Classifications

• • 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
• • 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
  • 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
  • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
• • 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
• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0048—Eye, e.g. artificial tears
• • 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
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• the described invention generally relates to the use of gene therapy to treat retinal disease, including age-related macular degeneration (AMD) and geographic atrophy (GA).
• AMD age-related macular degeneration
• GA geographic atrophy
• Age-related macular degeneration is a slow and progressive disease of the macula. AMD is the leading cause of blindness in patients over 60 years of age in developed countries (Friedman, DS, et al. Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol. 2004;122:564-572; van Leeuwen, R, Klaver, CC, Vingerling, JR, Hofman, A, de Jong, PT. Epidemiology of age-related maculopathy: a review. Eur J Epidemiol. 2003;18:845-854).
• AMD Globally, AMD accounts for approximately 9% of all blindness and is predicted to affect approximately 196 million people by 2020 (Wong WL, Su X, Li X, et al. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Glob Health. 2014 Feb;2(2):el06-l 16). [5] The most significant loss of vision occurs in advanced AMD. Advanced AMD is divided into 2 categories: (1) 'wet' or exudative AMD; and (2) “dry” AMD, also referred to as geographic atrophy (GA).
• GA geographic atrophy
• VEGF vascular endothelial growth factor
• the terminal component of the complement pathway is formation of the membrane attack complex (MAC), a complex of proteins on the cell membrane which has cytolytic functions.
• MAC membrane attack complex
• Multiple lines of evidence suggest that the MAC complex is involved in the pathogenesis of AMD including 1) MAC deposition has been found to be increased in AMD patient samples and 2) individuals with a R95X nonsense mutation in C9, a key component of the MAC complex, results in a 4.7-fold decreased risk of wet AMD (Nichiguchi KM, Yasuma TR, Tomida D, et al. C9-R95X polymorphism in patients with neo vascular age-related macular degeneration. Invest Ophthalmol Vis Sci. 2012 Jan 31 ;53(1): 508-512).
• preventing formation of the MAC complex may serve as a therapeutic strategy for AMD.
• a natural inhibitor of the MAC complex is CD59, a glycosylphosphatidylinositol (GPI) anchored membrane bound protein which prevents formation of the MAC complex.
• GPI glycosylphosphatidylinositol
• Preclinical studies have shown that intravitreal injection of an AAV2 viral vector expressing a soluble form of CD59 (sCD59), could decrease MAC deposition and demonstrated efficacy in a rodent model of wet AMD (Cashman SM, Ramo K, Kumar-Singh R.
• a non membrane-targeted human soluble CD59 attenuates choroidal neovascularization in a model of age related macular degeneration. PLoS One. 2011 Apr 28;6(4):el9078) suggesting that expression of sCD59 can decrease MAC deposition in vivo, and may have therapeutic efficacy in AMD.
• the described invention provides a method for treating age- related macular degeneration (AMD) in a subject, the method comprising administering a pharmaceutical composition into an AMD-affected eye of a subject by ocular injection, wherein the composition comprises a nucleic acid encoding a soluble CD59 (sCD59) protein operably linked to a promoter, wherein the nucleic acid encoding sCD59 is packaged into a delivery vector and wherein the administering results in expression and secretion of the sCD59 protein by cells of the AMD-affected eye and the expression results in treatment of AMD-affected cells in the AMD-affected eye.
• AMD age- related macular degeneration
• the AMD is geographic atrophy (GA).
• the ocular injection is an intravitreal injection.
• the intravitreal injection is a single injection.
• the delivery vector is an adeno-associated virus (AAV) vector.
• AAV vector is AAV2.
• the promoter is a CAG promoter.
• the pharmaceutical composition comprises a dose of viral particles selected from the group consisting of about 3.56x10 10 DNAse-resistant particles (DRP), about 1.071x10 11 DRP, about 3.56xl0 n DRP and about 1.07xl0 12 DRP.
• DRP DNAse-resistant particles
• the described invention provides a method of regulating a complement activity disorder in a subject, the method comprising contacting an affected cell of the subject with a pharmaceutical composition comprising a vector carrying a nucleotide sequence encoding a recombinantly engineered human soluble CD59 (sCD59) protein operably linked to a promoter sequence causing expression of the protein in the affected cell, wherein the sCD59 protein comprises at least one mutation resulting in loss of function of glycosylphosphatidylinositol (GPI) anchoring domain resulting in loss of membrane targeting and observing a physiological indicium of the complement activity disorder after the contacting, in comparison to an abnormal amount of the physiological indicium observed prior to the contacting, wherein a decrease after the contacting compared prior to the contacting is a positive indication that the affected cell is treated.
• a pharmaceutical composition comprising a vector carrying a nucleotide sequence encoding a recombinantly engineered human soluble CD59 (sCD59) protein oper
• the complement activity disorder is GA.
• the contacting is by intravitreal injection.
• the intravitreal injection is a single injection.
• the affected cell is a retinal cell.
• the vector is AAV2.
• the physiological indicium is best corrected visual acuity (BCVA).
• BCVA is measured as mean change from baseline.
• the mean change from baseline is -7.100 letters.
• the pharmaceutical composition comprises a dose of viral particles selected from the group consisting of about 3.56x10 10 DNAse-resistant particles (DRP), about 1.071x10 11 DRP, about 3.56xl0 n DRP and about 1.07xl0 12 DRP.
• DRP DNAse-resistant particles
• the described inventio provides a method of treating a complement disorder comprising contacting a cell with a therapeutically effective amount of a pharmaceutical composition having as an active agent a nucleic acid encoding a human sCD59 protein or a source of expression of a human sCD59 protein comprising administering the pharmaceutical composition to a subject in need thereof.
• the complement disorder is GA.
• the contacting is by intravitreal injection.
• the intravitreal injection is a single injection.
• the affected cell is a retinal cell.
• the therapeutically effective amount is a dose of viral particles selected from the group consisting of about 3.56x10 10 DNAse-resistant particles (DRP), about 1.071x10 11 DRP, about 3.56xl0 n DRP and about 1.07xl0 12 DRP.
• DRP DNAse-resistant particles
• Figure 1 shows a schematic diagram depicting a dose-escalation study conducted to establish the safety of a single intravitreal injection of gene therapy vector AAVCAGsCD59 for the treatment of patients with advanced dry age-related macular degeneration (AMD) with geographic atrophy (GA).
• DRP DNAse-resistant particles.
• GA geographic atrophy
• mm lesion area
• Figure 3 shows a plot depicting least square (LS) mean with 95% confidence interval (CI) for Change from Baseline in Square root transformed of geographic atrophy (GA) lesion area (mm) measured by fundus autofluorescence (FAF) over time in Study Eye, labeled by Cohort.
• G geographic atrophy
• mm lesion area
• FAF fundus autofluorescence
• Figure 4 shows a plot depicting least square (LS) mean with 95% confidence interval (CI) for Change from Baseline in Square root transformed of geographic atrophy (GA) lesion area (mm) measured by fundus autofluorescence (FAF) over time in Study Eye, (Pooled Cohort).
• G geographic atrophy
• mm lesion area
• FAF fundus autofluorescence
• Figure 5 shows a plot depicting least square (LS) mean with 95% confidence interval (CI) for Change from Baseline in geographic atrophy (GA) lesion area (mm 2 ) measured by fundus autofluorescence (FAF) over time in Study Eye, labelled by Cohort.
• LS least square
• CI 95% confidence interval
• Figure 6 shows a plot depicting least square (LS) mean with 95% confidence interval (CI) for Change from Baseline in geographic atrophy (GA) lesion area (mm 2 ) measured by fundus autofluorescence (FAF) over time in Study Eye, (Pooled Cohort).
• LS least square
• CI 95% confidence interval
• compositions can be administered systemically either orally, buccally, parenterally, topically, by inhalation or insufflation (i.e., through the mouth or through the nose), or rectally in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired, or can be locally administered by means such as, but not limited to, injection, implantation, grafting, topical application, or parenterally.
• CD59 refers to a membrane-bound glycoprotein found associated with membranes of cells including both human hematopoietic and non-hematopoietic 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.
• CD59 protein inhibits assembly of functional membrane attack complexes (MACs) and thus protects cells from complement- mediated activation and/or lysis.
• MACs functional membrane attack complexes
• the protein structure of CD59 includes a single cysteine-rich domain, a hydrophobic core with three loops and a small fourth helical loop (Yu et al. 1997 Journal of Experimental Medicine 185(4): 745-753).
• Human CD59 includes 26 amino acids located at the C terminus, which specifies a signal sequence for attachment of a glycosyl phosphatidyl inositol anchor (GPI anchor) at amino acid asparagine at position 77.
• GPI anchor glycosyl phosphatidyl inositol anchor
• condition refers to a variety of health states and is meant to include disorders or diseases caused by any underlying mechanism or injury.
• disease or “disorder,” as used herein refers to an impairment of health or a condition of abnormal functioning.
• doctor unit form refers to a physically discrete unit of active agent appropriate for the patient to be treated.
• drug refers to a therapeutic agent or any substance used in the prevention, diagnosis, alleviation, treatment, or cure of disease.
• drusen refers to yellow deposits under the retina.
• a polypeptide functionally equivalent to SEQ ID NO: 3 may have a biologic activity, e.g., an inhibitory activity, kinetic parameters, salt inhibition, a cofactor-dependent activity, and/or a functional unit size that is substantially similar or identical to the expressed polypeptide of SEQ ID NO: 3.
• a biologic activity e.g., an inhibitory activity, kinetic parameters, salt inhibition, a cofactor-dependent activity, and/or a functional unit size that is substantially similar or identical to the expressed polypeptide of SEQ ID NO: 3.
• inhibitor and its various grammatical forms, including, but not limited to, “inhibiting” or “inhibition”, are used herein to refer to reducing the amount or rate of a process, to stopping the process entirely, or to decreasing, limiting, or blocking the action or function thereof.
• Inhibition can include a reduction or decrease of the amount, rate, action function, or process of a substance by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%.
• inhibitor refers to a second molecule that binds to a first molecule thereby decreasing the first molecule's activity.
• Enzyme inhibitors are molecules that bind to enzymes thereby decreasing enzyme activity. The binding of an inhibitor can stop a substrate from entering the active site of the enzyme and/or hinder the enzyme from catalyzing its reaction. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically, for example, by modifying key amino acid residues needed for enzymatic activity.
• reversible inhibitors bind non-covalently and produce different types of inhibition depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both. Enzyme inhibitors often are evaluated by their specificity and potency.
• injury refers to damage or harm to a structure or function of the body caused by an outside agent or force, which can be physical or chemical.
• membrane attack complex and “MAC” are used interchangeably herein to refer to an effector of the immune system comprising a complex of proteins typically formed on the surface of pathogen cell membranes as a result of activation of a host's complement system.
• Antibody-mediated complement activation leads to MAC deposition on the surface of infected cells, leading to pores that disrupt the cell membrane of the infected cells, resulting in cell lysis and death.
• the MAC is composed of complement components C5b, C6, C7, C8 and several C9 molecules.
• modify means to change, vary, adjust, temper, alter, affect or regulate to a certain measure or proportion in one or more particulars.
• modulate means to regulate, alter, adapt, or adjust to a certain measure or proportion.
• nucleic acid is used herein to refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).
• nucleotide is used herein to refer to a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups.
• the base is a derivative of purine or pyrimidine
• the sugar is the pentose deoxyribose or ribose.
• Nucleotides are the monomers of nucleic acids, with three or more bonding together in order to form a nucleic acid.
• Nucleotides are the structural units of RNA, DNA, and several cofactors, including, but not limited to, CoA, FAD, DMN, NAD, and NADP.
• Purines include adenine (A), and guanine (G); pyrimidines include cytosine (C), thymine (T), and uracil (U).
• A adenine
• G guanine
• pyrimidines include cytosine (C), thymine (T), and uracil (U).
• reference sequence refers to a sequence used as a basis for sequence comparison.
• a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
• comparison window refers to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• the comparison window is at least 20 contiguous nucleotides in length, and optionally can be at least 30 contiguous nucleotides in length, at least 40 contiguous nucleotides in length, at least 50 contiguous nucleotides in length, at least 100 contiguous nucleotides in length, or longer.
• a gap penalty typically is introduced and is subtracted from the number of matches.
• the BLAST family of programs which can be used for database similarity searches, includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences.
• sequence identity/similarity values refer to the value obtained using the BLAST 2.0 suite of programs using default parameters.
• Altschul et al. Nucleic Acids Res. 25:3389-3402 (1997).
• Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information.
• This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra).
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Set. USA 90:5873-5787 (1993)).
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• P(N) the smallest sum probability
• BLAST searches assume that proteins may be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids.
• Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar.
• a number of low-complexity filter programs may be employed to reduce such low-complexity alignments. Lor example, the SEG (Wooten and Lederhen, Comput. Chem., 17: 149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993)) low-complexity filters may be employed alone or in combination.
• sequence identity or “identity” in the context of two nucleic acid or polypeptide sequences is used herein to refer to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
• sequence identity When percentage of sequence identity is used in reference to proteins it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, i.e., where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
• Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Set., 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif, USA).
• the term “percentage of sequence identity” is used herein mean the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
• polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity and at least 95% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters.
• sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters.
• One of skill will recognize that these values may be adjusted appropriately to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.
• Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, or at least 70%, at least 80%, at least 90%, or at least 95%.
• nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. However, nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
• One indication that two nucleic acid sequences are substantially identical is that the polypeptide that the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
• substantially identical of protein sequences refers 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%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% identity.
• parenteral refers to introduction into the body by way of an injection (i.e., administration by injection), including, for example, intraocularly (also known as intravitreally) (i.e., an injection into the vitreous of the eye), subretinally (i.e., an injection into the subretinal space which exists between the photoreceptors of the retina and the retinal pigment epithelium (RPE) layer), subcutaneously (i.e., an injection beneath the skin), intramuscularly (i.e., an injection into a muscle); intravenously (i.e., an injection into a vein), intrathecally (i.e., an injection into the space around the spinal cord), intrasternal injection, or infusion techniques.
• intraocularly also known as intravitreally
• subretinally i.e., an injection into the subretinal space which exists between the photoreceptors of the retina and the retinal pigment epithelium (RPE) layer
• subcutaneously i.e.
• a parenterally administered composition of the described invention is delivered using a needle, e.g., a surgical needle.
• a surgical needle refers to any needle adapted for delivery of fluid (i.e., capable of flow) compositions of the described invention into a selected anatomical structure.
• injectable preparations such as sterile injectable aqueous or oleaginous suspensions, can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• the term “pharmaceutically acceptable carrier” refers to any substantially non-toxic carrier conventionally useable for administration of pharmaceuticals in which the isolated polypeptide of the present invention will remain stable and bioavailable.
• the pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the mammal being treated. It further should maintain the stability and bioavailability of an active agent.
• the pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with an active agent and other components of a given composition.
• pharmaceutically acceptable salt means those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio.
• polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
• the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
• the essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids.
• polypeptide and protein also are used herein in their broadest sense to refer to a sequence of subunit amino acids, amino acid analogs, or peptidomimetics. The subunits are linked by peptide bonds, except where noted.
• the polypeptides described herein may be chemically synthesized or recombinantly expressed. Polypeptides of the described invention also can be synthesized chemically. Synthetic polypeptides, prepared using the well-known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids.
• Amino acids used for peptide synthesis may be standard Boc (N-a-amino protected N-a-t-butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or the base-labile N-a-amino protected 9-fluorenylmethoxy carbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J. Org. Chem. 37:3403-3409). Both Fmoc and Boc N-a-amino protected amino acids can be obtained from Sigma, Cambridge Research Biochemical, or other chemical companies familiar to those skilled in the art.
• polypeptides can be synthesized with other N-a-protecting groups that are familiar to those skilled in this art.
• Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, for example, in Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, Ill.; Fields and Noble, 1990, Int. J. Pept. Protein Res. 35: 161-214, or using automated synthesizers.
• the polypeptides of the invention may comprise D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), a combination of D- and L-amino acids, and various “designer” amino acids (e.g., P-methyl amino acids, C-a- methyl amino acids, and N-a- methyl amino acids, etc.) to convey special properties.
• D-amino acids which are resistant to L-amino acid-specific proteases in vivo
• various “designer” amino acids e.g., P-methyl amino acids, C-a- methyl amino acids, and N-a- methyl amino acids, etc.
• synthetic amino acids include ornithine for lysine, and norleucine for leucine or isoleucine.
• the polypeptides can have peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties.
• a peptide may be generated that incorporates a reduced peptide bond, i.e., RI-CH2-NH- R2, where R1 and R2 are amino acid residues or sequences.
• a reduced peptide bond may be introduced as a dipeptide subunit.
• Such a polypeptide would be resistant to protease activity, and would possess an extended half-live in vivo. Accordingly, these terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
• the essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, the protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids.
• polypeptide also are inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. It will be appreciated, as is well known and as noted above, that polypeptides may not be entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslational events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well. In some embodiments, the peptide is of any length or size.
• prefferve refers to maintaining, keeping safe from harm or injury, protecting, sparing or maintaining function.
• prevent refers to the keeping, hindering or averting of an event, act, or action from happening, occurring or arising.
• the term "recombinant” refers to a cell or vector that has been modified by the introduction of a heterologous nucleic acid or the cell that is derived from a cell so modified.
• Recombinant cells express genes that are not found in identical form within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of deliberate human intervention.
• the term "recombinant” as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation transduction/transposition) such as those occurring without deliberate human intervention.
• recombinant expression cassette refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a host cell.
• the recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, virus, or nucleic acid fragment.
• the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, a promoter, and a transcription termination signal such as a poly-A signal.
• the term "recombinant host” refers to any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned genes, or gene of interest, in the chromosome or genome of the host cell.
• recombinant protein refers to a protein produced by genetic engineering, for example, by manipulation of genetically modified organisms such as microorganisms.
• the term “reduce” or “reducing” as used herein refers to the limiting of an occurrence of a disorder in individuals at risk of developing the disorder.
• the term “regulate” as used herein means to control or maintain a process, function or mechanism, for example, a biological process.
• solution refers to a homogeneous mixture of two or more substances. It is frequently, though not necessarily, a liquid. In a solution, the molecules of the solute (or dissolved substance) are uniformly distributed among those of the solvent.
• soluble CD59 refers to a CD59 amino acid sequence that lacks a glycosylphosphatidylinositol (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 glycosylphosphatidylinositol
• stimulation in any of its grammatical forms as used herein refers to inducing activation or increasing activity.
• suspension refers to a dispersion (mixture) in which a finely- divided species is combined with another species, with the former being so finely divided and mixed that it doesn't rapidly settle out.
• the most common suspensions are those of solids in liquid.
• the terms “subject” or “individual” or “patient” or “participant” are used interchangeably to refer to a member of an animal species of mammalian origin, including humans.
• the term “a subject in need thereof’ is used to refer to a subject having, or at risk of progression to heart failure, including a subject having an AMI that leads to a disease manifestation of left ventricular remodeling.
• subject in need of such treatment refers to a patient who suffers from a disease, disorder, condition, or pathological process.
• the term “subject in need of such treatment” also is used to refer to a patient who (i) will be administered at least one dose of the adenovirus vector construct expressing human soluble CD59 of the described invention; (ii) is receiving at least one dose of the adenovirus vector construct expressing human soluble CD59 of the described invention; or (iii) has received at least one dose of the adenovirus vector construct expressing human soluble CD59 of the described invention, unless the context and usage of the phrase indicates otherwise.
• substantially similar means that a first value, aspect, trait, feature, number, or amount is of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of a second value, aspect, trait, feature, number, or amount.
• a polypeptide substantially similar to (SEQ ID NO: 3) would have at least 70% amino acid sequence identity, at least 75% amino acid sequence identity, at least 80% amino acid sequence identity, at least 90% sequence identity, or at least 95% amino acid sequence identity to amino acid sequence (SEQ ID NO: 3).
• substitution is used herein to refer to a situation in which a base or bases are exchanged for another base or bases in a DNA sequence. Substitutions may be synonymous substitutions or nonsynonymous substitutions. As used herein, “synonymous substitutions” refer to substitutions of one base for another in an exon of a gene coding for a protein, such that the amino acid sequence produced is not modified. The term “nonsynonymous substitutions” as used herein refer to substitutions of one base for another in an exon of a gene coding for a protein, such that the amino acid sequence produced is modified.
• symptom refers to a phenomenon that arises from and accompanies a particular disease or disorder and serves as an indication of it.
• therapeutic agent refers to a drug, molecule, nucleic acid, protein, metabolite, composition or other substance that provides a therapeutic effect.
• active refers to the ingredient, component or constituent of the compositions of the described invention responsible for the intended therapeutic effect.
• therapeutic agent and “active agent” are used interchangeably herein.
• therapeutic component refers to a therapeutically effective dosage (i.e., dose and frequency of administration) that eliminates, reduces, or prevents the progression of a particular disease manifestation in a percentage of a population.
• An example of a commonly used therapeutic component is the ED50 which describes the dose in a particular dosage that is therapeutically effective for a particular disease manifestation in 50% of a population.
• terapéuticaally effective amount refers to an amount that is sufficient to provide the intended benefit of treatment.
• An effective amount of the active agent(s) that can be employed according to the described invention generally ranges from about 1x10 10 DNAse-resistant particles (DRP) to 1x10 12 DRP per dose.
• DRP DNAse-resistant particles
• dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular active agent employed. Thus the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods.
• compositions of the described invention includes prophylactic or preventative amounts of the compositions of the described invention.
• pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and/or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes presenting during development of the disease, disorder or condition. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to some medical judgment.
• dose and “dosage” are used interchangeably herein.
• therapeutic effect refers to a consequence of treatment, the results of which are judged to be desirable and beneficial.
• a therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation.
• a therapeutic effect can also include, directly or indirectly, the arrest, reduction or elimination of the progression of a disease manifestation.
• the therapeutically effective amount may be initially determined from preliminary in vitro studies and/or animal models.
• a therapeutically effective dose may also be determined from human data.
• the applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan.
• Pharmacokinetic principles provide a basis for modifying a dosage regimen to obtain a desired degree of therapeutic efficacy with a minimum of unacceptable adverse effects. In situations where the drug's plasma concentration can be measured and related to the therapeutic window, additional guidance for dosage modification can be obtained.
• Drug products are considered to be pharmaceutical equivalents if they contain the same active ingredients and are identical in strength or concentration, dosage form, and route of administration. Two pharmaceutically equivalent drug products are considered to be bioequivalent when the rates and extents of bioavailability of the active ingredient in the two products are not significantly different under suitable test conditions.
• the term "therapeutic window” refers to a concentration range that provides therapeutic efficacy without unacceptable toxicity. Following administration of a dose of a drug, its effects usually show a characteristic temporal pattern. A lag period is present before the drug concentration exceeds the minimum effective concentration (“MEC") for the desired effect. Following onset of the response, the intensity of the effect increases as the drug continues to be absorbed and distributed. This reaches a peak, after which drug elimination results in a decline in the effect's intensity that disappears when the drug concentration falls back below the MEC. Accordingly, the duration of a drug's action is determined by the time period over which concentrations exceed the MEC. The therapeutic goal is to obtain and maintain concentrations within the therapeutic window for the desired response with a minimum of toxicity.
• Drug response below the MEC for the desired effect will be subtherapeutic, whereas for an adverse effect, the probability of toxicity will increase above the MEC.
• Increasing or decreasing drug dosage shifts the response curve up or down the intensity scale and is used to modulate the drug's effect.
• Increasing the dose also prolongs a drug's duration of action but at the risk of increasing the likelihood of adverse effects. Accordingly, unless the drug is nontoxic, increasing the dose is not a useful strategy for extending a drug's duration of action.
• treat or “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms.
• Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).
• variants are used herein to refer to nucleotide or polypeptide sequences with substantial identity to a reference nucleotide or polypeptide sequence.
• the differences in the sequences may be the result of changes, either naturally or by design, in sequence or structure. Natural changes may arise during the course of normal replication or duplication in nature of the particular nucleic acid sequence. Designed changes may be specifically designed and introduced into the sequence for specific purposes. Such specific changes may be made in vitro using a variety of mutagenesis techniques. Such sequence variants generated specifically may be referred to as “mutants” or “derivatives” of the original sequence.
• a skilled artisan likewise can produce polypeptide variants of polypeptide SEQ ID NO: 3 having single or multiple amino acid substitutions, deletions, additions or replacements, but functionally equivalent to SEQ ID NO: 3.
• These variants may include inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids; (b) variants in which one or more amino acids are added; (c) variants in which at least one amino acid includes a substituent group; (d) variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at conserved or non-conserved positions; and (d) variants in which a target protein is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the target protein, for example, an epitope for an antibody.
• mutation refers to a change of the DNA sequence within a gene or chromosome of an organism resulting in the creation of a new character or trait not found in the parental type, or the process by which such a change occurs in a chromosome, either through an alteration in the nucleotide sequence of the DNA coding for a gene or through a change in the physical arrangement of a chromosome.
• Three mechanisms of mutation include substitution (exchange of one base pair for another), addition (the insertion of one or more bases into a sequence), and deletion (loss of one or more base pairs).
• the term “vector” as used herein refers to a carrier that is genetically engineered to deliver a gene to a cell.
• the term “viral vector” as used herein refers to a virus used as a vector to deliver a gene of interest by infecting a cell. Such viruses are modified so they cannot cause disease when used in humans. Types of viruses include, but are not limited to, retroviruses, which integrate their genetic material (including the gene interest) into a chromosome in a cell, and adenoviruses, which introduce their DNA (including the gene of interest) into the nucleus of a cell without integrating the genetic material into a chromosome.
• the term “vehicle” as used herein refers to a substance that facilitates the use of a drug or other material that is mixed with it.
• the described invention provides a nucleotide sequence encoding human soluble CD59 (sCD59) protein.
• the nucleotide sequence is a complementary DNA (cDNA) sequence.
• cDNA sequences encoding human CD59 are known in the art. For example, cDNA sequences have been reported by Sawada, R. et al. 1989 Nucleic Acids Res 17(16): 6728 and are available from the American Type Tissue Culture Collection (ATCC, Manassas, Va.). A cDNA encoding CD59 has also 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).
• YT human T-cell leukemia
• K562 human erythroleukemia
• the human sCD59 lacks the primary amino acid sequence for a functional glycosylphosphatidylinositol (GPI) anchor.
• the human sCD59 comprises a modified GPI anchor domain amino acid sequence that is functionally defective and lacks the ability to target a membrane.
• the modified GPI anchor domain amino acid sequence comprises a variation. Such variations include, but are not limited to, substitution and deletion of nucleic acids encoding amino acids at omega positions used to reduce or eliminate the attachment of the GPI anchor or reduce or eliminate the effective functionality of the GPI anchor. Omega amino acids are amino acids to which GPI is transferred.
• such a variation includes, but is not limited to, 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 may include substituting the omega residue with another amino acid, such as a glycine for a tyrosine.
• the human sCD59 protein of the described invention includes conservative sequence modifications.
• Conservative sequence modifications are amino acid modifications that do not significantly affect or alter the characteristics of the human sCD59 protein containing the amino acid sequence, i.e., amino acid sequences of sCD59 that present these side chains at the same relative positions will function in a manner similar to human sCD59.
• conservative modifications include amino acid substitutions, additions and deletions.
• Methods of modifying amino acid sequences are known in the art e.g., site-directed mutagenesis or PCR based mutagenesis.
• the human sCD59 protein of the described invention comprises conservative amino acid substitutions.
• Conservative amino acid substitutions are ones in which an 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.
• 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
• aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
• the human sCD59 amino acid sequence is an amino acid sequence that is substantially identical to that of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 70% identical to that of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 75% identical to that of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 80% identical to that of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 90% identical to that of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 95% identical to that of the wild-type sequence.
• the human sCD59 comprises SEQ ID NO: 3.
• the human sCD59 consists essentially of SEQ ID NO: 3.
• the human sCD59 is SEQ ID NO: 3. [110] According to some embodiments, the human sCD59 of the described invention is a recombinant protein.
• a variety of commercially available expression vector/host systems are useful to contain and express a CD59 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., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.
• Types of cells used in recombinant protein expression include, but are not limited to, prokaryotic and eukaryotic cells.
• Prokaryotic cells include, but are not limited to, bacterial cells.
• a non-limiting example of a bacterial cell includes Escherichia coli.
• Eukaryotic cells include, but are not limited to, mammalian, insect, yeast and algae cells.
• Non-limiting examples of mammalian cells include human embryonic kidney cells (e.g., HEK293, HEK293T), baby hamster kidney cells (e.g., BHK21), Chinese hamster ovary (CHO) cells, mouse myeloma cells (e.g., NSO) and murine non-producing hybridoma cells (e.g., SP2/O-Agl4).
• Non-limiting examples of insect cells include, Spodoptera frugiperda pupa ovarian cells (e.g., Sf9, Sf21).
• a non-limiting example of a yeast cell includes Saccharomyces cerevisiae.
• a non-limiting example of an algae cell includes Chlamydomonas reinhardtii.
• Methods for expressing recombinant proteins also include cell-free systems.
• Cell-free protein expression includes the in vitro production of recombinant proteins in solution (i.e., cell lysate) using biomolecular translation machinery extracted from cells.
• the human sCD59 is a synthetic protein.
• Methods of preparing synthetic proteins are well-known in the art. See, for example, Peptide Synthesis Protocols, Methods in Molecular Biology, vol. 35, Pennington, M. W. and Dunn, B. M., 1995, XII, Humana Press, Inc. Totowa, New Jersey.
• Synthetic proteins, prepared using well-known techniques such as solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids.
• Amino acids used for peptide synthesis may be standard Boc amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc.
• the nucleotide sequence encoding human sCD59 is used to construct an expression vector.
• the described invention provides a human sCD59 expression construct.
• Methods used to construct expression vectors are well known to those skilled in the art. For example, such methods can be used to construct expression vectors containing the nucleotide sequence encoding the human sCD59 protein operably linked to appropriate transcriptional and translational control elements. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination or genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989.
• the nucleotide sequence encoding human sCD59 is operably linked to a promoter.
• the promoter is a constitutive promoter.
• the promoter is a cell cycle-specific promoter.
• the promoter is a ubiquitous promoter.
• the promoter is a tissue-specific promoters. Examples of tissue-specific promoters include, but are not limited to, human rhodopsin kinase (hRK) promoter and retinal pigmemt epithelium specific promoter (e.g., RPE65 promoter).
• the promoter is a metabolically regulated promoter.
• the promoter is an inducible promoter.
• the promote is a hybrid promoter.
• a non-limiting example of a hybrid promoter is cytomegalovirus (CMV) early enhancer element/the first exon and the first intron of chicken beta-actin gene/the splice acceptor of the rabbit beta-globin gene (CAG).
• CMV cytomegalovirus
• Non-limiting examples of promoters are shown in Evans et al. U.S. Pat. No. 6,677,311 Bl issued Jan. 13, 2004; Clark et al. U.S. Pat. No. 7,109,029 B2 issued Sep. 19, 2006; and Hallenbeck et al. U.S. Pat. No. 5,998,205 issued Dec. 7, 1999, each of which is incorporated herein by reference in its entirety.
• the nucleotide sequence encoding human sCD59 operably linked to a promoter is packaged into a delivery vector.
• the human sCD59 expression construct is packaged into a delivery vector.
• the delivery vector is a virus vector.
• Virus vectors include, but are not limited to, adenovirus vectors, lentivirus vectors, adeno- associated virus (AAV) vectors, and helper-dependent adenovirus vectors.
• adenovirus vectors include, but are not limited to, adenovirus vectors, lentivirus vectors, adeno- associated virus (AAV) vectors, and helper-dependent adenovirus vectors.
• Adenovirus vectors are commercially available from American Type Tissue Culture Collection (Manassas, Va.). Methods of constructing adenovirus vectors and using adenovirus vectors are described in Klein et al. 2007 Ophthalmology 114: 253-262, and van Lecuwen et al. 2003 Eur. J. Epidemiol. 18: 845-854. Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al. 1991 Gene, 101: 195-202) and vaccine development (Graham et al. 1991 Methods in Molecular Biology: Gene Transfer and Expression Protocols 7, (Murray, Ed.), Humana Press, Clifton, N.J., 109-128). Further, recombinant adenovirus vectors are used for gene therapy (Wu et al. U.S. Pat. No. 7,235,391 issued Jun. 26, 2007 which is incorporated herein by reference in its entirety).
• Recombinant adenovirus vectors are generated, for example, from homologous recombination between a shuttle vector and a provirus vector (Wu et al., U.S. Pat. No. 7,235,391 issued Jun. 26, 2007).
• the adenovirus vectors used herein are replication defective.
• the adenovirus vectors are conditionally defective, lacking adenovirus El region.
• a polynucleotide encoding a protein of interest, human sCD59 for example, is introduced at the position from which the El -coding sequences have been removed.
• the polynucleotide encoding the protein of interest e.g., human sCD59
• Defective adenovirus vectors can be generated and propagated using a helper cell line.
• Helper cell lines may be derived from human cells such as, 293 human embryonic kidney cells (HEK293), 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 adenovirus, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. Generation and propagation of these replication defective adenovirus vectors using a helper cell line is described in Graham et al 1977 J. Gen. Virol. 36: 59-72.
• Lentiviral packaging vectors are commercially available from Invitrogen Corporation (Carlsbad Calif).
• An HIV-based packaging system for the production of lentiviral vectors is prepared using constructs described in Naldini et al. 1996 Science 272: 263-267; Zufferey et al. 1997 Nature Biotechnol. 15: 871-875; and Dull et al. 1998 J. Virol. 72: 8463-8471.
• a number of vector constructs are available to be packaged using a system, based on third-generation lentiviral SIN vector backbone (Dull et al. 1998 J. Virol. 72: 8463-8471).
• 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 green fluorescent protein (GFP) driven by the CMV promoter, and the woodchuck hepatitis virus PRE element, which appears to enhance nuclear export.
• the GFP marker gene allows quantitation of transfection or transduction efficiency by direct observation of UV fluorescence microscopy or flow cytometry (Kafri et al. 1997 Nature Genet. 17: 314-317; and Sakoda et al. 1999 J. Mol. Cell. Cardiol. 31 : 2037-2047).
• Retroviral nucleic acids to construct a retroviral vector containing a gene of interest (e.g., gene that encodes for human sCD59 protein) and packaging cells is accomplished using techniques known in the art (See, e.g., Ausubel, et al., 1992, Volume 1, Section III (units 9.10.1-9.14.3); Sambrook, et al., 1989. Molecular Cloning: A Laboratory Manual. Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Miller, et al., Biotechniques. 7:981-990, 1989; Eglitis, et al., Biotechniques. 6:608-614, 1988; U.S. Pat. Nos.
• a retroviral vector can be constructed and packaged into non-infectious transducing viral particles (virions) using an amphotropic packaging system. Examples of such packaging systems are described in Miller et al. 1986 Mol. Cell Biol. 6 : 2895-2902; Markowitz et al. 1988 J. Virol. 62: 1120-1124; Cosset et al. 1990 J. Virol. 64: 1070-1078; U.S. Pat. Nos.
• producer cells can be accomplished by introducing retroviral vectors into the packaging cells. Examples of such retroviral vectors are found in, for example, Korman et al. 1987 Proc. Natl. Acad. Sci. USA. 84: 2150-2154; Morgenstern et al. 1990 Nucleic Acids Res.
• Herpesvirus packaging vectors are commercially available from Invitrogen Corporation, (Carlsbad, Calif).
• Exemplary herpesviruses include, but are not limited to, an a-herpesvirus, such as Varicella-Zoster virus or pseudorabies virus; a herpes simplex virus such as HSV-1 or HSV-2; or 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 human sCD59 nucleotide or polynucleotide sequence, in the absence of a helper virus that is capable to most herpesviruses is described in Fraefel et al. (U.S. Pat. No. 5,998,208, issued Dec. 7, 1999 which is incorporated by reference in its entirety).
• the herpesvirus DNA vector can be constructed using techniques known 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. 1989 Science 244: 1307- 1313).
• cosmids Evans, et al., Gene 79, 9-20, 1989
• yeast artificial chromosomes YACS
• E. coli F element plasmids O'Conner et al. 1989 Science 244: 1307- 1313.
• 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.
• Epstein-Barr virus Varicella-Zoster virus
• pseudorabies virus pseudorabies virus
• HSV-1 herpesviruses
• Adeno-associated virus is a dependent parvovirus in that it depends on coinfection with another virus (either adenovirus or a member of the herpes virus family) to undergo a productive infection in cultured cells (Muzyczka 1992 Curr. Top. Microbiol. Immunol., 158:97 129).
• recombinant AAV (rAAV) virus can be made by cotransfecting a plasmid containing a gene of interest (e.g., human sCD59 gene), flanked by the two AAV terminal repeats (McLaughlin et al. 1988 J. Virol., 62(6): 1963-1973; Samulski et al. 1989 J.
• Virol, 63: 3822-3828 and an expression plasmid containing wild-type AAV coding sequences without terminal repeats.
• Cells are also contacted or transfected with adenovirus or plasmids carrying the adenovirus genes required for AAV helper function.
• AAVs are innately nonpathogenic, poorly immunogenic, and broadly tropic, making them attractive gene delivery candidates for virus-based gene therapies.
• Most naturally occurring AAVs utilize glycan moieties for initial attachment to a cell surface, and these interactions have been well characterized for a number of serotypes.
• the interacting glycan moieties identified include AAV serotype 2 (AAV2), AAV3, AAV6 and AAV8; N- terminal galactose for AAV9; and specific N- or O-linked sialic acid moieties for AAV1, -4, -5, and -6.
• Serotypes differ by the types of cells they infect, making AAV a very useful system for preferentially transducing specific cell types.
• Adeno-associated virus (AAV) packaging vectors are commercially available from GeneDetect (Auckland, New Zealand). AAV has a broad host range for infectivity (Tratschin et al. 1984 Mol. Cell. Biol. 4: 2072-2081; Laughlin et al. 1986 J. Virol., 60(2): 515-524; Lebkowski et al. 1988 Mol. Cell. Biol. 8(10): 3988-3996; McLaughlin et al. 1988 J. Virol. 62(6):1963- 1973).
• the nucleotide sequence encoding human sCD59 operably linked to a promoter is packaged into an adeno-associated virus (AAV) vector.
• AAV vector is AAV2.
• the AAV vector is AAV5.
• the AAV vector is AAV8.
• the human sCD59 expression vector is packaged into an adeno-associated virus (AAV) vector.
• AAV vector is AAV2.
• the AAV vector is AAV5.
• the AAV vector is AAV8.
• the nucleotide sequence encoding human sCD59 is packaged between inverted terminal repeat (ITR) sequences within an AAV vector.
• the nucleotide sequence encoding human sCD59 operably linked to a promoter is packaged between inverted terminal repeat (ITR) sequences within an AAV vector.
• the human sCD59 expression vector is packaged between inverted terminal repeat (ITR) sequences within an AAV vector.
• the ITR sequences are AAV2 sequences.
• the ITR sequences are AAV5 sequences.
• the ITR sequences are AAV8 sequences.
• the AAV vector is a hybrid vector.
• Hybrid vectors contain ITR sequences from one AAV serotype and a capsid protein from a different AAV serotype.
• the hybrid vector comprises ITR sequences from AAV2 and a capsid protein from AAV5 (AAV2/5).
• the hybrid vector comprises ITR sequences from AAV2 and a capsid protein from AAV8 (AAV2/8).
• the hybrid vector comprises ITR sequences from AAV5 and a capsid protein from AAV2 (AAV5/2).
• the hybrid vector comprises ITR sequences from AAV5 and a capsid protein from AAV8 (AAV5/8). According to some embodiments, the hybrid vector comprises ITR sequences from AAV8 and a capsid protein from AAV2 (AAV8/2). According to some embodiments, the hybrid vector comprises ITR sequences from AAV8 and a capsid protein from AAV5 (AAV8/5).
• the delivery vectors are non-viral vectors.
• the delivery vectors are 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, but are not limited to, liposomes, peptides, nanoparticles, emulsions, or encapsulated two or more phase systems or other suitable preparation.
• the described invention provides a non-viral vector with nucleic acid that is loaded and contacted to a tissue or cell.
• a liposome containing naked DNA encoding a human sCD59 protein having a modified GPI anchor that does not target a membrane, or a gene encoding a human sCD59 protein having no GPI anchor 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.
• the described invention provides a pharmaceutical composition.
• the pharmaceutical composition comprises a human sCD59 protein comprising a full-length nucleic acid of CD59 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 human sCD59 protein 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 GPI anchor location, such that the protein is unable to attach to a membrane of a cell.
• the described invention provides a pharmaceutical composition that comprises a CD59-encoding nucleic acid or a source of human sCD59 protein expression.
• the CD59 protein includes a membrane-independent (i.e., soluble) CD59 protein.
• the pharmaceutical composition is compounded as an ophthalmologic formulation for administration to the eye.
• the pharmaceutical composition is compounded to enhance delivery to the fundus.
• the pharmaceutical composition is compounded to provide sustained release locally to the retina.
• the pharmaceutical composition is formulated to provide effective treatment of vessels and/or tissue involved in ocular diseases.
• the ocular disease is age-related macular degeneration (AMD).
• AMD age-related macular degeneration
• the AMD is wet or exudative AMD.
• the AMD is dry AMD or geographic atrophy (GA).
• the pharmaceutical composition of the described invention is formulated sufficiently pure for administration to a human subject, e.g., to the eye of a human subject.
• the pharmaceutical composition includes one or more additional therapeutic agent(s).
• the additional therapeutic agent or agents are selected from the group consisting of growth factors, antiinflammatory agents, vasopressor agents, including, but not limited to, nitric oxide and calcium channel blockers, collagenase inhibitors, steroids (e.g., prednisolone), matrix metalloproteinase inhibitors, ascorbates, angiotensin H, angiotensin III, calreticulin, tetracyclines, fibronectin, collagen, thrombospondin, transforming growth factors (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), epidermal growth factor (EGF), platelet derived growth factor (PDGF), neu differentiation factor (NDF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), heparin-binding EGF (HBEGF), thrombospondins, von Willebrand Factor
• TGF
• the additional therapeutic agent or agents include, without limitation, anti-tumor, antiviral, antibacterial, anti- mycobacterial, anti-fungal, anti-proliferative or anti-apoptotic agents.
• Therapeutic agents that are included in the pharmaceutical composition of the described invention are well known in the art. See for example, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman, et al., eds., McGraw-Hill, 1996, the contents of which are herein incorporated by reference herein.
• the additional therapeutic agent or agents is a compound, composition, biologic or the like. According to some embodiments, the additional therapeutic agent or agents potentiate, stabilize, synergize or substitute for the ability of human sCD59 protein to protect cells from MAC deposition. According to some embodiments, the additional therapeutic agent or agents are provided at the same time as the pharmaceutical composition that comprises the human sCD59 protein. According to some embodiments, the additional therapeutic agent or agents are provided after the pharmaceutical composition that comprises the human sCD59 protein. According to some embodiments, the additional therapeutic agent or agents are provided before the pharmaceutical composition that comprises the human sCD59 protein. According to some embodiments, the additional therapeutic agent or agents are used to treat the same, a concurrent or a related symptom, condition or disease.
• the pharmaceutical composition of the described invention comprises a pharmaceutically acceptable carrier.
• Pharmaceutical acceptable carriers include, but are not limited to, 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 carriers 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, according to the judgment of the formulator.
• sugars such as glucose and sucrose
• the described invention provides a method for treating a complement disorder (e.g., AMD).
• the method comprises contacting cells or tissue with a pharmaceutical composition comprising a source of human sCD59 protein.
• the pharmaceutical composition comprises a nucleotide sequence encoding human sCD59 operably linked to a promoter.
• the pharmaceutical composition comprises a human sCD59 expression construct.
• the pharmaceutical composition comprises a nucleotide sequence encoding human sCD59 operably linked to a promoter that is packaged into a delivery vector.
• the pharmaceutical composition comprises a human sCD59 expression construct that is packaged into a delivery vector.
• the human sCD59 protein is administered as a recombinant protein.
• CD59 plasma membranes of cells are normally protected from the effects of complement by cell-surface proteins, e.g., CD59, that specifically inhibit activation of the C5b-9 pore upon C9 complement protein binding to membrane C5b-8
• CD59 competes with C9 complement protein for binding to C8 complement protein in the C5b-8 complex, thereby decreasing or preventing the formation of the C5b-9 membrane attack complex. CD59 thus acts to reduce both cell activation and cell lysis by terminal complement MACs.
• Diseases associated with uncontrolled complement activity include: bacterial infection such as with Haemophilus influenza, Streptococcus pnemoniae, Neisseria meningitidis; angiodema; renal disease for example atypical haemolytic uremic syndrome; paroxysmal nocturnal hemoglobinuria; systemic lupus erythematosus; central nervous system diseases including Alzheimer's disease, Huntington's disease and diseases of the retina including, but not limited to, age-related macular degeneration (AMD).
• AMD age-related macular degeneration
• human soluble CD59 is effective to inhibit MAC formation.
• MAC formation is inhibited by delivering into a cell a vector containing a human sCD59-encoding nucleic acid.
• human sCD59 is effective to treat AMD.
• AMD is treated by delivering into a cell a vector containing a human sCD59-encoding nucleic acid.
• human sCD59 is effective to prevent the onset of AMD.
• the onset of AMD is prevented by delivering into a cell a vector containing a human sCD59-encoding nucleic acid.
• human sCD59 is effective to prevent the progression of AMD.
• the progression of AMD is prevented by delivering into a cell a vector containing a human sCD59-encoding nucleic acid.
• human sCD59 is effective to reverse the progression of AMD.
• the progression of AMD is reversed by delivering into a cell a vector containing a human sCD59-encoding nucleic acid.
• the AMD is wet or exudative AMD.
• the AMD is dry AMD or geographic atrophy (GA).
• human sCD59 is effective to attenuate choroidal neovascularization (CNV).
• human sCD59 is delivered by an approach using methods of gene therapy that is effective to attenuate choroidal neovascularization (CNV).
• human sCD59 is effective to reduce the extent of MAC deposition on CNV spots.
• the extent of MAC deposition on CNV spots is reduced by delivering into a cell a vector containing a human sCD59-encoding nucleic acid.
• human sCD59 prevents lysis of retina cells.
• the lysis of retinal cells is prevented by delivering into a cell a vector containing a human sCD59-encoding nucleic acid.
• human sCD59 is delivered by an adeno-associated virus (AAV) vector.
• AAV adeno-associated virus
• the adeno-associated virus vector is AAV2.
• the adeno-associated virus is AAV5.
• the adeno-associated virus vector is AAV8.
• the AAV vector is a hybrid vector comprising ITR sequences from AAV2 and a capsid protein from AAV5 (AAV2/5).
• the AAV vector is a hybrid vector comprising ITR sequences from AAV2 and a capsid protein from AAV8 (AAV2/8).
• the AAV vector is a hybrid vector comprising ITR sequences from AAV5 and a capsid protein from AAV2 (AAV5/2).
• the AAV vector is a hybrid vector comprising ITR sequences from AAV5 and a capsid protein from AAV8 (AAV5/8).
• the AAV vector is a hybrid vector comprising ITR sequences from AAV8 and a capsid protein from AAV2 (AAV8/2).
• the AAV vector is a hybrid vector comprising ITR sequences from AAV8 and a capsid protein from AAV5 (AAV8/5).
• the AAV vector is administered by injection.
• the injection is subretinal.
• the injection is intravitreal.
• the injection is a single injection.
• the injection is multiple injections.
• contacting cells with a vector containing a human sCD59-encoding nucleic acid produces a subset of cells that are 'factories' for local production and secretion of sCD59, which may protect adjacent ocular cells including retinal pigment epithelium (RPE) cells and choroidal blood vessels.
• RPE retinal pigment epithelium
• compositions and methods using nucleotide sequences encoding human sCD59 offer additional advantages over protein-based delivery methods.
• Peptides have limited half-lives in vivo and need to be re-administered on a regular basis.
• Current treatments for wet AMD include, for example, intraocular ranibizumab antibody injections every four to six weeks. This method of treatment exposes patients to complications and associated pathologies such as endophthalmitis.
• the incidence of endophthalmitis is relatively low (0.16% per dose) in the presence of a robust immune system.
• the rate of endophthalmitis increases substantially due to the cumulative effect of an attenuated complement system and serial injections over many years, such as for the treatment of chronic diseases such as AMD.
• Viral vectors such as adenovirus vectors have been used to provide lifetime expression of transgenes in vivo in mice.
• AAV vectors for example, have facilitated transgene expression in dogs for more than seven years.
• AAV has been found to have therapeutic transgene expression for over 3.7 years, the longest time periods studied.
• Adenovirus has been found to be an efficient vector for delivery of transgenes to ocular tissue and has been found to be safe in several ocular gene therapy trials.
• Adenovirus vectors engineered for long-term transgene expression and the technology for scaled production of such vectors are known in the art.
• AAV vectors have been shown to be safe for use in humans and are generally considered less immunogenic than adenovirus vectors.
• delivery of human sCD59 to the eyes of AMD patients using an AAV vector is effective for long-term transgene expression.
• the described invention provides a pharmaceutical composition for treating AMD comprising a vector carrying a nucleotide sequence encoding a recombinantly engineered human sCD59 protein operably linked to a promoter sequence causing expression of the protein in a cell, such that the nucleotide sequence carries at least one mutation conferring loss of a glycosylphosphatidylinositol (GPI) anchoring function, such that the protein is expressed as a recombinant membrane-independent (i.e., soluble) CD59 protein and is not membrane targeting.
• the pharmaceutical composition further comprises a pharmaceutically acceptable buffer.
• the AMD is wet or exudative AMD.
• the AMD is dry or GA.
• the pharmaceutical composition is formulated sterile for ocular delivery.
• the pharmaceutical composition formulated for sterile ocular delivery is in a dose effective to treat AMD.
• the pharmaceutical composition formulated for ocular delivery further includes at least one of a pharmaceutically acceptable buffer, a pharmaceutically acceptable salt and a pharmaceutically acceptable emollient suitable for delivery by at least one route selected from: intra-ocular injection, subconjunctival injection, subtenon injection, eye drop, and ointment.
• the vector is at least one of: an engineered viral vector recombinantly linked to the nucleotide sequence encoding the sCD59 protein; and a synthetic gene delivery vector for delivery of the nucleotide sequence encoding human sCD59.
• the viral vector is selected from the group consisting of adenovirus, adeno-associated virus, a herpesvirus, a poxvirus, and a lentivirus.
• the synthetic gene delivery vector is selected from the group consisting of a liposome, a lipid/polycation (LPD), a peptide, a nanoparticle, a gold particle, and a polymer.
• the pharmaceutical composition further includes a peptide for overall delivery (POD), the pharmaceutical composition operably linked to the compound to obtain a conjugated compound, such that the POD includes a protein transduction domain (PTD).
• POD protein transduction domain
• the POD composition is one shown in Kumar-Singh et al. PCT7US2008/010179 filed Aug. 28, 2008 or Kumar-Singh et al. U.S. publication 2010/0209447 published Aug. 19, 2010, each of which is incorporated herein by reference in its entirety.
• the pharmaceutical composition comprises a dose of viral vector particles administered to an affected eye.
• the dose of viral particles ranges from about IxlO 7 to about IxlO 9 .
• the dose of viral particles ranges from about IxlO 8 to about IxlO 10 .
• the dose of viral particles ranges from about IxlO 9 to about IxlO 11 .
• the dose of viral particles ranges from about IxlO 11 to about IxlO 12 .
• the dose of viral particles ranges from about IxlO 11 to about IxlO 13 .
• the pharmaceutical composition further includes at least one therapeutic agent selected from the group consisting of an anti-inflammatory, an antitumor, an antiviral, an antibacterial, an anti-mycobacterial, an anti-fungal, an anti-proliferative and an anti-apoptotic.
• the dose of viral particles ranges from about IxlO 10 DNAse-resistant particles (DRP) to about IxlO 12 DRP.
• the dose of viral particles is about 3.56xl0 10 DRP.
• the dose of viral particles is about 1.071xl0 n DRP.
• the dose of viral particles is about 3.56xl0 n DRP.
• the dose of viral particles is about 1.07xl0 12 DRP.
• the pharmaceutical composition comprises a promoter sequence.
• the promoter sequence is a ubiquitous promoter for general for expression in a mammalian cell.
• the promoter is a promoter from a gene encoding actin, polyhedron, or hydroxyl-methylglutaryl CoA reductase (HMGCR).
• HMGCR hydroxyl-methylglutaryl CoA reductase
• Such promoters include, but are not limited to, a chicken beta-actin promoter or a human beta-actin promoter.
• the promoter sequence is a tissue specific promoter for expression in a specific cell-type. Specific cell-type promoters include, but are not limited to, a rhodopsin promoter or tissue specific promoter for the eye or liver.
• the described invention provides a method for formulating a composition for treating age-related macular degeneration (AMD) in a subject, the method comprising engineering a vector to deliver and express a human sCD59 nucleotide sequence encoding an amino acid sequence corresponding to human sCD59, such that the nucleotide sequence includes a mutation encoding for amino acids of a glycosyl phosphatidyl inositol (GPI) anchoring domain of the protein, such that the resulting vector encodes an engineered recombinant membrane- independent (i.e., soluble) CD59 (sCD59) protein, and the vector is a viral vector or a synthetic gene delivery vector; and, contacting at least one ocular tissue of the subject with the composition, such that the cells of the tissue express and secrete the CD59 locally, thereby treating the subject for AMD.
• AMD age-related macular degeneration
• the viral vector is derived from a genetically engineered genome of at least one virus selected from the group consisting of an adenovirus, an adeno-associated virus, a herpesvirus, and a lentivirus.
• the synthetic gene delivery vector is selected from the group consisting of a liposome, a lipid/poly cation (LPD), a peptide, a nanoparticle, a gold particle, and a polymer.
• contacting at least one ocular tissue of the subject further includes injecting by a route selected from the group consisting of intravitreal, subretinal, subconjunctival, subtenon; subcutaneous and intravenous.
• the tissues contacted by the pharmaceutical composition comprises at least one tissue selected from the group consisting of retinal pigment epithelium, retina, choroid, sclera, Bruch's membrane and choroidal blood vessels.
• the described invention provides a method of regulating complement activity or treating a complement activity disorder in a subject, the method comprising contacting an affected tissue or organ of the subject at risk for or suffering from the complement activity disorder with a composition including a vector carrying a nucleotide sequence encoding a recombinantly engineered human sCD59 protein operably linked to a promoter sequence causing expression of the protein in a cell, such that the protein includes at least one mutation resulting in loss of function of glycosylphosphatidylinositol (GPI) anchoring domain, such that the protein is recombinant membrane-independent (i.e., soluble) CD59 (sCD59) and is not membrane targeting; and, observing a physiological indicium of the complement activity disorder after contacting, in comparison to an abnormal amount of the physiological indicium observed prior to contacting, such that a decrease after contacting compared prior to contacting is a positive indication that the affected tissue or organ is treated.
• a composition including a vector carrying
• the affected tissue is selected from the group consisting of epithelial tissue, endothelial tissue and vascular tissue.
• the affected organ is selected from the group consisting of eye, heart, kidney, lung, liver, pancreas and vascular system.
• the subject is a tissue or organ donor or recipient.
• the subject is an immunocompromised patient that is an organ recipient.
• the described method comprises treating a disorder selected from the group consisting of age-related macular degeneration (AMD), bacterial infection, toxic shock syndrome (TSS), atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, dense deposit disease, peroximal nocturnal hemoglobinurea, systemic lupus erythromatosis, atherosclerosis and the like.
• AMD age-related macular degeneration
• TSS toxic shock syndrome
• atypical hemolytic uremic syndrome membranoproliferative glomerulonephritis
• dense deposit disease peroximal nocturnal hemoglobinurea
• systemic lupus erythromatosis atherosclerosis and the like.
• the AMD is wet or exudative AMD.
• the AMD is dry AMD or geographic atrophy (GA).
• the disorder is AMD.
• the AMD is dry AMD (GA).
• the observing further includes measuring the indication selected from the group consisting of visual acuity
• the described invention provides a method of treating a complement disorder comprising contacting a tissue or a cell with a pharmaceutical composition.
• the method comprises administering a therapeutically effective amount of a pharmaceutical composition having as an active agent a nucleic acid encoding a human sCD59 protein or a source of expression of a human sCD59 protein, to a subject in need thereof, in such amounts and for such time as is necessary to achieve the desired result.
• the method comprises treating AMD by contacting an ocular tissue or cell with huma sCD59 protein or a vector encoding human sCD59 protein.
• the pharmaceutical composition is administered using any amount and any route of administration effective for treating AMD or other complement- related diseases and conditions.
• amount effective for treating AMD refers to a sufficient amount of the pharmaceutical composition to beneficially prevent or ameliorate the symptoms of AMD.
• the exact dosage of the pharmaceutical composition may be 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 AMD; 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 may be administered one time, hourly, twice hourly, every 3 to four hours, once daily, twice daily, every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition.
• the active agents of the described invention can be formulated in dosage unit form for ease of administration and uniformity of dosage. 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. Such information can then be used to determine useful doses and routes of administration for humans. 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.
• a therapeutically effective dose refers to that amount of active agent that ameliorates the symptoms or condition or prevents progression of AMD.
• 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.
• the daily dosage of a pharmaceutical composition may be varied over a wide range, such as from 0.001 to 100 mg per adult human per day.
• pharmaceutical compositions may be provided 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 (pg) of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
• a unit dose typically contains from about 0.001 micrograms to about 500 micrograms of the active ingredient, from about 0.1 micrograms to about 100 micrograms of active ingredient, or from about 1.0 micrograms to about 10 micrograms of active ingredient.
• An effective amount of a drug may be supplied at a dosage level of from about 0.0001 mg/kg to about 25 mg/kg of body weight per day.
• the range maybe 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.
• Pharmaceutical compositions may be administered on a regimen of, for example, one to four or more times per day.
• a unit dose may be divided and administered, for example, in two or more divided doses.
• a source of expression of a human sCD59 protein is administered as a dose of a viral vector or a nucleic acid vector, such that the dose contains at least about 50, 100, 500, 1000, or at least about 5000 particles per cell to be treated. Cell number can be calculated from retinal area in need of treatment by methods known to one of skill in the art.
• the dose ranges from about IxlO 10 DNAse-resistant particles (DRP) to about 1x10 12 DRP.
• the dose is about 3.56xl0 10 DRP.
• the dose is about 1.071xl0 n DRP.
• the dose is about 3.56xl0 n DRP.
• the dose is about 1.07xl0 12 DRP.
• the source of expression of human sCD59 protein is administered by ocular injections.
• Ocular injections include, but are not limited to, intra-ocular injection into the aqueous or the vitreous humor, or injection into the external layers of the eye, such as via subconjunctival injection or subtenon injection.
• injectable preparations such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• the injectable preparation is a sterile injectable preparation.
• the sterile injectable preparation may 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, but not limited to, 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.
• the sterile injectable preparation includes excipients.
• excipients include, without limitation, suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia), dispersing or wetting agents including, a naturally-occurring phosphatide (e.g., lecithin), or condensation products of an alkylene oxide with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyl-eneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexito
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable or diluent or solvent, for example, as a solution in 1, 3- butanediol.
• a solution generally is considered as a homogeneous mixture of two or more substances; it is frequently, though not necessarily, a liquid.
• the molecules of the solute (or dissolved substance) are uniformly distributed among those of the solvent.
• a suspension is a dispersion (mixture) in which a finely- divided species is combined with another species, with the former being so finely divided and mixed that it does not rapidly settle out. In everyday life, the most common suspensions are those of solids in liquid water.
• Suitable vehicles and solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• suitable vehicles consist of solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants.
• Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
• Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
• the suspension also may contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
• the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
• liquid dosage forms for ocular injection.
• Such liquid dosage forms 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 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, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
• ocular- delivered pharmaceutical compositions can also include adjuvants such as wetting agents and emulsifying and suspending agents.
• the pharmaceutical composition of the described invention may be in the form of a sterile injectable aqueous or oleaginous suspension.
• injectable preparations such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• the described invention comprises 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 the pharmaceutical composition described herein.
• the described invention provides administering the pharmaceutical composition to a subject.
• the step of administering comprises oral administration, topical administration or parenteral administration.
• parenteral administration is selected from the group consisting of intravitreal injection and subretinal injection.
• the administering step comprises administering the pharmaceutical composition as a single dose or as multiple doses.
• the administering step comprises administering the pharmaceutical composition as a single dose.
• the single dose is administered to the eye of a subject in need thereof.
• the subject in need thereof is suffering from AMD.
• the subject in need thereof is suffering from wet or exudative AMD.
• the subject in need thereof is suffering from GA.
• the composition is administered in a pharmaceutically acceptable solution, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic agents.
• the pharmaceutical composition is an aqueous suspension or emulsion in admixture with excipients suitable for the manufacture of aqueous suspensions and emulsions.
• excipients include, but are not limited to, suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide such as lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of
• Solutions or suspensions used for parenteral, intradermal, subcutaneous, intrathecal, or topical application may include, but are not limited to, a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• the parenteral preparation may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Administered intravenously, particular carriers are physiological saline or phosphate buffered saline (PBS).
• PBS physiological saline or phosphate buffered sa
• the injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that may be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
• 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 also may be a sterile injectable solution, suspension or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as a solution in 1,3-butanediol.
• Suitable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
• sterile, fixed oils conventionally are employed or as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono- or diglycerides.
• fatty acids such as oleic acid are used in the preparation of injectables.
• Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain anti-oxidants, buffers, bacteriostats and solutes, which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which may include suspending agents and thickening agents.
• the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-inj ection, immediately prior to use.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
• Suspensions in addition to the active compounds, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.
• suspending agents for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.
• the pharmaceutical composition of the described invention may further include conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral application which do not deleteriously react with the active compounds.
• suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil; fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc.
• the pharmaceutical composition of the described invention may be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
• suitable vehicles include solutions, such as oily or aqueous solutions, as well as suspensions, emulsions, or implants.
• Aqueous suspensions may contain substances which increase the viscosity of the suspension and include, for example, but not limited to, sodium carboxymethyl cellulose, sorbitol and/or dextran.
• the suspension also may contain stabilizers.
• These compositions also may contain adjuvants including preservative agents, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It also may be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
• the pharmaceutical composition of the described invention comprises a therapeutically effective amount of human sCD59 and optionally other therapeutic agents included in a pharmaceutically-acceptable carrier.
• the components of the pharmaceutical composition also are capable of being commingled in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
• the pharmaceutical composition of the described invention includes a pharmaceutically acceptable salt.
• Pharmaceutically acceptable salts are those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio.
• Pharmaceutically acceptable salts are well-known in the art. For example, P. H. Stahl, et al. describe pharmaceutically acceptable salts in detail in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley VCH, Zurich, Switzerland: 2002).
• Example 1 Adenovirus Vector Constructs Expressing CD59
• Adenovirus vector constructs expressing human soluble CD59 were prepared as described in U.S. Patent Nos. 8,324,182 and 10,351,617. Briefly, human CD59 cDNA was obtained from the American Type Tissue Culture Collection (ATCC, Manassas, Va.).
• Human CD59 lacking the sequence coding for the C terminal 26 amino acids, which includes a signal sequence for attachment of the glycosylphosphatidylinositol (GPI) anchor was PCR amplified using a forward primer containing an Xhol site (5'ccccctcgagtggacaatcacaatggg3'; SEQ ID NO: 1) and a reverse primer with an EcoRV site (5'taaggagatatcttaattttcgttta3'; SEQ ID NO: 2).
• the reverse primer introduced a stop codon following Asparagine 77 resulting in a sequence that encodes a soluble form of human CD59 (sCD59) (MGIQGGSVLFGLLLVLAVFCHSGHSLQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQ VYNKCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLEN; SEQ ID NO: 3).
• sCD59 human CD59
• the PCR product was gel purified and XhoI/EcoRV digested.
• the XhoI/EcoRV digested PCR product was cloned into XhoI/EcoRV digested pShCAG and the resulting plasmid pShCAGsCD59 was used to produce adenovirus AAVCAGsCD59 using protocols known in the art (e.g., Klein et al. 2007 Ophthalmology 114: 253-262, and van Leeuwen et al. 2003 Eur. J.
• Example 2 Phase l/2a, Open-label., Single-site, Dose-escalating, Safety and Tolerability Study of a Single Intravitreal Injection of AAVCAGsCD59 in Patients with Advanced Non-exudative (Dry) Age-related Macular Degeneration with Geographic Atrophy
• the study population consisted of adult men or women, 50 years of age or older, with advanced dry AMD with GA in the study eye. Participants had a BCVA Snellen equivalent of 20/200 or worse in the study eye for the first 3 participants, and then a BCVA Snellen equivalent of 20/80 or worse in the study eye after the first 3 participants.
• Total GA lesion size was 5 mm 2 (2 disc areas (DA)) to 20 mm 2 (8 DA) in the study eye, and BCVA of 20/800 or better in the fellow eye.
• DRP DNase-Resistant Particles
• FA CNV fluorescein angiography choroidal neovascularization
• OCT features were consistent with participants with GA.
• the mean OCT central subfield thickness was 182.41 pm across all participants.
• 4 participants had pigment epithelial detachments (PEDs), with mean thickness of 143.25 pm.
• PEDs pigment epithelial detachments
• No participants had intraretinal, subretinal fluid, or CNV by OCT.
• Mean drusen volume within a 5 mm circle was 0.05 mm 3 across all participants.
• the mean OCT central subfield thickness was 211.96 pm across all participants. Eight participants had PED with mean thickness of 150.63 um. One (1) participant had subretinal fluid and 2 participants had intraretinal fluid. Twelve (70.6%) participants did not have CNV on optical coherence tomography angiography (OCTA). Four (4) participants had CNV Type 1 on OCTA and 1 participant had CNV which could not be graded. Mean drusen volume by OCT in the central 5 mm circle was 0.11 mm 3 across all participants.
• the most common prior therapeutic categories by WHO ATC classification included vitamins (94.1%), diuretics (58.8%), lipid modifying agents (58.8%), and antithrombotic agents (52.9%).
• the most common prior ocular therapy was Ophthalmologicals (29.4%) with the PT macrogol 400; propylene glycol (17.6%).
• the most common concomitant therapeutic categories by WHO ATC classification included therapies for the Cardiovascular system (58.8%) (such as diuretics, lipid modifying agents and renin-angiotensin system anti-hypertensives), Systemic anti-infectives (52.9% such as systemic antibacterials), and Ophthalmologicals (52.9% with the most common PT of Artificial Tears [umbrella Term] [23.5%]).
• the study intervention(s) administered to participants is outlined in Table 6.
• the study intervention was administered to the eye which met the I/E criteria and had the worse visual acuity.
• Three participants received Dose 1 (3.56 x 10 10 DRP), 3 participants received Dose 2 (1.07 x 10 11 DRP), and 11 participants received Dose 3 (3.56 x 10 11 DRP).
• Table 6 Study Intervention(s) Administered a Highest dose of AAVCAGsCD59 was not administered to any study participants as sponsor ended enrollment after Cohort 3 due to slow recruitment. b Labels contained information to meet the applicable regulatory requirements.
• PCR Quantitative polymerase chain reaction
• AAV2 neutralizing antibody titers were detected in all participants at baseline and were highly variable (range 1: 5.10 to 1: 50819.74). Seven (7) participants had baseline titers less than 1: 100, 5 participants had titers between 1:100 and 1: 10000, and 5 participants had titers greater than 1: 10,000. Nine (9) out of 17 participants had a 4-fold increase in baseline with titers at any time point (termed “treatment-boosted NAbs”). One participant had one timepoint over 4-fold from baseline, but subsequent tiers were below 4-fold of baseline (termed “transient positive”). There was no apparent relationship between incidence of intraocular inflammation and either high baseline AAV2 neutralizing titers or change from baseline AAV2 neutralizing titers after study intervention.
• Clinical laboratory measures including hematology, liver function tests, renal function tests, blood chemistry, urinalysis, and pregnancy test;
• IOP Intraocular Pressure
• SD-OCTA Spectral domain optical coherence tomography angiography
• Table 7 summarizes the overall systemic treatment emergent adverse events (TEAEs) by dose level. Overall, 16 (94.1%) participants experienced 1 or more TEAEs, 1 participant (5.9%) died during the study, and 9 (52.9%) participants experienced 1 or more SAEs. None of the systemic TEAEs, systemic SAEs, or the death were considered related to treatment intervention.
• Nervous system disorders 2(66.7%) 0 0 2(11.8%)
• the PTs for the related events were anterior chamber inflammation (1 participant), optic nerve disorder (1 participant) and vitritis (4 participants) (Table 11).
• the anterior chamber inflammation (reported term: mild post-injection anterior inflammation) and optic nerve disorder (reported term: worsening of cup to disc ratio oculus uterque [OU]) were in the same participant. All TEAEs in the study eye were mild, except 1 moderate TEAE on the study eyelid (basal cell carcinoma).
• Intraocular pressure 1 2 increased 0 (33.3%) 1 (9.1%) (11.8%)
• Neoplasms benign, malignant and unspecified incl cysts and 1 polyps (33.3%) 0 0 1 (5.9%)
• AEs are coded using MedDRA Version 23.0.
• Intraocular pressure 1 2 increased 0 (33.3%) 1 (9.1%) (11.8%)
• Adverse events are coded using MedDRA Version 23.0.
• the PTs of the remaining AEs occurring in one participant each were: retinal artery embolism, neovascular age-related macular degeneration, retinopathy and cataract.
• One (1) participant had an AE of retinal artery embolism (reported term: Hollenhorst plaque) with an onset at Day 457. The event was mild in severity, not considered related by the investigator and participant had not recovered by the end of the study. No cases of endophthalmitis in the fellow eye were reported. [287] Clinical Laboratory Evaluation
• IOP Intraocular Pressure
• Baseline IOP ranged from 8 to 21.50 mmHg for the study eye, and 9.5 to 21.50 mm Hg for the fellow eye. Mean change in IOP from baseline was less than 2 mm Hg for all cohorts in the study eye at Week 26 and Week 104. Mean change in IOP from baseline was less than
• MMRM Mixed model repeated measures
• Baseline GA lesion area was similar across dosing arms. Mean baseline GA lesions area was 11.72 mm 2 (range 8.15; 14.39) for Cohort 1, 11.467 mm 2 (9.08; 12.84) for Cohort 2, and 10.865 mm 2 (5.47; 19.50) for Cohort 3. Percent change from baseline at Week 26 was 9.235 for Cohort 1, 16.037 for Cohort 2, and 14.888 for Cohort 3. Percent change from baseline at Week 104 was 32.572 for Cohort 1, 35.830 for Cohort 2, and 34.406 in Cohort 3. Due to small numbers, there was no formal analysis of differences in dosing arms with rate of growth of GA. The MMRM estimating change in GA lesion size for each dosing cohort as well as for the pooled cohorts is shown in Figure 5 and Figure 6.
• the mean baseline BCVA in the fellow eye was lower for Cohort 1 compared with Cohort 2 and 3.
• the mean baseline BCVA for the fellow eye for Cohort 1 was 44.000 letters (28.50 to 71.00), 59.167 letters (50.50 to 70.00) for Cohort 2 and 59.591 letters (35.50 to 78.00) for Cohort 3.
• the mean baseline BCVA change from baseline at Week 26 was -5.333 letters, - 7.056 letters and -0.205 letters in Cohorts 1, 2, and 3, respectively.
• the mean BCVA change from baseline at Week 104 was -21.333 letters, -11.167 letters and -7.100 letters in Cohorts 1, 2 and 3, respectively.
• Individual participant data for visual acuity scores over time for the fellow eye is shown in (Figure 8).
• Aqueous levels of sCD59 were determined using a custom Western Blot assay using a commercially available anti-CD59 rabbit polyclonal antibody (Abeam, Cambridge UK, cat. no. abl24396). Aqueous was collected at baseline prior to injection as well as Week 8. No participants had detectable levels of aqueous sCD59 protein prior to study intervention administration. Aqueous sCD59 protein was detected in 5/17 participants at Week 8. Levels of protein were highly variable with a range of 250 ng/ml to 5719.4 ng/ml. All participants with detectable sCD59 protein were in the high dose cohort. Western Blot analysis was not available for 1 participant (unable to electrophorese for unknown reasons).

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