGCD55 and ADCAGGFP and incubated with NHS showed reduced MAC deposition compared to eyecups injected with a mixture of AdCAGpA and ADCAGGFP.
  • the expression of hCD55 in the eyes caused reduction of MAC deposition.
  • Figure 20 panel B is a bar graph of quantification of percentage of MAC staining intensity relative to the uninjected region (ordinate) as a function of adenovirus vector pretreatment of flatmount eyecups (abscissa). Eyecups were pretreated with a mixture of AdCAGpa and AdCAGGFP (left bar) or a mixture of AdCAGCD55 and AdCAGGFP (right bar). Data show that adenovirus-delivered hCD55 (AdCAGCD55) resulted in a significant reduction (55.7%) in flatmount MAC deposition on mouse RPE compared to adenovirus- delivered AdCAGpA. ***p ⁇ 0.0001 (paired t test).
  • Figure 21 is a drawing showing a nucleotide sequence of a gene encoding a STAC protein having a genetic fusion of amino acid sequences of human CD46, human CD55, and human CD59 proteins.
  • the nucleotide sequence encodes a start codon, a secretory signal and complementary regulatory domains of the CD59 protein amino acid sequence of 104 amino acid open reading frame (ORF).
  • the nucleotide sequence further encodes a linker having five glycines and the CD46 protein amino acid sequence of 300 amino acids including four short consensus repeat (SCR) domains/motifs and a serine/threonine/proline (STP) rich domain.
  • SCR short consensus repeat
  • STP serine/threonine/proline
  • the nucleotide sequence further encodes a second linker of five glycines and the CD55 protein amino acid sequence of 322 amino acids including four SCR domains and a STP rich domain followed by two stop codons (TGA).
  • Figure 22 panel A is a drawing showing construct AdCAGSTAC serotype 5 adenovirus vector expressing a gene encoding STAC protein under control of the CAG promoter, and construct AdCAGGFP expressing GFP under control of the CAG promoter.
  • CAG cytomegalovirus chicken ⁇ -actin ⁇ -globin promoter
  • A polyadenylation signal
  • LITR left inverted terminal repeat
  • RJTR right inverted terminal repeat
  • ⁇ Ad packaging signal
  • MLT major late transcript
  • E early region labels.
  • Figure 22 panel B is a photograph of a Western blot of media of human RPE (ARPE19) cells contacted with AdCAGSTAC (right columns) or AdCAGGFP (left columns) and analyzed using a monoclonal antibody specific for binding to either human CD59, human CD46, or human CD55.
  • the Western blot shows presence of antigen from each of human CD59 protein, human CD46 protein, and human CD55 protein in medium of cells contacted with AdCAGSTAC. Dark bands were observed at approximately 130kD and faint bands were observed at approximately 150kD in the medium of the ARPE19 cells ( Figure 22 panel B right three columns). Human CD59, human CD46, and human CD55 signal were not detected in medium from ARPE19 cells contacted with AdCAGGFP control vector ( Figure 22 panel B left three columns).
  • Figure 23 is a set of photomicrographs and data graphs showing that medium conditioned by AdCAGSTAC-contacted ARPE19 cells protected mouse hepa-lclc7 cells from complement-mediated injury. Images and data are representative of three independent experiments performed in duplicate each time.
  • Figure 23 panel A is a set of photomicrographs of mouse hepa-lclc? cells contacted with media conditioned by ARPE19 cells contacted by either AdCAGGFP (top row) or AdCAGSTAC (bottom row) and then incubated with 10% (v/v) normal human serum (NHS) and visualized by antibody stain for deposition of membrane attack complex (MAC).
  • the left column shows hepa-lclc? cells visualized by bright field (DIC) microscopy.
  • the right column shows the same hepa-lclc7 cells stained for both for DAPI, which stains the nucleus, and MAC. Increased magnifications of the highlighted portions of the photomicrographs are also shown.
  • FIG. 23 panel B is a graph of quantification of MAC pixel (staining) intensity, ordinate, as a function of source of media from adeno virus-contacted ARPE-19 cells and serum to which mouse hepa-lclc7 cells were contacted, abscissa.
  • the hepa-lclc7 cells were contacted with medium conditioned by ARPE19 cells contacted by either AdCAGGFP (left most bars) or AdCAGSTAC (right most bars) and then contacted with NHS (on the left in each pair) or heat inactivated (Hi) NHS (on the right in each pair).
  • the treated hepa-lclc7 cells were contacted with: AdCAGGFP medium and NHS (left), AdCAGGFP medium and HI-NHS (right), AdCAGSTAC medium and NHS (left), or AdCAGSTAC medium and HI-NHS (right).
  • AdCAGSTAC contacted ARPE19 cells compared to cells contacted with medium conditioned by AdCAGGFP contacted ARPE19 cells.
  • Figure 23 panel C is a line graph showing cell lysis monitored by percent propidium iodide (PI) uptake (ordinate) in contacted mouse hepa-lclc7 cells as a function of
  • AdCAGGFP-contacted ARPE19 cells contact with medium from AdCAGSTAC-contacted ARPE19 cells resulted in 4.5-fold less PI uptake in cells compared to contact with medium from ARPE19 cells contacted with AdCAGGFP vector. Cells contacted with the medium from AdCAGGFP-contacted ARPE19 cells showed 70% PI uptake lysis of cells even at lower percent NHS.
  • Figure 24 is a set of photomicrographs and a bar graph showing that endothelial cells of liver vasculature of C57/B16J mice pre-injected intraperitoneally with AdCAGSTAC vector seven days prior to left ventricle injection of anti-mPECAMl/NHS were observed to have less MAC staining compared to liver vasculature from mice pre-injected with AdCAGGFP vector.
  • Figure 24 panel A are photographs of DIC staining (left photomicrograph) and GFP fluorescence (right photomicrograph) of liver sections of a subject seven days after injection with AdCAGGFP vector. The images show transduction of the adenovirus on the liver capsule.
  • Figure 24 panel B are photographs of DIC staining (left photomicrograph) and MAC staining (right photomicrograph) of cross sections taken from the center of the left liver lobes of subjects injected with AdCAGGFP vector (top row) or AdCAGSTAC vector (bottom row), then seven days later injected with anti-mPECAMl/NHS into the left ventricle.
  • Figure 24 panel C is a bar graph showing mean pixel intensity (ordinate) of MAC staining in subjects injected with an adenovirus prior to injection with anti-mPECAMl/NHS (abscissa).
  • the injected adenovirus vector strain used was control AdCAGGFP (left bar), or AdCAGSTAC (right bar).
  • Dysregulation of the complement system is considered to be one of the major factors contributing towards the etiology of AMD, one of the leading causes of blindness in the elderly (Gehrs et al. 2010 Arch Ophthalmol 128: 349-358).
  • the most devastating form of the disease affects approximately 10% of patients (Klein 2008 Ophthalmology 115: 1026-1031), and involves the growth of attenuated blood vessels from the choroidal vasculature through Bruch's membrane and into the retina.
  • the plasma released by these "ill-formed” vessels damages photoreceptors and other retinal cells, eventually leading to a severe loss of vision.
  • Haplotype variants in both Factor B and complement component 2 (C2) result in a significantly reduced risk of developing AMD (Gold et al. 2006 Nat Genet 38: 458-462), and an R80G substitution in complement component 3 (C3) increased the risk of having AMD to as much as 22% (Yates et al. 2007 N Engl J Med 357: 553-561).
  • the factor B (32Q) variant has been shown to have a 4- fold lower binding affinity for C3b, with a reduced ability to form the convertase (Montes et al. 2009 Proc Natl Acad Sci USA 106: 4366-4371).
  • Complement components have also been observed in the epiretinal membranes of patients suffering from proliferative vitreoretinopathy (PVR), and upregulation of the classical pathway initiator protein, C 1 q, as well as altered expression of other proteins of the cascade have been observed in glaucomatous eyes (Baudouin et al. 1990 Am J Ophthalmol 110: 593-598; Stasi et al. 2006 Invest Ophthalmol Vis Sci 47: 1024-1029; and Tezel et al. 2010 Invest Ophthalmol Vis Sci 51(11): 5697-707).
  • PVR proliferative vitreoretinopathy
  • C3 One of three distinct complement pathways (classical, lectin or alternative) initiates the complement cascade (Markiewski et al. 2007 Am J Pathol 171: 715-727) and these pathways converge at the point in the pathway of the breakdown of C3 into C3a and C3b.
  • the breakdown of C3 initiates the final part of the pathway that culminates in the formation of the membrane attack complex (MAC), a pore-like structure which inserts in the membranes of self- or non-self cells causing their lysis.
  • MAC membrane attack complex
  • activation of C3 generates the anaphylatoxins, C3a and C5a, both of which are powerful and pleiotropic effectors of inflammation.
  • the alternative pathway is constitutively active with small amounts of C3 hydrolysis and conversion to the convertase occurring in the serum.
  • the effects of complement over-expression in mouse retina, specifically by determining the consequences of an increased local expression of C3 in mouse retina on retinal anatomy and function are analyzed in Examples herein. Without being limited by any particular theory or mechanism of action, it is here envisioned that a local increase in C3 expression resulted in a local increase in C3 hydrolysis and conversion to the convertase.
  • Methods, compositions and kits described herein increased expression of C3 by injection into the sub-retinal space of an adenovirus expressing murine C3 regulated by the cytomegalovirus (CMV) promoter. Scotopic electroretinograpy, fluorescein angiography and histological analyses in Examples herein showed that that increased expression of C3 in murine RPE caused significant functional and anatomical changes in the murine retina.
  • CMV cytomegalovirus
  • Inhibitors of complement such as factor H and CD59 have been shown to impact the development of choroidal neovascularization in response to laser-induced damage of Bruch's membrane (Bora et al. 2005 J Immunol 174: 491-497; Bora et al. 2006 J Immunol 177: 1872- 1878; Bora et al. 2007 J Immunol 178: 1783-1790; and Rohrer et al. 2009 Invest Ophthalmol Vis Sci 50: 3056-3064).
  • Aged Factor H-deficient mice were observed to have deposition of C3 and C3b in retinal vasculature, which resulted in attenuation of retinal vessels and reduced blood flow (Lundh von Leithner 2009 Am J Pathol 175: 412-421). Examples herein show consequences of local C3 over-expression in the RPE of murine retina.
  • Adenovirus expressing either murine C3 or GFP from a CMV promoter were injected into the mouse sub-retinal space and eight days or 14 days post- injection, eyes were analyzed to detennine the effect of local exogenous C3 production. Without being limited by any particular theory or mechanism of action, it is envisioned that an increased expression of C3 in murine RPE increased local C3 hydrolysis, convertase production, and complement activation.
  • AMD patients generally initially present with pigmentaiy changes in the RPE, manifesting as areas of both hyper- and hypo-pigmentation (Klein et al. 2007 Ophthalmology 114: 253-262).
  • the pigmentary changes progress in approximately 40% of advanced AMD cases to geographic atrophy in which areas of RPE cell loss through which choroidal vessels and an overlying area of photoreceptor degeneration are observed.
  • Previous studies have observed transdifferentiating and migrating RPE cells in intimate association with choroidal neovascular membranes of AMD patients (Lopez et al. 1996 Invest Ophthalmol Vis Sci 37: 855-868).
  • RPE cells have also been shown to migrate into the retina in proliferative vitreoretinopathy (Fisher et al. 2005 Prog Retin Eye Res 24: 395-431; and Hiscott et al. 1999 Prog Retin Eye Res 18: 167-190).
  • Retinas contacted with adenovirus expressed C3 showed a loss of RPE cells and pigmentation, and a loss of photoreceptor outer segments in the region of endothelial cell proliferation.
  • the C3-contacted retinas were observed to have pigmentary deposits in close association with cells staining positive for the endothelial cell marker, GSL I, and in some cases these cells could be observed penetrating the retina.
  • An increase in endothelial cell staining was also observed within the region of injection in the choroid (and in some cases the retina) of GFP-contacted eyes, however these proliferating endothelial cells did not penetrate the RPE.
  • Ad5 expressing red fluorescent protein from a CMV promoter has been shown not to cause CNV when injected into the subretinal space (Cashman et al. 2006 Invest Ophthalmol Vis Sci 47: 3496-3504).
  • a titer of adenovirus was used in Examples herein that was approximately 20-fold less than was used in this previous analysis.
  • VEGF Increased VEGF has been observed in the eyes of AMD patients, and inhibitors of VEGF and VEGF receptors are highly effective in controlling and reducing exudative disease (Ozkiris 2010 Expert Opin Ther Pat 20: 103-118).
  • the complex relationship between CNV development and angiogenic factors such as VEGF, bFGF and HGF has been investigated in the laser-induced model of neovascularization (Hu et al. 2009 Exp Eye Res 88: 79-91).
  • VEGF may have a role in development of the phenotype or the adenovirus itself may have contributed to the development of the pathology of AdcmvC3 -injected retinas as activation of mouse complement has been observed previously for adenovirus (Tian et al. 2009 J Virol 83 : 5648-5658). Patients with either exudative AMD or with geographic atrophy show reduced cone and rod function as measured by electroretinography (Gerth 2009 Doc Ophthalmol 118: 63-68). The reduction in scotopic a- and b-wave amplitudes was observed to correlate with increasing light intensity (Walter et al.
  • C3- contacted eyes were observed herein to have significantly reduced a- wave amplitudes, as measured by scotopic ERG, at the higher light intensity but not at the lower light intensity.
  • B- wave amplitudes were also observed in Examples herein to be reduced at both light intensities compared to both GFP-contacted eyes or uninjected murine subjects.
  • the observed reduction in retinal function indicates the loss of photoreceptor segments observed in C3-injected mice. Loss of outer segments is caused by vascular leakage and/or deposition of the MAC complex, which was observed on the remnant segments.
  • Muller cells reactive gliosis
  • retinal detachment Activation of Muller cells (reactive gliosis) is a common feature of damage, e.g. retinal detachment, to the retina (Bringmann et al. 2006 Prog Retin Eye Res 25: 397-424).
  • Muller cells have been shown to play a pivotal role in retinal detachment associated with
  • fibrocontractive disorders such as proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy, PDR (Guidry 2005 Prog Retin Eye Res 24: 75-86).
  • PVR proliferative vitreoretinopathy
  • PDR proliferative diabetic retinopathy
  • increased vascular permeability is a concern during reactive gliosis.
  • C3a receptor is consistent with presence of the receptor on Muller cells (Vogt et al. 2006 Exp Eye Res 83: 834-840). Examples herein show C3-contacted eyes had altered expression and distribution of the intermediate filament protein GFAP, indicating activation of Muller cells.
  • a system and assay are provided herein involving local increased expression of C3 in mouse retina using an adenovirus that models many of the pathologies observed in AMD eyes - neovascularization, increased vascular permeability, RPE atrophy, hypo-pigmentation, retinal detachment, photoreceptor degeneration, and reduced retinal function, but does so within one to two weeks of virus administration.
  • the systems, methods, compositions and kits described herein are useful in further elucidating the role of complement not only in AMD, and in other retinal diseases such as PVR and PDR, and in the development of therapies for complement-mediated retinal diseases, such as AMD.
  • Examples herein show systems, compositions, methods and kits for assaying potential therapeutic agents for treatment of complement-based ocular diseases.
  • the systems, methods and kits include assays showing an animal model of complement-based ocular diseases through the overactivation of natural complement takeover.
  • the assays are for example in vitro or in vivo.
  • the natural complement takeover is overactivated using adenovirus vectors having a nucleic acid that encode C3.
  • C3 in ocular cell or on ocular tissues increases C3 hydrolysis, C3-convertase production, and complement activation.
  • the activation of the complement cascade forms different products including anaphylatoxins such as C3a and C5a, and membrane attack complex that causes cell lysis and significant functional and anatomical negative changes to ocular cells and tissue including damaged photoreceptor cells and vascular leakage in the retina.
  • Complement-associated ocular disease as used herein and in the claims includes without limitation a "complement-based ocular disease” and refers to an ocular disease, pathology or condition associated with complement activation or overactivation or natural complement takeover.
  • ocular tissue as used herein and in the claims includes without limitation an "ocular surface” and refers to a tissue or surface of the eye or the ocular mucosa.
  • ocular tissues examples include retinal pigment epithelium, pupil, cornea, iris, lens, aqueous humor, retina, choroid, sclera, fovea, eye muscles such as ciliary muscles or orbital muscles, glands such as the lacrimal glands, and the conjunctiva.
  • a complement-based ocular disease as used herein refers to a disease characterized by function of complement proteins or overactivation of natural complement takeover in an ocular cell or tissue, for example complement-based ocular diseases include age-related macular degeneration, glaucoma, retinal pigmentosa, and proliferative vitreoretinopathy.
  • the methods and kits herein include a C3 protein or a vector having a nucleic acid that encodes C3 exemplified by amino acid sequence shown. See Bednarczyk, J.L. et al.1988 Scand J Immunol 27: 83-95; and Van den Berg, C.W. et al, 1989 J Immunol Methods 122: 73-78. Additional exemplary amino acid sequences are obtained by mutating the nucleic acid sequence encoding C3 to obtain point mutations, substitutions or deletions having a nucleic acid sequence that encodes a modified amino acid sequence, encoding a protein that retains the binding function capable of binding the therapeutic agent or imaging agent to the ocular cell or ocular tissue.
  • the C3 herein is envisioned to include conservative sequence modifications in residues of the protein or in residues modified by conservative amino acid changes that do not reduce the overactivation of natural complement takeover function.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the functional characteristics of the protein or peptide. Such conservative modifications include amino acid substitutions, additions and deletions.
  • Modification of the amino acid sequence of C3 is achieved using any known technique in the art e.g., site-directed mutagenesis or PCR based mutagenesis. Such techniques are described in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989 and Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.
  • Conservative amino acid substitutions are changes in the C3 in which an amino acid residue is replaced with a different amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g.. threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • the amino acid sequence of the C3 is substantially identical to that of a wild type sequence/The term "substantially identical" is used herein to refer to a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are identical to aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity.
  • amino acid sequences that contain a common structural domain having at least about 30% identity, or at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%o, or 99% identity.
  • sequence identity between sequences are performed as follows. To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison memeposes (e.g., gaps can be introduced in one or both of a first and a second amino acid sequence for optimal alignment). The amino acid residues 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 proteins 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 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.
  • Percent identity between two amino acid sequences is determined using an alignment software program using the default parameters. Suitable programs include, for example, CLUSTAL W by Thompson et al., Nuc. Acids Research 22:4673, 1994 (www.ebi.ac.uk/clustalw), BL2SEQ by Tatusova and Madden, FEMS Microbiol. Lett. 174:247, 1999 (www.ncbi.nlm.nih. gov blast bl2seq/bl2.html), SAGA by Notredame and Higgins, Nuc. Acids Research 24: 1515, 1996 (igs-server.cnrs- mrs.fr/ ⁇ cnotred), and DIALIGN by Morgenstern et al., Bioinformatics 14:290, 1998
  • An aspect of the invention herein provides a model system for diagnosing or prognosing an ocular pathology or for screening potential therapeutic agents including: a first sample of cells or tissue contacted with a vector including a nucleic acid encoding complement component 3 (C3) to overactivate natural complement takeover in the cells or the tissue; a second sample of cells or tissue contacted with a control nucleic acid encoding a detectable protein, such that the vector and the control vector include the same promoter sequence operably linked to nucleic acids encoding the C3 and the detectable protein, such that the detectable protein is a control that does not overactivate the natural complement takeover, such that the second sample of the cells or tissue are otherwise identical to the first sample of the cells or tissue contacted with the vector; a control sample of cells or a tissue from a normal subject, such that the normal subject is not affected by a complement-based ocular disease or the ocular pathology; and a marker associated with complement activation for measuring in each of the first sample, the second sample and
  • the C3 is a mammalian C3 for example C3 derived from a human, a mouse, a cow, a pig, a rabbit, a goat, or a dog.
  • the vector includes a viral vector, for example an adenovirus, an adeno-associated virus, a retrovirus, a herpesvirus, and a lentiviras.
  • the vector includes a plasmid vector.
  • the detectable protein is at least one selected from: a fluorescent protein, an enzyme having a colorimetric assay, and a chemifluorescent protein
  • the fluorescent protein is at least one selected from the group consisting: green fluorescent protein, enhanced green fluorescent protein, aequorin, cyan fluorescent protein, DsRed fluorescent protein, and yellow fluorescent protein.
  • the marker includes at least one selected from: a membrane attack complex protein, glial fibrillary acidic protein, vascular endothelial growth factor, and Griffonia simplicifolia lectin I.
  • the marker is a complement component or complement protein such as an anaphylatoxin, such as C3a or C5a.
  • the cells or the tissues are cultured in vitro.
  • the cells or tissue are contacted in an animal model in vivo.
  • the animal model is a mouse model.
  • the cells or the tissue includes ocular cells or ocular tissues, respectively.
  • the cells or tissue are contacted by injection, for example by at least one route of administration selected from: intra-ocular, invitreal, subconjunctival, subretinal, and subtenon.
  • the cells or tissue are contacted topically.
  • the marker includes at least one selected from the group of: a membrane attack complex protein, glial fibrillary acidic protein, vascular endothelial growth factor, and Griffonia simplicifolia lectin I.
  • measuring includes observing cellular staining or tissue staining, for example staining of a cellular membrane or nucleus.
  • measuring includes observing at least one from the group of: increased vascular permeability, increased endothelial cell proliferation and migration, RPE atrophy, loss of photoreceptor outer segments, reactive gliosis, increased drasen formation, Muller cell activation, formation of membrane attack complex, retinal detachment, and reduced retinal function.
  • the first sample of the cells or the tissue is characterized as having loss of integrity of retinal vessels.
  • An aspect of the invention herein provides a method for diagnosing a complement- based ocular disease in cells or tissue from a subject, the method including: contacting a first sample of the cells or tissue from the subject with a vector including a nucleic acid encoding complement component 3 (C3), such that the C3 overactivates natural complement takeover in the cells or the tissue; measuring an amount of a marker in the first sample, such that the marker is characteristic of the disease; and comparing amounts of the marker in the first sample to amount of the marker in a second sample of cells or tissue from the subject not so contacted to the vector and otherwise identical, such that amount of the marker in the first sample of cells is compared to that in the cells or the tissue from the subject, such that a substantially similar amount or even greater amount of the marker in the second sample compared to the amount of the marker in first sample is an indication of the diagnosis of the complement-based ocular disease in the subject.
  • the cells or the tissue includes ocular cells or ocular tissues, respectively.
  • the method further includes obtaining a control sample of cells or tissue from a normal subject, such that the normal subject is not affected by the complement-based ocular disease, and measuring an amount of the marker in the control sample, such that a greater amount of the marker in the second sample and/or first sample compared to the amount of the marker in the control sample is an indication of presence of the complement-based ocular disease or ocular pathology in the subject.
  • the method indicates the presence of age-related macular degeneration.
  • An aspect of the invention herein provides a method of identifying in a model system a potential therapeutic agent for treating or preventing a complement based-ocular disease, the method including: contacting a first sample of cells or tissue with a vector including a nucleic acid encoding complement component 3 (C3) to overactivate natural complement takeover in the cells or the tissue, contacting a second sample of the cells or tissue with a control nucleic acid encoding a detectable protein, such that the vector and the control vector include the same promoter sequence operably linked to nucleic acids encoding the C3 and the detectable protein, such that the detectable protein is a control that does not overactivate the natural complement takeover, and contacting at least a third sample of cells or tissue with the nucleic acid encoding the C3 and at least one of a plurality of potential therapeutic agents; and measuring in the first sample, the second sample and the third sample, an amount of the marker, such that the marker is characteristic of the disease, such that the amount of the marker in the third sample
  • the complement based-eye disease includes age- related macular degeneration (AMD) condition, for example wet AMD or dry AMD.
  • AMD age- related macular degeneration
  • the detectable protein is at least one selected from: a fluorescent protein, an enzyme having a colorimetric assay, and a chemifluorescent protein.
  • the fluorescent protein is at least one selected from the group of: green fluorescent protein, enhanced green fluorescent protein, aequorin, cyan fluorescent protein, DsRed fluorescent protein, and yellow fluorescent protein.
  • the promoter includes a cytomegalovirus (CMV) promoter, for example a human CMV promoter or mouse CMV promoter.
  • CMV cytomegalovirus
  • the vector includes a viral vector, for example the viral vector is an adenovirus, an adeno- associated virus, a retrovirus, a herpesvirus, or a lentivirus.
  • the marker includes a protein, an enzyme, a lipid, a carbohydrate, or a nucleic acid.
  • the vector is selected from the viral vector encoding a DNA or an RNA, a naked DNA vector, and a microencapsulated DNA or R A.
  • the marker includes at least one selected from the group of: membrane attack complex (MAC) or a MAC protein, glial fibrillary acidic protein, vascular endothelial growth factor, and Griffonia simplicifolia lectin I.
  • MAC membrane attack complex
  • glial fibrillary acidic protein glial fibrillary acidic protein
  • vascular endothelial growth factor vascular endothelial growth factor
  • Griffonia simplicifolia lectin I Griffonia simplicifolia lectin I.
  • measuring further includes observing a localization of the marker in the cells or the tissue contacted by at least one of the first sample, the second sample, and the third sample.
  • the method includes observing the localization of the marker within the cytoplasm or in a nucleus.
  • the cells or the tissues are cultured in vitro.
  • the cells or tissue are cultured in a tube, a flask, or a plate.
  • contacting is in an animal model in vivo.
  • contacting includes injecting the cells or the tissue, for example by at least one route of administration selected from: intra-ocular, invitreal, subconjunctival, subretinal, and subtenon.
  • contacting includes administering topically, for example to an ocular tissue or mucosal lining.
  • the method prior to contacting, includes engineering the vector having the nucleic acid encoding the C3 to express the C3 as a soluble protein.
  • measuring further includes observing cellular staining or tissue staining, for example staining of a cellular membrane or nucleus.
  • measuring further includes observing the marker specifically located on the cells or the tissue, for example retinal pigment epithelium or retina.
  • measuring further includes observing cellular morphology, cell viability, or tissue functionality in the samples.
  • the method includes observing decreased ocular functionality (e.g., retinal functionality) in the first sample compared to the second sample and/or third samples.
  • measuring further includes observing in the samples at least one selected from the group consisting of: retinal detachment, cellular pathology, and tissue pathology. In related embodiment of the method, measuring further includes performing electroretinography.
  • kits for diagnosing or prognosing an ocular pathology and/or identifying a potential therapeutic agent for treating or preventing a complement based-ocular disease including: a vector including a nucleic acid encoding a component 3 (C3) that overactivates natural complement takeover in cells or tissue;
  • the instructions for use include methods described herein for identifying the potential therapeutic agent or for diagnosing or prognosing an ocular pathology.
  • the kit further includes an anti- C3 antibody or an anti-MAC antibody.
  • the kit further includes a control vector including a control nucleic acid encoding a detectable protein absent the C3, such that the vector and the control vector include the same promoter sequence operably linked to nucleic acids encoding the C3 and the detectable protein, such that the detectable protein does not overactivate the natural complement takeover in the cells or the tissue.
  • the instructions for use in kits herein include any of the methods described herein.
  • the methods and kits herein identifying a potential therapeutic agent for exudative choroidal diseases, herododystrophic diseases, retinal vascular disorders, and other diseases that associated with complement protein or the immune response. Methods and kits herein are useful for identifying a therapeutic agent that treats any of these diseases or conditions resulting in vision loss and ocular disease dysfunction.
  • CD46 prevents formation of C3 convertase by acting as a cofactor for factor I mediated decay of C3b, an important regulator of complement.
  • CD55 acts by dissociating the C3 convertase.
  • CD59 regulates the complement pathway by preventing formation of the MAC complex.
  • Data herein show that CD46, CD55 and CD59 individually and in combination were therapeutic agents that effectively eliminated and modulated complement-based cell lysis and disease.
  • CD46 also known as MCP (complement membrane cofactor protein)
  • MCP complement membrane cofactor protein
  • Mature human CD46 protein is composed of 392 amino acids and has a molecular weight of about 48kD to about 68 kD.
  • CD46 is found in leukocytes, platelets, epithelial cells, sperm cells, and fibroblasts. Numerous transcript variants encoding different isoforms have been identified for this gene. Structures of different CD46 proteins are shown for example in U.S. patent number 5,846,715 issued December 8, 1998 to Purcell et al. which is incorporated by reference herein in its entirety.
  • Isoforms of human CD46 CI, C2, BC1, and BC2 have distinct molecular weights.
  • CI and C2 have a molecular weight of about 56 kD
  • BC1 and BC2 have a molecular weight of about 66 kD
  • Isoforms ABC1 and ABC2 have a molecular weight of about 76 kD (358 amino acids and 365 amino acids respectively).
  • CD46 has four N-terminal short consensus (SCR) modules that are referred to as Sushi domains which are known motifs in protein-protein interactions. Sushi domains contain four cysteines forming two disulfide bonds in a 1-3 and 2-4 pattern. SCR modules 2-4 function in C3b/C4b binding and regulatory activity. CD46 is post-transcriptionally modified by glycosylation. See Stern et al. 1986 J Immunol 137: 1604-1609; Seya et al. 1986 J Exp Med 163: 837-855; Post et al. 1991 J Exp Med 174: 93-102; Russell et al. 1992 Eur J Immunol 22: 1513-1517; Okada et al. 1995 Proc Natl Acad Sci USA 137: 3689-3695; and Thorley et al. 1997 Eur J Immunol 27: 726-734.
  • SCR short consensus
  • CD46 has been identified as a major risk factor for developing diseases associated with chronic alternative pathway activity, such as aHUS (Kavanagh et al. 2008 Annu Rev Med 59: 293-309). Although the importance of CD46 expression has been defined for aHUS, its role in AMD has not been fully evaluated. Analysis of the expression pattern for CD46 in normal human eyes revealed that it is expressed on the basal and lateral surfaces of RPE cells (Vogt et al. 2006 Exp Eye Res 83(4): 834-840). As the basal surface of RPE cells are exposed to drusen, CD46 is in a prime location to dampen alternative pathway activity.
  • the levels of endogeneous CD46 may not be sufficient to protect the RPE cells from complement mediated attack or a state of chronic inflammation, such as occurs with AMD. Without being limited by any particular theory or mechanism of action, it is here envisioned that increasing the expression of CD46 in RPE cells by contact with CD46 or a source of CD46 protein restores the balance between alternative complement activation and regulation in AMD patients.
  • a novel murine model of complement activation using C3 to determine the capacity for adenoviral delivery of a human complement regulator to protect against human MAC deposition is shown in Examples herein. See also Ramo, K., S.M. Cashman, and R. Kumar- Singh, Invest Ophthalmol Vis Sci, 2008. 49(9): p. 4126-4136, hereby incorporated herein by reference.
  • This and other humanized murine models provide convenient platforms to evaluate the role of CD46 protein, CD55 protein and STAC protein in the murine retina. Data herein show that complement activation occurred primarily through the alternative pathway and that human CD46 protected murine RPE cells from alternative pathway mediated complement attack.
  • CD46 Despite robust expression on the membrane of mouse hepatocytes transduced with a CD46-expressing adenovirus vector, displayed no inliibition of complement-mediated cell lysis conferred in the presence of all pathways, and offered 39 ⁇ 0.88% protection against lysis that was specifically mediated by the alternative pathway. Without being limited by any particular theory or mechanism of action, it is here envisioned that CD46 has a higher binding affinity for proteins of the alternative pathway component C3b relative to the classical component C4b.
  • CD46 may take longer for CD46 to cleave C3b and C4b than does a regulator such as CD59 to disrupt the MAC complex due to requirement of CD46 as a co-factor for cleavage, requiring binding of the serine protease factor I to cleave C3b and C4b. Therefore, limiting the process of MAC deposition to the alternative pathway allows enough time for CD46 to bind C3b and recruit factor I before the convertase is formed.
  • CD46 no longer binds to either C3b or C4b (Gao et al. 2009 BMC Cancer 9: 384; Brodbeck et al. 2000 J Immunol 165(7): 3999-4006), so that once convertase has assembled on the cell membrane, the rate of C3 cleavage is rapid and the amount of C3b exceeds that of CD46.
  • CD46 in human retinal tissue is localized to the basal and lateral surface of RPE cells (Vogt et al. 2006 Exp Eye Res 83(4): 834-840).This places CD46 close to the site of complement activation in AMD patients in which MAC deposition has been observed in RPE cells, drusen, and choroidal endothelial cells. Also CD46 expression is reduced on the RPE of AMD patients. Polymorphisms in CD46 have been linked to the kidney disease aHUS (Richards et al. 2003 Proc Natl Acad Sci USA 100(22): 12966-12971), another disease resulting from over-activation in the alternative arm of complement (Kavanagh et al.
  • Renal allographs which provide wild type expression of CD46, have been a viable therapy for patients suffering from aHUS due to CD46 polymorphisms (Caprioli et al. 2006 Blood 108(4): 1267-1279; Kavanagh et al. 2006 Semin Thromb Hemost, 32(2): 155-159).
  • Examples herein show that expression of CD46 from an engineered adenovirus vector in vivo in mice resulted in 24 ⁇ 4.5% protection against an acute insult from human complement attack mediated by the alternative pathway.
  • Figure 14 shows that CD46 is most abundantly localized to the basal surface of mouse RPE cells after injection of
  • AdCAGCD46 AdCAGCD46, with some expression also on the apical and lateral surfaces. This pattern of expression is normal for human RPE (Vogt et al. 2006 Exp Eye Res, 2006. 83(4): 834-840). The apical side of the RPE is exposed directly to emmprin antibody and human serum in Examples herein such that MAC deposition and CD46 protection occured almost exclusively on the apical surface. Further assays showed that CD46 blocked MAC deposition on the basal surface.
  • CD46 in fact can compensate for the loss or reduction in factor H activity in human serum (Barilla-LaBarca, M.L., et al.,. J Immunol, 2002. 168(12): 6298- 6304), a situation analogous to that proposed for AMD patients with factor H polymorphisms.
  • CD46 compositions including a STAC protein that includes amino acid sequences of CD46 protein are useful to treat ocular diseases including macular degeneration, by inhibiting MAC deposition and preventing lysis of retina cells.
  • ocular tissues is used interchangeably with the phrase “ocular surfaces” and refers to any tissue or surface of the eye or the ocular mucosa. Examples of ocular tissues include retinal pigment epithelium, pupil, cornea, iris, lens, aqueous humor, retina, choroid, sclera, fovea, eye muscles such as ciliary muscles or orbital muscles, glands such as the lacrimal glands, and the conjunctiva.
  • the CD46 composition or STAC protein composition that includes amino acid sequences of CD46 protein provided used herein lacks the primary amino acid sequence for a functional hydrophobic transmembrane spanning domain.
  • a functionally equivalent protein includes a modified hydrophobic transmembrane spanning domain amino acid sequence hence is defective in the ability to target a membrane.
  • a sCD46 is an example of a recombinant membrane-independent CD46 (rmiCD46). Additional methods of obtaining membrane-independent CD46 include non-recombinant methods such as using an inhibitor of membrane association, for example, synthesizing CD46 in vivo or in vitro such that the transmembrane domain is lacking. Methods of obtaining the membrane-independent CD46 are shown in examples herein. Additional recombinant techniques for altering the nucleic acid sequence and amino acid sequence of a molecule are well known in the art of genetics and molecular biology.
  • the composition includes a CD46 protein that includes a full length nucleic acid of CD46 that was recombinantly modified to remove the signal sequence for attachment of a hydrophobic transmembrane spanning domain.
  • the nucleic acid sequence of CD46 is modified by point mutations, substitutions or deletions to obtain a nucleic acid sequence that encodes an amino acid sequence that has a modified amino acid sequence at the a hydrophobic transmembrane spanning domain location, such that the protein is unable to attach to a membrane of a cell.
  • membrane independent refers to a CD46 amino acid sequence that lacks a a hydrophobic transmembrane spanning domain or has a modified a hydrophobic transmembrane spanning domain that lacks functional ability to bind to a cell membrane or a cell-membrane-associated structure such as a membrane-bound protein.
  • CD46 protein herein is envisioned to include conservative sequence modifications.
  • conservative sequence modifications refers to amino acid modifications that do not significantly alter the characteristics of the CD46 protein or membrane-independent CD46 containing the amino acid sequence, i.e., amino acid sequences of CD46 that present amino acids having chemically-related side chains at the same relative positions and that will function in a manner similar to human CD46. Such conservative modifications include amino acid substitutions, additions and deletions. Modification of the amino acid sequence of CD46 is achieved using any known technique in the art e.g., site- directed mutagenesis or PCR based mutagenesis.
  • amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspart
  • the CD46 amino acid sequence is substantially identical to that of the wild type sequence.
  • substantially identical is used herein to refer to a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are identical to aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity.
  • amino acid sequences that contain a common structural domain having at least about 60% identity, or at least 75%, 85%, 95%, 96%, 98%, or 99% identity.
  • sequence identity between sequences are performed as follows. To determine the percent identity of two amino acid sequences, 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 sequence for optimal alignment). The amino acid residues 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 proteins 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.
  • Percent identity between two amino acid sequences is determined using an alignment software program using the default parameters. Suitable programs include, for example, CLUSTAL W by Thompson et al, Nuc. Acids Research 22:4673, 1994 (www.ebi.ac.uk/clustalw), BL2SEQ by Tatusova and Madden, FEMS Microbiol. Lett. 174:247, 1999 (www.ncbi.nlm.nih.gov blast/bl2seq/bl2.html), SAGA by Notredame and Higgins, Nuc. Acids Research 24:1515, 1996 (igs-server.cnrs- mrs.fr/ ⁇ cnotred), and DIALIGN by Morgenstern et al, Bioinformatics 14:290, 1998
  • CD55 is known also as complement decay-accelerating factor or decay-accelerating factor (DAF), and is a protein complement regulator. See Lublin et al. 1989 Annu Rev Immunol 7: 35-58, Sims et al. U.S. patent number 5,955,441 issued September 21, 1999 and Martens et al. U.S. patent number 7,008,775 issued March 7, 2006, each of which is incorporated by reference herein in its entirety.
  • CD55 is a glycosyl phosphatidyl inositol (GPI)-anchored glycoprotein of molecular weight of approximately 70 kDa expressed on the plasma membrane of all cell types that come into contact with plasma complement proteins. The protein is a constituent of the extracellular matrix.
  • GPI glycosyl phosphatidyl inositol
  • CD55 variants include soluble forms of CD55 secreted after glycosylation.
  • the human CD 55 protein is uniformly GPI-anchored, which is referred to as DAF-GPI or GPI-DAF or CD55a.
  • Mice possess two closely related genes, termed decay-accelerating factor 1 (DAF1) and decay-accelerating factor 2 (DAF2), also referred to as CD55b.
  • DAF1 decay-accelerating factor 1
  • DAF2 decay-accelerating factor 2
  • CD55b decay-accelerating factor 2
  • the genes encoding Cd55a and CD55b share 93 % identity in their coding regions. See Clift et al. 2009 Journal of Reproductive Immunology Vol. 81(1): 62-73; and Yamada et al. 2004 The Journal of Immunology vol. 172 (6): 3869-3875.
  • membrane independent CD55 refers to a CD55 amino acid sequence that lacks a GPI anchor or has a modified GPI anchor that lacks function and ability to bind to a cell membrane or a cell-membrane-associated structure such as a membrane-bound protein.
  • the CD55 protein herein used to engineer the CD55 composition or STAC protein composition including CD55 protein is envisioned to include conservative sequence modifications including deletions, substitutions, and additions as has been described herein.
  • Inflammatory processes and specifically activated complement has been implicated in the pathogenesis of a number of diseases including Alzheimer's disease (Tenner et al. 2001 Neurobiol Aging 22: 849-861), atherosclerosis (Niculescu et al. 2004 Immunol Res 2004 30: 73-80), and glomerular basement membrane kidney disease (Fang et al. 2008 Blood 111: 624- 632; and Licht et al. 2009 Thromb Haemost 101 : 271-278).
  • a role for complement has been recently established in the pathogenesis of AMD. Consequently, a number of phase I/II clinical trials aimed at attenuating complement activation in AMD patients have recently been initiated.
  • C3 compstatin/POT-4 peptide
  • C5 eculizumab antibody, ARC1905 aptamer
  • Factor D TNX-234 antibody
  • compositions, methods and kits using CD55 to attenuate complement activation by accelerating the concomitant decay of the classical and alternative C3 convertases on RPE cells of mice are developed. Without being limited by any particular theory or mechanism of action, it is here envisioned that a perturbation upstream in complement activation through C3 convertase activity using CD55 significantly altered levels of MAC assembled in the terminal pathway.
  • MAC is a major component of complement-mediated cellular damage, levels of MAC deposition on RPE cell surfaces, a tissue intimately involved in AMD pathogenesis, were quantified. Examples herein show that expression of hCD55 mediated reduction of MAC deposition on ocular tissues.
  • Adenovirus vectors are engineered to express transgenes for extended periods of up to 1 year— the longest time period examined (Kim et al. 2001 Proc Natl Acad Sci USA 98:13282-13287; Kreppel et al. 2002 Invest Ophthalmol Vis Sci 43: 1965-1970; and Lamartina et al. 2007 J Gene Med 9: 862-874).
  • adenovirus vectors carrying a gene encoding CD55 as described herein are able to provide long-term expression of the protein on ocular tissues including the RPE, a tissue where CD55 is not normally found at high levels.
  • CD55b and CD59b are exclusively expressed in the mouse testis whereas CD55a and CD59a are expressed broadly. Accordingly, Examples herein show that adenovims-mediated delivery of hCD55 to murine RPE protects those cells against human complement mediated lysis and MAC formation. The protection conferred by attenuating the C3 convertases through hCD55 was as potent an approach as blocking the assembly of MAC directly on the cell surface via the expression of hCD59.
  • An aspect of the invention herein provides a method for treating AMD in a subject, the method involving contacting retinal pigment epithelium (RPE) of the subject with a CD55 protein composition, in which the retina is treated for AMD.
  • RPE retinal pigment epithelium
  • contacting the RPE is delivering at least one composition selected from the group consisting of: a nucleic acid vector with a gene encoding CD55 protein; CD55 protein; or CD55 expressed directly from naked nucleic acid.
  • the vector is a viral vector or a plasmid; for example, the viral vector is derived from a genetically engineered genome of at least one virus selected from the group consisting of adenovirus, adeno-associated virus, a herpesvirus, and a lentivirus.
  • the lentivirus is a retrovirus.
  • delivery of protein or nucleic acid is by at least one injection route selected from the group consisting of intravenous, intra-ocular, intramuscular, subcutaneous, and intraperitoneal.
  • the macular degeneration is dry.
  • An aspect of the invention provides a method of assaying extent of human MD in a model cell system or a method in a model cell system of assaying a serum complement component for prognosis or diagnosis of macular degeneration (MD), the method including: exposing a first sample of cells to serum and measuring resulting lysis, and comparing extent of lysis to that in a second sample of control cells not so exposed to serum, such that the extent of lysis in the first sample compared to that in the second sample is a measure of complement- induced MD.
  • An aspect of the invention provides a method of assaying in a model cell system potential therapeutic agents for human MD, the method including: contacting a first sample of cells to serum and measuring resulting lysis, and contacting a second sample of otherwise identical control cells with serum and a source of human CD55 and measuring resulting lysis; and contacting at least a third sample of cells to a candidate therapeutic composition and otherwise identically to serum, such that the extent of lysis of the third sample compared to that in the first and second sample is a measure of protection by the candidate composition, thereby providing the method of assaying for potential therapeutic agents.
  • a related embodiment of the above methods further includes contacting cells or tissues with a recombinant vector having a gene capable of expressing CD55. Lysis is measured for example by propidium iodide uptake and cell sorting.
  • the cells are hepatocytes.
  • the cells are of murine origin.
  • the source of CD55 is human.
  • the serum is normal human serum. Alternatively, the serum is from a diseased subject, for example, the diseased subject has MD.
  • An aspect of the invention provides a method of diagnosing or prognosing presence or progression of macular degeneration, the method including determining extent of membrane attack complex (MAC) deposition on retina.
  • determining extent of MAC deposition is analyzing by immunohistochemistiy with antibodies that are specific for human MAC.
  • An aspect of the invention provides a pharmaceutical composition for treating macular degeneration including CD55 protein or a source of expression of CD55 protein in vivo, in which the composition is formulated for ocular delivery, in a dose effective to treat macular degeneration.
  • the CD55 protein or source of expression of CD55 protein is at least one selected from the group consisting of: a nucleic acid vector with a gene encoding CD55 protein; a viral vector with a gene encoding CD55 protein; and a CD55 protein.
  • the composition formulated for ocular delivery is at least one selected from the group consisting of: injection, eye drop, and ointment.
  • injection is at least one selected from the group consisting of: intra-ocular injection, subconjunctival injection, and subtenon injection.
  • ⁇ H plo torl f consisting of: anti-tumor, antiviral, antibacterial, anti-mycobacterial, anti-fungal, antiproliferative and anti-apoptotic.
  • the CD55 protein is expressed as a soluble protein.
  • the CD55 protein has a deletion encoding a glycosyl phosphatidyl inositol (GPI) anchoring domain.
  • GPI glycosyl phosphatidyl inositol
  • kits for assaying MAC deposition on ocular tissue or cells and for screening agents that inhibit deposition includes anti-MAC antibody, a container, and instructions for use with normal human serum.
  • the kit further includes anti-emmprin antibody and/or normal human serum.
  • the kit further includes CD55 protein as a positive control and the CD55 protein is a soluble form or a membrane-bound form, the latter for example embedded in a liposome preparation.
  • at least one of the antibody, the serum, and the CD55 protein is a lyophil.
  • An aspect of the invention provides a method in a model cell system of assaying a serum complement component for prognosis or diagnosis of macular degeneration (MD), the method including: contacting detectably labeled cells with serum from a subject and measuring amount of extracellular and/or intracellular detectable agent for contacted cells; and comparing extracellular and/or intracellular agent in the cells to that in detectably labeled control cells not exposed to the serum and otherwise identical, such that amount of extracellular and/or intracellular agent in the contacted cells is compared to that in the control cells, such that a greater amount of extracellular detectably labeled agent in cells contacted with serum compared to the control cells is an indication of prognosis or diagnosis of MD.
  • An aspect of the invention provides a method of assaying in a model cell system a potential therapeutic agent for efficacy in treatment of human macular degeneration (MD), the method including: contacting a first sample of detectably labeled cells with serum from a subject and measuring amount of extracellular and/or intracellular detectable agent, and contacting a second sample of otherwise identical detectably labeled control cells with serum and a source of human CD55 protein and measuring amount of extracellular and/or intracellular detectable agent; and contacting at least a third sample of detectably labeled cells to at least one candidate therapeutic composition and otherwise identically to serum and measuring amount of extracellular and/or intracellular detectable agent, such that the amount of extracellular and/or intracellular detectable agent of the third sample compared to that in the first sample and the second sample is a measure of protection by the candidate composition, such that a greater amount of extracellular detectably labeled agent is an indication of MD, thereby assaying for a potential therapeutic agent for efficacy in treatment of human MD.
  • MD human macular
  • the detectable agent is at least one composition selected from the group consisting of a recombinant vector having a gene capable of expressing a detectable protein, a fluorescent agent, a colorimetric agent, an enzymatic agent, and a radioactive agent.
  • the detectable protein is at least one fluorescent protein selected from the group consisting: green fluorescent protein, aequorin, cyan fluorescent protein, DsRed fluorescent protein, enhanced green fluorescent protein, and yellow fluorescent protein.
  • the detectable agent is not a protein, for example, the detectable agent is at least one fluorescent agent selected from the group consisting of: Indocyanine Green, Doxorubicin, Riboflavin, Chlorophyll, and Porphyrin.
  • the detectable protein is enzyme, for example, ⁇ -galactosidase or alkaline phosphatase.
  • the cells are hepatocytes.
  • the source of CD55 protein is human.
  • the serum is normal human serum.
  • the serum is from a diseased subject. In general, the subject is in need of diagnosis or prognosis of MD.
  • the CD55 protein is soluble. In other embodiments the protein is membrane- bound.
  • a membrane bound CD59 was observed to protect cells from complement- mediated disease, however the site of expression of the regulator, yielded only a "patch" of protection in the ocular tissue such as the RPE.
  • a secreted regulator of CD59 sCD59 or rmiCD59 is here engineered, which was capable of diffusing through the retina and offer protection to the entire affected region (Kumar-Singh international application
  • Soluble sCD59 was previously considered an inefficient regulator of complement in vivo unless it was fused with a membrane targeting moiety (Mizuno et al. 2001 Arthritis Rheum 44: 2425-2434; Bora 2010 J Biol Chem 285: 33826-33833; Song et al. 2003 J Clin Invest 111: 1875-1885; and Zhang et al. 1999 J Clin Invest 103: 55-61).
  • membrane-independent sCD59 expressed in vivo in murine ocular tissue via an adenovirus or AAV vector significantly reduced MAC deposition and laser-induced choroidal
  • neovascularization in a mouse model of neo ascular AMD (Cashman et al. 2011 A Non Membrane-Targeted Human Soluble CD59 Attenuates Choroidal Neovascularization in a Model of Age Related Macular Degeneration PLoS ONE 6(4): el9078, which is hereby incorporated by reference in its entirety).
  • Adeno virus-delivered sCD59 was observed to inhibit human MAC deposition even on murine liver vasculature.
  • a recombinant fusion protein containing at least two of CD59 protein, CD46 protein and CD55 protein is a potent regulator of a number of complement pathways and proteins.
  • Examples herein provi methods for engineering a novel chimeric soluble terminator of activated complement (STAC) having small functional units of each of CD46 protein, CD55 protein, and CD59 protein that are effective for treating complement-related conditions by modulating the complement cascade, and provide the composition.
  • STAC protein composition includes functional units of CD46 protein, CD55 protein, and CD59 protein that in certain embodiments are operably linked.
  • the functional units are connected by amino acid linkers that do not affect the function of the components or the structural stability of the protein.
  • the protein in certain embodiments is mutated to remove or delete a sequence encoding a protein membrane anchor.
  • an exemplary STAC protein includes a secretory signal at the N-terminus.
  • the STAC protein is
  • the recombinant STAC protein engineered herein is differs from naturally occurring regulators because it includes multiple complement regulatory domains from different combinations of CD59, CD46, and CD55 proteins and is membrane independent. Hence STAC protein is capable of diffusing and blanketing a large group of affected cells or tissue for treatment after a single administration at one time.
  • the STAC protein includes an amino acid sequence from at least two of a CD46 protein, a CD55 protein, and a CD59 protein.
  • the STAC protein includes at least one of: the CD46 protein and the CD59 protein, the CD46 protein and the CD55 protein, and the CD55 protein and the CD59 protein.
  • the STAC protein includes each of CD59 protein, CD46 protein and CD55 protein, operably linked and expressed for example in a soluble form.
  • the CD46 protein, the CD55 protein, and the CD59 protein are derived from mammalian proteins (e.g., human, mouse, and rabbit).
  • the STAC protein comprises a CD46 protein and a CD59 protein that are human proteins and a CD55 protein that is a murine protein, or comprises each of CD46, CD55, and CD59 that are human proteins.
  • the STAC protein comprises proteins that are from the same mammal type, or from different types of mammals.
  • the STAC protein synergistically blocks complement activation at mutiple steps in the complement pathway, including each of the complement pathways regulated by each of CD59 protein, CD46 protein, and CD55 protein.
  • the STAC protein is shown in Examples herein to inhibit MAC deposition in vivo when delivered by an adenovirus vector, and is therefore potentially effective as an anti-complement therapy for treating or even preventing complement-associated diseases or conditions.
  • the STAC protein or composition includes a CD46 protein encoded by a full length nucleic acid of CD46 which was modified to remove the amino acid sequences for signal sequence and hydrophobic transmembrane spanning domains.
  • nucleic acid sequence of CD46 protein is modified by point mutations, substitutions or deletions to obtain a nucleic acid sequence that encodes a modified amino acid sequence with the modification located in the hydrophobic transmembrane spanning domain, such that the resulting protein fails to attach to cell membranes.
  • membrane independent CD46 refers to a CD46 amino acid sequence that lacks a hydrophobic transmembrane spanning domain or has a modified hydrophobic transmembrane spanning domain that lacks functional ability to bind to a cell membrane or a cell-membrane-associated structure such as a membrane-bound protein.
  • the scope of the CD46 protein herein is envisioned to include conservative sequence modifications including deletions, substitutions, and additions as has been described herein.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the characteristics of the CD46 protein containing the amino acid sequence, i.e., amino acid sequences of CD46 protein that present these side chains at the same relative positions will function in a manner similar to human CD46 protein. Such conservative modifications include amino acid substitutions, additions and deletions. Modification of the amino acid sequence of CD46 protein is achieved using any known technique in the art e.g. , site-directed mutagenesis or PCR based mutagenisis.
  • Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains
  • the CD46 amino acid sequence is an amino acid sequence that is substantially identical to that of the wild type sequence.
  • substantially identical is used herein to refer to a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are identical to aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity.
  • amino acid sequences that contain a common structural domain having at least about 60% identity, or at least 75%, 85%, 95%, 96%, 98%, or 99% identity.
  • the STAC protein or composition includes a CD55 protein and/or a CD59 protein.
  • the CD55 protein includes a full length nucleic acid of CD55.
  • the CD55 protein is a portion or homologue of full length nucleic acid sequence or amino acid sequence as described herein.
  • the CD55 protein includes conservative sequence modifications CD59 protein.
  • Mature human CD59 protein is composed of 77 amino acids and has a molecular weight of about 18kD to about 21 kD.
  • Precursor human CD59 protein includes an amino- terminal signal peptide of 25 amino acids and a carboxyl-terminal peptide of 26 amino acids which allows for attachment of a membrane anchor.
  • Amino acid sequences of precursor human CD59 protein, a mature human CD59 protein, and CD59 protein of other mammals e.g., baboon, African green monkey, owl monkey, marmoset, HVS-15, pig, rabbit, rat, and mouse, are shown in Sims et al. U.S. patent number 7,166,568 issued January 23, 2007 which is incorporated herein by reference in its entirety.
  • CD59 The protein structure of CD59 is characterized as a single cysteine-rich domain, having a hydrophobic core with three loops and a small fourth helical loop (Yu et al., Journal of Experimental Medicine, 185(4):745-753, 1997). Disulfide-bonded cysteine pairs connect each of these loops (Yu et al., 1997).
  • the structure of the gene encoding CD59 has been characterized (Fodor et al. U.S. patent number 5,624,837, issued April 29, 1997).
  • the gene is located on the short arm of chromosome 11 in humans, specifically chromosome 1 lpl3 and 1 lpl4 (Online Mendelian Inheritance in Man accession number andl07271), and consists of 4 exons spanning 20 kb (Petranka et al. Proc. Nat. Acad. Sci. 89:7876-7879, 1992).
  • An untranslated first exon is preceded by a G and C-rich promoter region that lacks a consensus TATA or CAAT motif.
  • the second exon encodes the hydrophobic leader sequence of the protein, and the third exon encodes the N-terminal portion of the mature protein.
  • the fourth exon encodes the remainder of the mature protein, including the hydrophobic sequence for glycophosphoinosital anchor attachment to a cell membrane.
  • CD59 is a glycosylphosphatidylinositol-anchored glycoprotein that is expressed on human peripheral blood leukocytes, erythrocytes, and many cell lines.
  • the protein is expressed on both hematopoietic and non-hematopoietic non-hemopoietic cells, for example on endothelial cells, peripheral nerve fibers, neurons, microglia, oligodendrocytes, astrocytes, ependymal cells, epithelial cells, acinar cells of the salivary glands, bronchial epithelium, renal tubules and squamous epithelium. See Nose, M. et al. 1990 Immunology 70(2): 145-149; Vedeler, C. et al. 1994 Immunology 82(4): 542-547; and Hidestima, T. et al. 1990
  • CD59 has been cloned from human T- cell leukemia (YT) and human erythroleukemia (K562) cell lines, and CD59 has been transiently expressed in COS cells (Walsh, L.A. et al. 1990 Eur J. Immol 21(3): 847-850).
  • Human CD59 is encoded by a nucleic acid sequence including 26 amino acids located at the C terminus, which contains a signal sequence for attachment of a GPI anchor at amino acid asparagine at position 77. The amino acid sequence of full length cDNA of CD59 is shown in Fodor et al, U.S. patent number 5,624,837 issued April 29, 1997.
  • the binding site for interactions of human CD59 with human C9 has been identified as amino acid residues 42 to 58 in the sequence of mature human CD59, that bind to the region of human C9 corresponding to human amino acid residues 334 to 418 of that protein, more particularly human C9 amino acid residues 359 to 384, immediately C-terminal to the predicted membrane-inserting domain of C9 (Sims et al. PCT/US96/17940 filed November 8, 1996, which is incoiporated herein by reference in its entirety).
  • the active surface exposed amino acid residue side chains that are available to bind C8/C9 identified from solution structure of mature human CD59 from published NMR data and the knowledge of the active portion of the CD59 molecule, are histidine at position 44, asparagine at position 48, aspartic acid at position 49, threonine at positions 51 and 52, arginine at position 55, and glutamic acid at position 58.
  • NMR structures for CD59 are described in deposits by Kieffer et al. Human Complement Regulatory Protein CD59
  • MMDB Id 891, PDB Id: 1ERH; Kieffer et al. Human Complement Regulatory Protein CD59 (Extracellular Region, Residues 1 70; NMR, Restrained), MMDB Id: 890, PDB Id: 1ERG; Fletcher et al, CD59 Complexed
  • a CD59 protein in certain embodiments used in construction of the STAC protein herein lacks the primary amino acid sequence for a functional GPI anchor.
  • a functional equivalent protein includes a modified GPI anchor domain amino acid sequence that is functionally defective and lacks the ability to target a membrane. Additional methods of obtaining a STAC protein having a membrane-independent CD59 protein include non- recombinant methods such as providing an inhibitor of membrane association, for example, synthesizing CD59 in vivo or in vitro such that the GPI anchor is lacking. Methods of obtaining the membrane-independent CD59 are shown in examples herein. Additional recombinant techniques for altering the nucleic acid sequence and amino acid sequence of a molecule are well known in the art of genetics and molecular biology.
  • the composition includes an amino acid sequence of a CD59 protein having a full length nucleic acid of CD59 protein that was modified to remove the signal sequence for attachment of the GPI anchor at the nucleotides encoding amino acid asparagine at position 77.
  • the nucleic acid sequence of CD59 is modified by one or more point mutations, substitutions or deletions to obtain a nucleic acid sequence that encodes an amino acid sequence that has a modified amino acid sequence at the GPI anchor location, such that the protein is unable to attach to a membrane of a cell.
  • membrane independent CD59 refers to a CD59 amino acid sequence that lacks a GPI anchor or has a modified GPI anchor that lacks function and ability to bind to a cell membrane or a cell-membrane-associated structure such as a membrane-bound protein.
  • GPI anchoring involves a multi-step pathway in the endoplasmic reticulum including the interaction of numerous gene products. Many proteins including CD59 require GPI to be expressed at the cell surface and to function effectively. The mechanism by which structure in a protein signal encodes for attachment of GPI anchors is reviewed by Orlean, P. et al. 2007 JLR 48:993-1011. GPI attachment generally involves an amino acid sequence that contains: a hydrophobic N-terminal secretion signal that targets the protein to the ER, and a C-terminal GPI signal anchor sequence.
  • the amino acid to which the GPI becomes linked is referred to as the omega (co) residue, with amino acids N-terminal to the omega residue referred to as omega- minus ( ⁇ -) and with amino acids C-terminal to the omega residue referred to as omega-plus ( ⁇ +).
  • the GPI anchor sequence includes a stretch of about ten polar amino acids (i.e., ⁇ -10 to ⁇ -l), for example arginine, lysine, aspartate, glutamate, asparagine, or glutamate, that form a flexible linker region.
  • the ⁇ residue has been observed to be one of: glycine, alanine, serine, asparagine, aspartic acid, or cysteine.
  • Mutation including substitution and deletion of nucleic acids encoding amino acids at omega positions are used to reduce or eliminate the attachment of the GPI anchor or reduce or eliminate the effective functionality of the GPI anchor.
  • such a variation includes substituting the nucleic acids encoding hydrophobic leucine (e.g., nucleic acids CTG) and alanine (e.g., nucleic acids GCA) with nucleic acids encoding glycine (e.g., nucleic acids CAG) and glutamate (e.g., nucleic acids GAA), which are less hydrophobic (i.e., more hydrophilic) amino acids.
  • a variation includes substituting the ⁇ residue with another amino acid, for example substituting a glycine for a tyrosine.
  • the STAC protein herein includes amino acid sequences from a CD59 protein having conservative sequence
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the characteristics of the CD59 protein or membrane-independent CD59 containing the amino acid sequence, i.e., amino acid sequences of CD59 that present these side chains at the same relative positions will function in a manner similar to human CD59. Such conservative modifications include amino acid substitutions, additions and deletions. Modification of the amino acid sequence of CD59 is achieved using any known technique in the art e.g., site-directed mutagenesis or PCR based mutagenesis.
  • amino acid residue is replaced with an amino acid residue having a similar side chain such as replacing a small amino acid with a different small amino acid, a hydrophilic amino acid with a different hydrophilic amino acid, etc..
  • Examples herein include methods, compositions and kits having a chimeric soluble terminator of activated complement (STAC) protein with amino acid sequences from each of a CD46 protein, a CD55 protein, and a CD59 protein, and a nucleic acid expressing the recombinant STAC protein, such that the STAC negatively modulates classical and alternative complement pathways and treats complement-related conditions.
  • the STAC protein includes an amino acid sequences from at least two of a CD46 protein, a CD55 protein, and a CD59 protein.
  • the phrase "complement-related” as used herein and in the claims includes without limitation "complement-associated”, and refers to any cell or tissue that is affected by a complement pathway. Examples of complement-related disorders or conditions include macular degeneration, lupus nephritis, Sjogren's syndrome, organ graft rejection, asthma, and chronic obstructive pulmonary disease.
  • the STAC protein composition has an amino acid sequence shown in SEQ ID NO: 1 or a portion thereof. Additional exemplary amino acid sequences are obtained by mutating the nucleic acid sequence encoding the STAC protein to obtain point mutations, substitutions or deletions having a nucleic acid sequence that encodes a modified amino acid sequence, encoding a protein that retains the binding and inhibitory functions and is thereby capable of treating or preventing a complement-related disorder or condition.
  • the STAC protein herein is envisioned to include conservative sequence modifications in residues of the protein or in residues modified by conservative amino acid changes that do not affect the therapeutic function resulting from inhibition of complement pathways, whether involved in binding the binding the protein to a target cell, a complement protein, a complement protein precursor, or involved in targeting in a complement pathway.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the functional characteristics of the protein. Such conservative modifications include amino acid substitutions, additions and deletions.
  • Modification of the amino acid sequence of the STAC protein is achieved using any known technique in the art e.g. , site-directed mutagenesis or PCR based mutagenesis. Such techniques are described in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.
  • Conservative amino acid substitutions are changes in the STAC protein in which an amino acid residue is replaced with a different amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the amino acid sequence of the STAC protein is substantially identical to SEQ ID NO: 1 or a portion.
  • substantially identical is used herein to refer to a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are identical to aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have common structural domains and/or common functional activities.
  • amino acid sequences that contain a common structural domain have at least about 30% identity, or at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% identity.
  • sequence identity between sequences are performed as follows. To determine the percent identity of two amino acid sequences, 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 sequence for optimal alignment). The amino acid residues 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 proteins 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 that is introduced for optimal alignment of the two sequences.
  • Percent identity between two amino acid sequences is determined using an alignment software program using the default parameters. Suitable programs include, for example, CLUSTAL W by Thompson et al., Nuc. Acids Research 22:4673, 1994 (www.ebi.ac.uk/clustalw), BL2SEQ by Tatusova and Madden, FEMS Microbiol. Lett. 174:247, 1999 (ww.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html), SAGA by Notredame and Higgins, Nuc. Acids Research 24: 1515, 1996 (igs-server.cnrs- mrs.fr/ ⁇ cnotred), and DIALIGN by Morgenstem et al., Bioinformatics 14:290, 1998
  • Methods herein for treating or preventing a complement-related disorder include contacting cells with a pharmaceutical composition including a C3 protein, a CD46 protein, a CD55 protein, or a STAC protein, which protein is recombinantly produced.
  • a pharmaceutical composition including a C3 protein, a CD46 protein, a CD55 protein, or a STAC protein, which protein is recombinantly produced.
  • recombinant refers to proteins produced by manipulation of genetically modified organisms, for example micro-organisms.
  • An exemplary source of the protein includes a polynucleotide sequence that encodes the protein, for example, a nucleotide sequence encoding the protein, or functional equivalent, is inserted into an appropriate expression vector, i. e. , a vector that contains the necessary nucleic acid encoding elements that regulate transcription and translation of the inserted coding sequence, operably linked to the nucleotide sequence encoding the amino acid sequence of the recombinant protein.
  • an appropriate expression vector i. e. , a vector that contains the necessary nucleic acid encoding elements that regulate transcription and translation of the inserted coding sequence, operably linked to the nucleotide sequence encoding the amino acid sequence of the recombinant protein.
  • a variety of commercially available expression vector/host systems are useful to carry and express a protein encoding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems contacted with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g. , Ti, pBR322, or pET25b plasmid); or animal cell systems. See Ausubel et al, Cuirent Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.
  • Virus vectors include, but are not limited to, adenovirus vectors, lentivirus vectors, adeno-associated virus (AAV) vectors, and helper-dependent adenovirus vectors.
  • virus vectors deliver a nucleic acid sequence that encodes a STAC protein that as shown herein that treat complement-related conditions.
  • Adenovirus packaging vectors are commercially available from American Type Tissue Culture Collection (Manassas, VA). Methods of constructing adenovirus vectors and using adenovirus vectors are shown in Klein et al, Ophthalmology, 114:253-262, 2007 and van Leeuwen et al, Eur. J. Epidemiol., 18:845- 854, 2003.
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, Gene, 101 : 195-202, 1991) and vaccine development (Graham et al, Methods in Molecular Biology: Gene Transfer and Expression Protocols 7, (Murray, Ed.), Humana Press, Clifton, NJ, 109- 128, 1991). Further, recombinant adenovirus vectors are used for gene therapy (Wu et al, U.S. patent number 7,235,391).
  • Recombinant adenovirus vectors are generated, for example, from homologous recombination between a shuttle vector and a provirus vector (Wu et al., U.S. patent number 7,235,391).
  • the adenovirus vectors herein are replication defective, for example, are conditionally defective, lacking an adenovirus region, and a polynucleotide encoding a peptide or protein is introduced at the position from which the coding sequences have been removed.
  • the polynucleotide encoding the gene of interest alternatively is inserted in the another region.
  • Helper cell lines may be derived from human cells, such as 293 human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.
  • the helper cells may be derived from the cells of other mammalian species that are permissive for human adenoviras, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. Generation and propagation of these replication defective adenoviras vectors using a helper cell line is described in Graham et al, J. Gen.
  • Lentiviral vector packaging vectors are commercially available from Invitrogen
  • HIV-based packaging system for the production of lentiviral vectors is prepared using constructs in Naldini et al., Science 272: 263-267, 1996; Zufferey et al, Nature Biotechnol., 15: 871-875, 1997; and Dull et al, J. Virol. 72: 8463-8471, 1998.
  • vector constructs are available to be packaged using a system, based on third-generation lentiviral SIN vector backbone (Dull et al, J. Virol. 72: 8463-8471, 1998).
  • the vector construct pRRLsinCMVGFPpre contains a 5' LTR in which the HIV promoter sequence has been replaced with that of Rous sarcoma virus (RSV), a self- inactivating 3' LTR containing a deletion in the U3 promoter region, the HIV packaging signal, RRE sequences linked to a marker gene cassette consisting of the Aequora jellyfish GFP driven by the CMV promoter, and the woodchuck hepatitis virus PRE element, which appears to enhance nuclear export.
  • RSV Rous sarcoma virus
  • the GFP marker gene allows quantitation of transfection or transduction efficiency by direct observation of UV fluorescence microscopy or flow cytometry (Kafri et al., Nature Genet, 17: 314-317, 1997 and Sakoda et al, J. Mol. Cell.
  • a retroviral vector is constructed and packaged into non-infectious transducing viral particles (virions) using an amphotropic packaging system. Examples of such packaging systems are found in, for example, Miller, et al, Mol. Cell Biol. 6:2895-2902, 1986;
  • producer cells is accomplished by introducing retroviral vectors into the packaging cells, a process of contacting referred to herein as “transducing", “transfecting”, or “infecting”.
  • retroviral vectors are found in, for example, Korman, et al., Proc. Natl. Acad. Sci. USA. 84:2150-2154, 1987; Morgenstern, et al., Nucleic Acids Res. 18:3587-3596, 1990; U.S. patent numbers 4,405,712, 4,980,289, and 5,112,767; and PCT patent publications numbers WO 85/05629, WO 90/02797, and WO 92/07943.
  • Herpesvirus packaging vectors are commercially available from Invitrogen Corporation, (Carlsbad, CA).
  • Exemplary herpesviruses are an a-herpesvirus, such as Varicella-Zoster virus or pseudorabies virus; a herpes simplex virus such as HSV-1 or HSV-2; and a herpesvirus such as Epstein-Barr virus.
  • a method for preparing empty herpesvirus particles that can be packaged with a desired nucleotide segment, for example a nucleotide or polynucleotide sequence, in the absence of a helper virus that is capable to most herpesviruses is shown in Fraefel et al. (U.S. patent number 5,998,208, issued December 7, 1999).
  • the herpesvirus DNA vector can be constructed using techniques familiar to the skilled artisan. For example, DNA segments encoding the entire genome of a herpesvirus is divided among a number of vectors capable of carrying large DNA segments, e.g., cosmids (Evans, et al., Gene 79, 9-20, 1989), yeast artificial chromosomes (YACS) (Sambrook, J. et al,
  • MOLECULAR CLONING A LABORATORY MANUAL, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989) or E. coli F element plasmids (O'Conner, et al, Science 244: 1307-1313, 1989).
  • sets of cosmids have been isolated which contain overlapping clones that represent the entire genomes of a variety of herpesviruses including Epstein-Barr virus, Varicella-Zoster virus, pseudorabies virus and HSV-1.
  • herpesviruses including Epstein-Barr virus, Varicella-Zoster virus, pseudorabies virus and HSV-1.
  • AAV is a dependent parvovirus in that it depends on co-infection with another virus (either adenovirus or a member of the herpes virus family) to undergo a productive infection in cultured cells (Muzyczka, Curr Top Microbiol Immunol, 158:97 129, 1992).
  • rAAV recombinant AAV virus
  • rAAV recombinant AAV virus
  • a plasmid containing the gene of interest for example, the a gene of interest, flanked by the two AAV terminal repeats
  • Cells are also contacted or transfected with adenovirus or plasmids carrying the adenovirus genes required for AAV helper function.
  • Recombinant AAV virus stocks made in such fashion include with adenovirus which must be physically separated from the recombinant AAV particles (for example, by cesium chloride density centrifugation).
  • Adeno-associated virus (AAV) packaging vectors are commercially available from AAV.
  • AAV GeneDetect (Auckland, New Zealand).
  • AAV has been shown to have a high frequency of integration and infects non-dividing cells, thus making it useful for delivery of genes into mammalian cells in tissue culture (Muzyczka, Curr Top Microbiol Immunol, 158:97 129, 1992).
  • AAV has a broad host range for infectivity (Tratschin et al, Mol. Cell. Biol., 4:2072 2081, 1984; Laughlin et al., J. Virol., 60(2):515 524, 1986; Lebkowski et al, Mol. Cell. Biol., 8(10):3988 3996, 1988; McLaughlin et al, J. Virol, 62(6): 1963 1973, 1988).
  • AAV vectors have been used successfully for in vitro and in vivo transduction of marker genes (Kaplitt et al., Nat Genet., 8(2):148 54, 1994; Lebkowski et al., Mol. Cell. Biol, 8(10):3988 3996, 1988; Samulski et al, EMBO J., 10:3941 3950,1991; Shelling and Smith, Gene Therapy, 1: 165 169, 1994; Yoder et al., Blood, 82 (Supp.): 1:347 A, 1994; Zhou et al, Exp. Hematol, 21 :928 933, 1993; Tratschin et al, Mol. Cell.
  • the vectors herein are non-viral vectors for example synthetic gene delivery vehicles or vectors that are not related to a virus particle and that specifically deliver the gene material to the target cells or tissue.
  • non-viral vectors include liposomes, peptides, nanoparticles, emulsions, or encapsulated two or more phase systems or other suitable preparation.
  • a method, kit, or composition involves a non-viral vector with nucleic acid that is loaded and contacted to a tissue or cell.
  • a liposome containing naked DNA encoding a protein is encapsulated in the liposome and the liposome is contacted to the tissue or cell such that the nucleic acid is effectively delivered to the tissue or cell for treatment of a complement-related disease.
  • compositions that include at least one of CD46 protein, a CD55 protein, and a STAC protein or a nucleic acid expressing the protein, for treating a complement-related disorder by negatively modulating complement proteins or pathways.
  • the pharmaceutical composition is compounded for systemic delivery to a subject, for example the composition is formulated as an injection.
  • the composition in another embodiment is formulated as an ophthalmologic formulation for administration to the eye and may be compounded for delivery to the fundus, or for release locally at the retina or otherwise formulated to provide effective treatment of the vessels and/or tissue involved in complement disorders negatively affecting the ocular tissues.
  • the pharmaceutical composition is formulated sufficiently pure for
  • compositions optionally further include one or more additional therapeutic agents.
  • the additional therapeutic agent or agents are selected from the group consisting of growth factors, anti-inflammatory agents, vasopressor agents including but not limited to nitric oxide and calcium channel blockers, collagenase inhibitors, topical steroids, matrix metalloproteinase inhibitors, ascorbates, angiotensin II, angiotensin III, calreticulin, tetracyclines, fibronectin, collagen,
  • TGF transforming growth factors
  • KGF keratinocyte growth factor
  • FGF fibroblast growth factor
  • IGFBPs insulin-like growth factors
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • NDF neu differentiation factor
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • HGF heparin-binding EGF
  • thrombospondins von Willebrand Factor-C
  • von Willebrand Factor-C heparin and heparin sulfates
  • hyaluronic acid transforming growth factors
  • TGF transforming growth factors
  • KGF keratinocyte growth factor
  • FGF fibroblast growth factor
  • IGFs insulin-like growth factors
  • IGFBPs IGF binding proteins
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • NDF neu differentiation factor
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • a plurality of therapeutic agents are include in the
  • the therapeutic agent is a drug that may include without limitation anti-coagulant, anti-tumor, anti-viral, anti-bacterial, anti-mycobacterial, anti-fungal, anti-proliferative or anti-apoptotic agents.
  • Drugs that are included in the compositions of the invention are well known in the art. See for example, Goodman & Oilman's The
  • the term "pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • Remington's Pharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, PA, 1995 provides various earners used in formulating pharmaceutical compositions and known techniques for the preparation thereof.
  • materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as glucose and sucrose; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, preservatives and antioxidants can also be present in the composition, the choice of agents and non-irritating concentrations to be determined according to the judgment of the composition
  • Methods provided herein involve contacting cells or tissues with a pharmaceutical composition, for example, administering a therapeutically effective amount of a
  • composition having as an active agent at least one of CD46 protein, CD55 protein, and STAC protein, a nucleic acid encoding a protein or a source of expression of the protein, to a subject in need thereof, in such amounts and for such time as is necessary to achieve the desired result including reduction or preventing of indicia of the complement- related condition.
  • compositions may be administered using any amount and any route of administration effective for treating the complement-related disorder.
  • amount effective for treating a complement-related disease or condition refers to a sufficient amount of composition to beneficially prevent or ameliorate the symptoms of the disease or condition.
  • the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, e.g., intermediate or advanced stage of macular degeneration; age, weight and gender of the patient; diet, time and frequency of administration; route of administration; drug combinations; reaction sensitivities; and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered hourly, twice hourly, eveiy three to four hours, daily, twice daily, every three to four days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition.
  • the active agents of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form refers to a physically discrete unit of active agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, as provided herein, usually mice, but also potentially from rats, rabbits, dogs, or pigs.
  • the animal cell model and in vivo model provided herein are also used to achieve a desirable concentration and total dosing range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active agent that ameliorates the symptoms or condition or prevents progression of the disease or condition.
  • Therapeutic efficacy and toxicity of active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population).
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.
  • the daily dosage of the products may be varied over a wide range, such as from 0.001 to 1000 mg per adult human per day.
  • the compositions are provided for example in the form of a solution containing 0.001, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, or 500.0 micrograms of the active ingredient for the
  • a unit dose typically contains from about 0.001 micrograms to about 500 micrograms of the active ingredient, preferably from about 0.1 micrograms to about 100 micrograms of active ingredient, more preferably from about 1.0 micrograms to about 10 micrograms of active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 25 mg/kg of body weight per day.
  • the range is from about 0.001 to 10 mg/kg of body weight per day, or from about 0.001 mg/kg to 1 mg/kg of body weight per day.
  • the compositions may be administered on a regimen of, for example, one to four or more times per day.
  • a unit dose may be divided for example, administered in two or more divided doses.
  • Administration of a source of expression of a protein is administration of a dose of a viral vector or a nucleic acid vector, for example the dose contains at least about 50, 100, 500, 1000, or at least about 5000 particles per cell to be treated.
  • the dose of a viral vector or a nucleic acid vector is at least about 10 4 to about 10 5 ; about 10 5 to about 10 6 ; 10 6 to about 10 7 ; 10 7 to about 10 8 ; about 10 s to about 10 9 ; about 10 9 to about 10 10 ; or at least about 10 10 to about 10 n .
  • the dose effective for treating a cell number can be calculated from the area in need of treatment by methods known to one of skill in the art.
  • the pharmaceutical composition provided herein is administered to humans and other mammals for example topically (as by powders, ointments, or drops), orally, rectally, mucosally, sublingually, parenterally, intracisternally, intravaginally, intraperitoneally, bucally, sublingually, ocularly, or intranasally, depending on preventive or therapeutic objectives and the severity and nature of a complement-related disorder or condition.
  • Injections include intravenous injection or intra-ocular injection into the aqueous or the vitreous humor, or injection into the external layers of the eye, such by subconjunctival injection or subtenon injection.
  • Liquid dosage forms for example for intravenous, ocular, mucosal, or other administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents
  • Dosage forms for topical or transdermal administration of an inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches.
  • the active agent is admixed under sterile conditions with a
  • ocular or cutaneous routes of administration are achieved with aqueous drops, a mist, an emulsion, or a cream.
  • Administration may be therapeutic or it may be prophylactic.
  • the invention includes ophthalmological devices, surgical devices, audiological devices or products which contain disclosed compositions (e.g., gauze bandages or strips), and methods of making or using such devices or products. These devices may be coated with, impregnated with, bonded to or otherwise treated with a composition as described herein.
  • Transdermal patches have the added advantage of providing controlled delivery of the active ingredients to the eye and body.
  • dosage forms can be made by dissolving or dispensing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
  • sterile injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • Delayed absorption of a parenterally administered active agent may be accomplished by dissolving or suspending the agent in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the agent in biodegradable polymers such as polylactide- polyglycolide. Depending upon the ratio of active agent to polymer and the nature of the particular polymer employed, the rate of active agent release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the agent in liposomes or
  • microemulsions which are compatible with body tissues.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the active agent(s) of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active agent(s).
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active agent(s).
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active agent is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin,
  • polyvinylpyrrolidinone, sucrose, and acacia c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.
  • disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate
  • e) solution retarding agents such
  • Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art.
  • the active agent(s) may be admixed with at least one inert diluent such as sucrose or starch.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • additional substances other than inert diluents e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active agent(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • Example 1 Adenovirus constructs carrying a nucleotide sequence encoding C3
  • MM fragment from pCMV-Sport6C3 (ATCC, Image clone ID 5134713; GenBank ID: BC043338; SEQ ID NO: 7) containing a murine C3 cDNA was cloned into the MM site of pShCMVMCS (a modified version of pShuttle containing the CMV promoter, an SV40 intron and polyA and an MM -inclusive multiple clomng site).
  • the C3-containing pShuttle was then recombined with pAd-Easyl and the rescued virus, AdCMVC3, was amplified (Cashman et al. 2002 Mol Ther 6: 813-823).
  • AdCMVGFP A control virus, AdCMVGFP, that expresses GFP was constructed as described in Cashman et al. 2004 Virology 324: 129-139.
  • human embryonic retinoblasts HERs
  • AdCMVC3 or AdCMVGFP Dulbecco's Modified Eagle's Medium supplemented with 2% fetal bovine serum (Invitrogen, Carlsbad, CA).
  • Cell lysates and cell media were loaded on a 10% Tris-HCl gel (Bio-Rad Criterion).
  • Membranes were probed with an anti-mouse C3 polyclonal antibody (1:1000; Cell Sciences, Canton, MA), followed by a 1: 10,000 dilution of HRP-conjugated goat anti-rabbit. Immunocytochemistry was performed by infecting 1.2 x 10 6 HERs at an MOI of 600 and fixed with 4% formalin 24 hours post-infection. Cells were incubated with a 1 :50 dilution of anti-mouse C3 (MP Biomedicals, Solon, Ohio) followed by a 1 :400 dilution of CY3 -conjugated donkey anti-goat (Jackson ImmunoResearch Laboratories; West Grove, PA). Cells were imaged using an Olympus 1X51 with appropriate filters, a Retiga 2000R FAST camera and QCapture Pro 5.0 (Qlmaging, British; Columbia, Canada) software.
  • Example 2 Subretinal administration of adenovirus
  • mice were purchased from Jackson Laboratories and maintained in 12-hour dark light cycles in accordance with federal, state, and local regulations.
  • Mouse subjects were anesthetized by intraperitoneal injection of O.lml/lOg of ketamine (10mg/ml)/xylazine (lmg/ml), followed by application of one drop of proparacaine hydrochloride (0.5%) to each eye.
  • Virus particles (2xl0 9 ) were injected into the sub-retinal space of 6-8 week old C57B16/J male mice using a trans-scleral/trans-choroidal approach. Both eyes were injected with the same virus in each subject (See also Cashman et al. 2002 Mol Ther 6: 813-823). A volumne of virus ( ⁇ ) was administered using a 33G needle and a 5 ⁇ glass syringe (Hamilton Inc.; Reno, NV).
  • Subjects were kept for overnight dark-adaptation, were anesthetized, and then one drop of 1% tropicamide (Akorn, Inc) was applied to each eye to dilate the pupil.
  • Body temperature of the mouse was maintained using an animal temperature controller (ATC 1000; World Precision Instruments; Sarasota, FL).
  • Electroretinograms (ERGs) were recorded at two different light intensities (-10 and OdB) using LKC UTAS Visual Diagnostic Test System with Big Shot LED Ganzfeld using contact lens electrodes (LKC). Ten flashes were recorded and averaged for each light intensity.
  • Eyes of the subjects were fixed overnight in 4% paraformaldehyde, and were dehydrated in 15-30% sucrose in 0.1M phosphate buffer. The cornea, lens and iris were removed and the eyecups were embedded in Tissue-Tek compound (Adwin Scientific
  • Sections (14 ⁇ ) were excised using a cryostat (Microm 550).
  • GSL I Sections were incubated at 37°C for ten minutes with 500 ⁇ g/ml BSA in PBS followed by 100 ⁇ / ⁇ FITC-conjugated Griff onia simplicifolia Lectin I (isolectin B4; Vector Labs, Burlingame, CA) in PBS for 1 hour at 37°C. Eyecups with retinas removed prior to fixation were stained as for sections, and blocking was performed for 30 minutes with 2.5mg/ml BSA in PBS. Four relaxing cuts were administered prior to mounting on slides.
  • Sections adjacent to those with representative GSL I stain for each of AdcmvC3 and AdcmvGFP-injected were stained for the following: DAPI: Sections were pre-treated with phosphate buffered saline and 0.05% Triton (PBST) for 15 minutes, followed by l,ug/ml DAPI in PBS for 5 minutes at room temperature (RT).
  • PBST phosphate buffered saline
  • PBST phosphate buffered saline and 0.05% Triton
  • Rhodopsin After blocking with 6% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in 0.25% PBST for one hour at RT, sections were incubated for 2.5 hours with a 1 :250 dilution of mouse monoclonal 1D4 in blocking buffer at RT, followed by a 1.5 hour incubation at RT with a 1 :400 dilution of CY3 -conjugated goat anti-mouse (Jackson ImmunoResearch).
  • GFAP Staining was performed as for rhodopsin with a 1 :500 dilution of rabbit polyclonal antibody anti-GFAP (Novus Biologicals Inc., Littleton, Colorado) followed by a 1 :500 dilution of CY3 goat anti-rabbit (Jackson ImmunoResearch), both in 6% normal goat serum.
  • MAC After blocking with 6% normal goat serum in 0.3% PBST for 30 minutes at RT, sections were stained for 2.5 hours at RT with a 1 :200 dilution of rabbit anti-mouse C9 antibody in PBST, followed by 1 :400 dilution of CY3-conjugated goat anti-rabbit (Jackson ImmunoResearch) at RT for 1 hour. In Examples herein, specificity of antibodies was determined using a control antibody of the same isotype. Sections and flatmounted eyecups were imaged using an Olympus 1X51 with appropriate filters, a Retiga 2000R FAST camera and QCapture Pro 5.0 (Qlmaging Inc.; British Columbia, Canada) software.
  • Murine subjects were anesthetized and a drop of 1% tropicamide was applied to each eye.
  • a volume (200 ⁇ 1) of 2.5% sodium fluorescein (Akorn) in IX PBS was injected intra- peritoneally. Eyes were coated with 2% methylcellulose and a commercially available coverslip (Corning, No. l; Corning Inc., Corning, NY) was used to image retinal vessels using a Nikon C-PS160 dissecting microscope and appropriate filters. Eyes were monitored for three to ten minutes and images and then injected with sodium fluorescein. Images were captured five minutes following the fluorescein injection using an Olympus DP20 camera.
  • Example 7 Quantitative RT-PC
  • RNA Stat-60 Tel-Test, Inc
  • VWR AHS200 VWR AHS200
  • iScript one-step RT-PCR kit Bio-Rad Inc., Hercules, CA
  • Applied Biosystems assays Mae C3: Mm00437858_ml
  • Mouse VEGF Mm00437304_ml
  • Mouse ⁇ -actin part # 4352663
  • C3 is a secreted protein, produced as a single polypeptide precursor, pro-C3, with a predicted molecular weight of 170 kDa.
  • Pro-C3 is cleaved intracellularly to form C3 and consists of an a chain (107 kDa) and ⁇ chain (62 kDa) connected by disulfide bonds
  • Example 10 Increased permeability of blood vessels in the presence of high levels of C3
  • Fluorescein angiography was performed on subjects injected with adenoviruses expressing either C3 or GFP into the sub-retinal space, at eight days and 14 days following injection.
  • Example 1 C3 over-expression results in extensive proliferation of endothelial cells
  • Neovascularization is typically preceded by proliferation and migration of vascular endothelial cells (Ucuzian et al. 2010 J Burn Care Res 31 : 158-175).
  • C3 adeno virus-injected or control GFP adeno virus-injected eyes were harvested nine days following injection. Cryosections were then stained using the FITC conjugated endothelial cell marker GSL I, isolectin B4. Prior to GSL I staining, cryosections of GFP adenovirus -injected eyes were analyzed for GFP expression (Figure 3 panel A).
  • Muller cells Retinal diseases are known to activate certain cells in the eye, specifically Muller cells are known to become activated (Brmgmann et al. 2006 Prog Retin Eye Res 25: 397-424).
  • Mulller cell activation in uninjected ocular tissues or ocular tissues injected with AdcmvC3 and AdcmvGFP was determined by analyzing expression of glial fibrillary acidic protein (GFAP). Data show detectable activation of Muller cells in C3-injected eyes. Activation of Muller glia was specifically determined because GFAP was detected throughout the Muller cell, extending from the inner to outer limiting membrane (Figure 6 panel C).
  • ERGs are non-invasive and are used to evaluate function of specific layers or neurons of the eye including the photoreceptors (rods and cones), retina including inner retinal cells such as bipolar and amacrine cells, and the ganglion cells.
  • Abnormal ERGs indicate loss of ocular tissue function and the presence of a disease or negative condition.
  • EMGs electroretinograms
  • b-wave amplitudes were observed to be significantly reduced in C3 -injected eyes relative to GFP-injected or uninjected eyes, p ⁇ 0.05 ( Figure 7 left graphs).
  • B-wave amplitudes of C3-injected mice were reduced by 29.8 ⁇ 7.4% (OdB) and 21.5 ⁇ 7.1% (-10dB) compared to GFP-injected mice.
  • A- wave amplitudes also were observed to be significantly reduced in C3 -injected eyes at the higher light intensity (OdB) compared to both uninjected eyes or GFP-injected eyes, p ⁇ 0.05 (Figure 7 top row right graph).
  • Example 15 C3 -injected and GFP-injected retinas differ in expression of C3 and VEGF
  • VEGF Vascular endothelial grow factor
  • C3-contacted eyes and GFP-contacted eyes were examined for levels of C3 expression and VEGF expression eight days following injection of each adenovirus vector. It was observed that expression of C3 mRNA was significantly increased in both C3 -contacted eyes (57.0 ⁇ 11.8-fold, p ⁇ 0.01) and GFP-contacted eyes (6.2 ⁇ 1.2-fold, pO.01) compared to control uninjected eyes (Figure 8 top row left graph). Surprisingly, it was observed that C3-contacted eyes showed little or no change in VEGF expression (0.97 ⁇ 0.14-fold above uninjected eyes), and that GFP-contacted eyes had a significant increase in VEGF expression, 2.6 ⁇ 0.5-fold above uninjected eyes, p ⁇ 0.05 ( Figure 8 top row right graph).
  • Example 16 Deposition of membrane attack complex (MAC) in AdcmvC3 -injected retinas
  • AdcmvC3 An adenovirus vector was constructed carrying a gene that encoded C3 (AdcmvC3) and analysis of cells showed that the adenovirus had mediated delivery of C3 to murine ocular tissues including RPE.
  • AdcmvC3 induced significant negative functional and anatomical changes associated with ocular and retinal diseases such as AMD.
  • Example 17 Cell lines and primary RPE cell culture
  • HEPA lclc7 and 293 cell lines were obtained from American Type Culture Collection
  • Cell culture reagents were purchased from Invitrogen Life Technologies. Hepalclc? cells were maintained in aMEM/10%FBS. The 293 cells and human embryonic retinoblast cell line 911 (911) cells were maintained in DMEM/10%FBS (Fallaux et al. 1996 Hum Gene Ther7(2): 215-22).
  • RPE cells Primary mouse RPE cells were obtained from six week old to ten week old C57B1/6 mice. Eyes were enucleated, and the lens, cornea, and retina were removed to reveal the RPE cell layer. Eye-cups were incubated for 1 hour at 37°C in 200 ⁇ 1 of 0.25% trypsin/EDTA. RPE cells were pulled off in sheets and homogenized in 20 ⁇ 1 aMEM/10%FBS. The suspension was placed in the center of an eight well poly-D-lysine coated chamber slide (Becton Dickenson, Franklin Lakes, NJ) for ten minutes for cells to adhere to the plate, and an additional 130 ⁇ 1 ⁇ /10% FBS was added to each well. Cells were kept in a humidified incubator at 37°C with 5% CO 2 for three days prior to use.
  • El/E3-deleted adenovirus serotype 5 was used to construct a vector to express human CD46 (hCD46), or as a control, to express no transgene.
  • hCD46 human CD46
  • ATCC MCC-26544;
  • GenBank ID: BC030594; SEQ ID NO: 3 was excised from pBluescriptR using EcoRI and Sspl and inserted into pCAGEN using EcoRI and EcoRV between a CMV enhancer/chicken ⁇ - actin promoter (CAG) and a rabbit globin polyadenylation (pA) termination sequence.
  • CAG CMV enhancer/chicken ⁇ - actin promoter
  • pA rabbit globin polyadenylation
  • the pShuttle was recombined with Adeasy-1 then was linearized and transfected into 293 cells and virus was produced (Cashman et al. 2004. 324(1): 129-39). Following initial transfection virus was amplified in 911 cells. Viral purification was performed using the adenopure purification kit (Puresyn Inc., Malvern, PA) and viral titer determined at OD260 using a spectrophotometer then plaque-purified (Kumar-Singh et al. 2000 Methods Enzymol 316: 724-743). The hCD46 or pA expressing viruses are identified herein as AdCAGCD46 and AdCAGpA, respectively (See also Ramo et al. 2008 Invest Ophthalmol Vis Sci 49(9): 4126- 36, which is incorporated herein in its entirety).
  • Hepalclc7 cells were contacted (MOI 1000) for three days with AdCAGCD46 or AdCAGpA in aMEM/2%FBS.
  • FACS analysis the cells were collected by trypsinization (0.25%/EDTA), were re-suspended in lx PBS containing 0.5% FBS, centrifuged at
  • MAC staining of Hepalclc7 cells were plated into an eight-well chamber slide (Becton Dickinson) and transfected with either AdCAGCD46 or AdCAGpA for three days (MOI 1000). Each well was incubated with 25 ⁇ g/ml emmprin for 30 min. at 4°C followed by either 10% NHS or 10% HINHS in GVB 2 at 37°C for five minutes with MgEGTA to de- activate the classical pathway. Cells were washed twice with cold lx PBS, fixed for 15 minutes in 10% neutral buffered formalin, and stored in lx PBS. Cell lysis was determined using PI exclusion.
  • Cells were transfected with either AdCAGCD46 or AdCAGpA at an MOI of 1000 for three days in aMEM/10%> FBS.
  • the medium was removed and 50 ⁇ g/ml rat anti mouse emmprin added and cells were incubated at room temperature for one hour.
  • 50% NHS or 50% HINHS containing MgEGTA was added to the cells which were then incubated at 37°C for one hour.
  • Cells were washed three times in cold lx PBS, fixed in 10% NBF for 15 minutes, and stored in lx PBS at 4°C.
  • Murine subjects were injected subretinally with adenovirus vector, and were sacrificed eight days later using carbon dioxide and each of the lens, cornea, and retina were removed.
  • Each eye-cup was incubated in GVB containing 140 ⁇ g/ml rat anti mouse emmprin for one hour at 4°C.
  • Either 50% NHS or 50% HINHS was added directly to the GVB 2+ /emmprin mixture and eyecups were incubated for four minutes at 37°C.
  • MgEGTA was added to each eyecup sample which was then incubated at 37°C for an additional 56 minutes. Samples were washed three times in cold lx PBS and fixed overnight at 4°C in 4% paraformaldehyde.
  • Example 24 Expression of human CP46 from adenovirus vectors in human embryonic retinoblasts and mouse hepatocytes
  • Human CD46 is a transmembrane protein ranging in molecular weight from about 48 kDa to about 68 kDa (Post et al.1991 J Exp Med 174(1): 93-102). There are at least four human isoforms, each containing four conserved short consensus sequences (SCR), an O- glycosylated serine/threonine/proline rich area, a hydrophobic transmembrane portion, and an intracellular domain.
  • SCR conserved short consensus sequences
  • hCD46 human CD46
  • CAG chicken ⁇ actin promoter
  • AdCAGCD46 protected hepatocytes from the alternative complement pathway Human CD46 has a high affinity for binding C3b than for C4b, resulting in increased inhibition of convertase formation by the alternative pathway (Barilla-LaBarca et al. 2002 J Immunol 168(12): 6298-6304).
  • Hepalclc7 cells were pre-treated with either AdCAGCD46 or AdCAGpA for three days and then treated with either 25 ⁇ g/ml emmprin antibody followed by 10% NHS to activate both classical and alternative pathways, or MgEGTA-treated NHS for inhibition of the classical pathway (i.e., activation of the alternative complement only).
  • Cell lysis was determined by FACs analysis using propidium iodide (PI) uptake.
  • PI an intercalating agent that fluoresces when bound to DNA, was obtained from Fluka BioChemica (Buchs,
  • PI is excluded from viable cells and identifies non-living cells in a mixed population.
  • Hepalclc7 cells were contacted for three days with either AdCAGCD46 or AdCAGpA, and then the alternative pathway was activated by incubating the cells with 25 ⁇ g ml emmprin followed by incubation in 10% NHS and MgEGTA. Amount of human MAC deposition was determined using a monoclonal antibody specific to the MAC C5b9 complex.
  • Example 26 AdCAGCD46 protected mouse primary RPE cells from alternative pathway mediated MAC deposition
  • RPE cells are more resistant to MAC deposition than Hepalclc7 cells (Mizuno et al. 2001 Arthritis Rheum 44: 2425-2434), and require higher concentrations of antibody and serum.
  • RPE cells were pre-incubated with 50 ⁇ g ml emmprin followed by incubation with MgEGTA and 50% NHS. MAC deposition on RPE cells was detected by
  • ADCAGCD46-mediated reduction in MAC staining for RPE cells was less than that observed for hepatocytes. Whether the difference in MAC staining between RPE cells and hepatocytes was due to a lower infection rate of RPE cells and/or reduced expression of hCD46 was determined. The expression of hCD46 from AdCAGCD46 in mouse primary RPE cells was analyzed by IHC.
  • AdCAGCD46 contacted cells showed almost 100% transduction and showed strong expression of hCD46 on the cell membrane, and AdCAGpA contacted cells had no detectable hCH46 expression (Figure 13 panel A). Thus, decreased MAC staining for AdCAGCD46 contacted RPE cells compared to AdCAD46 contacted hepatocytes was not a result of differential serum treatment effects, adenovirus infection rates, or hCD46 expression.
  • Example 27 AdCAGCD46 expression of hCD46 on basal and lateral surfaces of mouse RPE cells
  • CD46 is expressed in the human eye on both the basal and lateral surfaces of RPE cells
  • adenovirus vectors were injected into the sub- retinal space of adult mice. Either ⁇ of an empty vector control mixture containing 9 parts AdCAGpA (total 5xl0 7 particles) and 1 part AdCAGGFP (total lxlO 6 particles), or CD46 vector containing 9 parts AdCAGCD6 (total 5xl0 7 particles) and 1 part AdCAGGFP (total lxlO 6 particles) was injected subretinally. Unless indicated otherwise, assays herein were performed in duplicate at least 3 times. Error bars represent SD from the mean. Significance was calculated using student's i-test. Eyes were harvested eight days following injection and were examined for expression of hCD46.
  • hCD46 expressed from an adenovirus on mouse RPE cells in vivo offered protection from human MAC deposited by the alternative pathway was determined.
  • AdCAGCD46 contacted eyes compared to AdCAGpA contacted eyes ( Figure 15 panel C).
  • MAC staining assays were performed on un-injected eyes and cross sections through the RPE were prepared. Data show that MAC was deposited almost exclusively on the apical surface of the RPE cells ( Figure 15 panel D).
  • AdCAGCD46 specifically protected cells from alternative pathway mediated MAC damage and allowed the classical pathway to function unhindered.
  • a recombinant adenovirus carrying a gene encoding CD 55 was engineered, and was tested to determine protection of ocular cells from MAC staining.
  • Example 29 AdCAG55 construction and expression on ocular cells
  • adenovirus vector expressing human CD55 (hCD55) regulated by a chicken ⁇ -actin promoter was constructed and is shown in Figure 16 panel A.
  • Recombinant adenovirus serotype 5 (Ad5) expressing human CD55 (hCD55) was generated by cloning a Sal I/Not I fragment from a plasmid containing the human CD55 cDNA (ATCC 5830488; GenBank ID: BC001288; SEQ ID NO: 5) into pCAGEN generating pCAGCD55.
  • An Spe VBamHl fragment containing the entire CD55 expression cassette was inserted into pShuttle (He et al. 1998 Proc Natl Acad Sci USA 95: 2509-2514).
  • the pShuttle was co-transformed with pAdEasyl into BJ5183 cells to rescue the plasmid pAdCAGCD55 (Ibid.).
  • the hCD55-expressing virus was rescued by transfection of 911 cells with Pad linearized pAdCAGCD55 and virus was purified using the adenopure purification kit (Puresyn, Inc.). Viral titer was determined using a spectrophotometer set at 260 ⁇ .
  • Control recombinant Ad5 vector expressing either GFP (AdCAGGFP) and a vector Ad5 devoid of a transgene (AdCAGpA) were also constructed (Ramo et al. 2008 Invest Ophthalmol Vis Sci 49: 4126-4136).
  • hCD55 human embryonic retinoblasts
  • AdCAGCD55 or AdCAGpA at a multiplicity of infection (MOI) of 1000 for 24 hours.
  • MOI multiplicity of infection
  • the cell lysate and medium were collected and electrophoresed through a 12.5% tris HCL pre-cast gel (Biorad) under reducing conditions.
  • PVDF polyvinylidene fluoride
  • the membrane was probed for hCD55 with a 1 : 1000 goat anti-human CD55 antibody (R&D
  • Example 30 Adeno virus-delivered hCD 55 protects mouse hepalclc7 cells from complement- mediated lysis
  • AdCAGCD55-mediated to protect murine cells from complement-mediated damage was determined with a human serum-mediated cell lysis assay.
  • Hepal clc7 cells were contacted with each of AdCAGCD55 and AdCAGpA at an MOI of 1000 for 65 hours in cc- MEM/10%FBS.
  • Cells were washed with lx PBS and dissociated with TrypLE Express (GIBCO). Cells were collected by centrifugation at 1200 RPM/4°C and were re-suspended in ice-cold gelatin veronal buffer with Ca and Mg (GVB ) (Complement Technology, Tyler, TX).
  • FIG. 17 panel B A representative set of FACS printouts is shown in Figure 17 panel B for uninjected control cells (left graph) and cells contacted with either AdCAGpa (middle graph) or AdCAGCD55 (right graph) treated with NHS or HI-NHS.
  • AdCAGpa middle graph
  • AdCAGCD55 right graph
  • FACS analysis data of AdCAGCD55-contacted cells were comparable for treatment with either NHS or HI-NHS, indicating similar low levels of protection from cell lysis.
  • Example 31 Adenovirus-delivered hCD55 protects mouse cells from human MAC deposition
  • Complement-mediated cell lysis is caused by the formation of MAC on the plasma membrane.
  • Data herein show that hCD55 regulates all three complement pathways by binding and accelerating the decay of C3 convertase in the classical and alternative pathways, preventing downstream MAC deposition on biological surfaces.
  • Hepalclc7 were transfected with AdCAGCD55 or AdCAGpA at an MOI of 1000 for
  • AdCAGCD55-contacted cells showed significantly less staining for MAC compared to both AdCAGpA-contacted cells ( Figure 18 panel A) and uninfected cells. Images were captured using a microscope and camera and grayscale images were analyzed using Image J 1.41x (National Institute of Health, USA) for degree of MAC immunofluorescence in arbitrary units. Representative areas were selected with the polygon selection tool and mean fluorescence intensity/pixel was measured. Background fluorescence in the hepatocyte images was subtracted from the mean fluorescence measurements.
  • AdCAGCD55 inhibited C3 convertase and successfully attenuated downstream formation and deposition of MAC on cell membranes. Quantification of fluorescence intensity showed a 67.1% decrease in MAC on murine cells expressing hCD55 relative to those not expressing hCD55 ( Figure 18 panel B). Mean fluorescence intensity for AdCAGpA-contacted cells was 449.9 ⁇ 38.1 and for AdCAGCD55-contacted cells was 148.0 ⁇ 13.1, respectively (pO.0001). Thus, human CD55 protected murine hepatocytes from human MAC deposition.
  • Example 32 Adenovirus-delivered hCD55 protects murine ocular tissues from MAC deposition
  • Subretinal injections were performed on mouse subject as described herein using the transcleral-transchoroidal approach with a 32-gauge needle attached to a 5 iL glass syringe. Flatmounts of eyecups from eyes were pretreated by injection with a mixture of control vectors
  • AdCAGpA and AdCAGGFP (9:1 ratio), or a mixture of vectors AdCAGCD55 and
  • AdCAGGFP (9:1 ratio). A total of 1 ⁇ of each mixture containing a total 1x10 8 viral particles was injected into each eye.
  • hCD55 is generally localized at the nerve fiber layer in healthy human retinas (Vogt et al. 2006 Eye Res 83: 834-840), and is not observed on the RPE.
  • Expression and localization of hCD55 was analyzed in murine RPE cells. Subjects were sacrificed with carbon dioxide six days after subretinal injection and eyes were enucleated. The lens and cornea were removed and the eye cup incubated in GVB 2+ containing 140 ⁇ / ⁇ goat anti-mouse emmprin (R&D Systems) for one hour at 4°C. 50% NHS or HI-NHS was added to the GVB 2+ /emmprin solution and incubated for 15 minutes at 37°C.
  • Eye cups with intact RPE were immunostained with 1 : 100 goat anti-human CD55 and a 1 :200 secondary Cy3-conjugated donkey anti-goat (Jackson ImmunoResearch Laboratories, INC.) prior to flat mounting to detect successful infection of RPE by AdCAGCD55 and expression of hCD55.
  • Immunostained eye cups were embedded in Tissue-Tek (Sakura Finetek, Torrance, CA) prior to collecting 14 ⁇ frozen sections on glass slides.
  • Fixed eye cups were immunostained with 1:100 mouse anti-human C5b-9, flat-mounted and cover-slipped to visualize MAC deposition on mouse RPE. Image J was utilized to quantify MAC deposition on mouse RPE.
  • AdCAGCD55 and AdCAGGFP or a mixture of AdCAGpA and AdCAGGFP was injected into the sub-retinal space of adult mice.
  • AdCAGGFP was included in the mixture to identify the site of injection. Eyes were harvested six days following injection and the cornea, lens, and retina, were removed. Each resulting eyecup was then contacted with an anti-mouse emmprin antibody in 50% NHS for 15 minutes. Emmprin was shown above to be necessary for activation of complement on RPE cells (see also Ramo et al. 2008 Invest Ophthalmol Vis Sci 49: 4126-4136). Eyecups were stained for human MAC with the antibody directed against C5b-9.
  • Eye cups contacted with a mixture of AdCAGCD55 and AdCAGGFP showed significantly less MAC deposition on the RPE within the region of injection ( Figure 20 panel A) than the uninjected region of the same eyecup. It was observed that contacting eyes with a mixture containing AdCADCD55 resulted in eyes having no difference in the intensity of MAC deposition within the injected area compared to the uninjected regions of the eyecup.
  • AdCAGCD55-contacted RPE cells were relatively healthy and displayed normal cell morphology to the extent discernible at the magnifications (Figure 20 panel A top row).
  • adenovirus mediated delivery of hCD55 to murine RPE protected ocular tissues against human complement.
  • Expression of hCD55 using the methods and compositions described herein is an effective therapy for retinal disorders such as AMD, and hCD55 is an effective therapeutic agent.
  • a STAC protein was constructed as described herein having the complement regulatory domains of each of CD46, CD55, and CD59 proteins ( Figure 21), with the native secretory signal of CD59 at the N-terminus.
  • the STAC protein gene sequence was designed using a modified sequence of each complement regulator in an attempt to minimize overall size of the protein by eliminating domains considered not useful for complement regulation.
  • the SCR domains of CD46 and CD55 are involved in complement control and the serine threonine proline (STP) region is heavily glycosylated (Medof et al. 1987 Proc Natl Acad Sci USA 84: 2007-2011; and Xing et al. 1994 Immunology 83: 122-127), and is involved in protein stability.
  • the transgene encoding the STAC protein was placed into the deleted El region of an adenovirus vector under control of a chicken beta actin promoter (AdCAGSTAC).
  • a GFP negative control vector (AdCAGGFP) was also constructed. See Figure 22 panel A.
  • Human RPE cells (ARPE19) were contacted with these vectors herein to determine whether AdCAGSTAC would be expressed and secreted, and have the relevant CD46, CD55, and CD59 domains.
  • the conditioned-media were collected and were probed by western blot using individual antibodies specific to extra-cellular domains of each of human CD59, human CD46, and human CD55 respectively (See Figure 21 panel B).
  • ARPE19 cells were contacted with either AdCAGSTAC or AdCAGGFP and media were collected to test the ability of STAC protein to prevent MAC deposition.
  • the media were transferred to a plate having hepa-lclc7 cells. Complement was then activated by adding 10% normal human serum (NHS) or 10% heat inactivated (Hi)-NHS to the cells.
  • NHS normal human serum
  • Hi heat inactivated
  • Figure 23 panel A shows a representative photomicrograph of hepa-lclc7 cells treated with NHS and media conditioned with AdCAGGFP or AdCAGSTAC, respectively.
  • the morphology of the cells in AdCAGGFP-conditioned media changed dramatically compared to cells in AdCAGSTAC-conditioned media as viewed using DIC microscopy and an overlay of MAC/DAPI staining due to complement-mediated action in this system.
  • the photomicrographs of the AdCAGGFP-conditioned media show distinct indicia of cell lysis caused by complement activation ( Figure 23 panel A top row). Most important, the morphology of the cells contacted with AdCAGSTAC-conditioned media remained relatively unchanged with (See Figure 23 panel A bottom row inset). Cells contacted with
  • AdCAGSTAC-conditioned media appeared undamaged, and defined cell boundaries and normal hexagonal morphology were observed.
  • the permeability of the hepa-lclc7 cells following exposure to NHS was analyzed to determine the physiological relevance of decreased MAC deposition resulting from STAC inhibition.
  • ARPE-19 cells were contacted with either AdCAGSTAC or AdCAGGFP for three days, and the media were collected. Hepalclc7 cells were then incubated with the media and NHS at a concentration of l%-3% (v/v). The amount of PI uptake in the cells was measured using FACS analysis.
  • IP injection of the adenovirus resulted in a high degree of liver transduction because the adenovirus specifically transduces the Glisson's capsule that surrounds the liver ( Figure 24 panel A).
  • an anti mouse PEC AM antibody 200 ⁇ g in a volume of 200 ⁇ was injected into the left ventricle, causing binding of antibody to the endothelial lining of blood vessels.
  • NHS 90% in PBS containing calcium and magnesium
  • Liver sections obtained from subjects injected with control vector AdCAGGFP showed significant negative changes in cell morphology (Figure 24 panel B, left photomicrograph) compared to liver sections from subjects injected with AdCAGSTAC ( Figure 24 panel C, left photomicrograph).
  • MAC staining was observed in the liver sections from subjects injected with AdCAGGFP ( Figure 24 panel B right photomicrograph).
  • Liver sections of subjects injected with AdCAGSTAC showed little or no MAC staining ( Figure 24 panel B right photomicrograph).
  • AdCAGSTAC were observed to have reduced average MAC staining compared to the livers of mice injected with AdCAGGFP ( Figure 24 panels B and C respectively).
  • A.dCA.GSTAC injected mice compared with those injected with AdCAGGFP (See Figure 24 panel C), an extent of reduction that is statistically significant.

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Abstract

L'invention concerne des systèmes, des compositions, des procédés et des kits pour identifier des agents thérapeutiques potentiels pour le traitement de maladies oculaires basées sur le complément. Les procédés et kits comprennent une protéine ou un dérivé de composant du complément 3 (C3) qui est en contact avec les cellules ou le tissu oculaires. Un autre mode de réalisation de la présente invention concerne le diagnostic et/ou le pronostic d'une maladie oculaire associée au complément. L'invention concerne des compositions, des procédés et des kits pour réguler ou traiter une affection associée au complément au moyen d'au moins un élément parmi la protéine CD46, la protéine CD55, et une protéine de terminateur du complément activé (STAC) soluble chimérique recombiné ou une source de protéine de STAC. La protéine de STAC comprend une séquence d'acides aminés incluant au moins deux éléments parmi une séquence d'acides aminés d'une protéine CD59, une séquence d'acides aminés dérivée d'une protéine CD46 et une séquence d'acides aminés dérivée d'une protéine CD55, comprenant éventuellement en outre un lieur pour relier les séquences d'acides aminés.
PCT/US2011/045933 2008-02-15 2011-07-29 Compositions, procédés et kits pour modéliser, diagnostiquer et traiter des troubles du complément Ceased WO2012016162A2 (fr)

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JPWO2020204149A1 (fr) * 2019-03-29 2020-10-08
EP3956347A4 (fr) * 2019-04-13 2023-09-20 National Centre For Cell Science Protéines chimériques daf-mcp, leur procédé de fabrication et utilisation de la protéine chimérique pour traiter des affections pathologiques impliquant le système du complément
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CA2666843C (fr) * 2006-10-20 2015-06-16 Celldex Therapeutics, Inc. Traitement de la degeneration maculaire due au vieillissement et d'autres maladies oculaires
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ES2478820T3 (es) * 2008-02-15 2014-07-23 Tufts University Tratamiento de degeneración macular

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JP7275095B2 (ja) 2014-08-28 2023-05-17 トラスティーズ オブ タフツ カレッジ 補体関連障害を処置するための組成物、方法およびキット
JP2017527558A (ja) * 2014-08-28 2017-09-21 タフツ・ユニバーシティ 補体関連障害を処置するための組成物、方法およびキット
JP2020019813A (ja) * 2014-08-28 2020-02-06 タフツ・ユニバーシティ 補体関連障害を処置するための組成物、方法およびキット
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US20170209535A1 (en) * 2014-08-28 2017-07-27 Tufts University Compositions, methods and kits for treating complement related disorders
EP3892291A1 (fr) * 2014-08-28 2021-10-13 Tufts University Compositions, procédés et kits pour le traitement des troubles liés au complément
US20210138031A1 (en) * 2014-08-28 2021-05-13 Trustees Of Tufts College Compositions, methods and kits for treating complement related disorders
JP2021059560A (ja) * 2014-08-28 2021-04-15 タフツ・ユニバーシティ 補体関連障害を処置するための組成物、方法およびキット
WO2016033444A1 (fr) * 2014-08-28 2016-03-03 Tufts University Compositions, procédés et kits pour le traitement des troubles liés au complément
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EP3950953A4 (fr) * 2019-03-29 2023-04-19 Public University Corporation Yokohama City University Procédé de criblage et procédé d'évaluation de toxicité
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EP3956347A4 (fr) * 2019-04-13 2023-09-20 National Centre For Cell Science Protéines chimériques daf-mcp, leur procédé de fabrication et utilisation de la protéine chimérique pour traiter des affections pathologiques impliquant le système du complément
WO2025034741A1 (fr) * 2023-08-07 2025-02-13 Avirmax Biopharma Inc. Compositions et procédés d'expression d'agents thérapeutiques

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