Treating geographic atrophy with a gene therapy vector expressing soluble CD59

Abstract

The described invention provides a method for treating a complement disorder such as age-related macular degeneration (AMD) in a subject, comprising administering a pharmaceutical composition to an affected eye of the 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 administration results in expression and secretion of the sCD59 protein by cells of the affected eye, wherein expression results in treatment of the affected cells in the diseased eye.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Application No. 63 / 281,190, filed November 19, 2021, which is incorporated by reference herein in its entirety for all purposes.

[0002] (Reference to electronically submitted sequence listing) This application contains a computer readable sequence listing submitted herewith in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The sequence listing XML file submitted herewith is named "JBI6660WOPCT1_SL.xml", was created on November 9, 2022, and is 3843 bytes in size.

[0003] FIELD OF THEINVENTION The present invention relates generally to the use of gene therapy to treat retinal diseases, including Age-related Macular Degeneration (AMD) and geographic atrophy (GA).

[0004] BACKGROUND OF THEINVENTION Age-related macular degeneration (AMD) is a slowly progressive disease of the macula. AMD is the leading cause of blindness in developed countries in patients over 60 years of age (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). 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):e106-116).

[0005] In advanced AMD, the most significant vision loss occurs. Advanced AMD is divided into two categories: (1) "wet" or exudative AMD, and (2) "dry" AMD, also called geographic atrophy (GA). Wet AMD is associated with choroidal neovascularization (CNV) and can be successfully treated with Food and Drug Administration (FDA) approved vascular endothelial growth factor (VEGF) inhibitors, such as ranibizumab or aflibercept. Conversely, there is currently no FDA approved treatment for GA. Established risk factors that contribute to AMD include age, smoking, diet, and genetic risk factors (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; de Jong, PT. Age-related macular degeneration. N Engl J Med. 2006;355:1474-1485). Patients with AMD have morbidity beyond vision loss, including increased incidence of depression and anxiety, movement disorders, and dissociation (Dawson SR, Mallen CD, Gouldstone MB, Yarham R, Mansell G. The prevalence of anxiety and depression in people with age-related macular degeneration: a systematic review of observational study data. BMC Ophthalmol. 2014 Jun 12;14:78).

[0006] Human genetic studies suggest that overactivation of the complement system may play a role in the pathogenesis of AMD. Single nucleotide polymorphisms in multiple components of the complement pathway, including complement factor H, complement factor I, factor B, and C3, are associated with an increased risk of advanced AMD (Schramm EC, Clark SJ, Triebwasser MP, Raychaudhuri S, Seddon J, Atkinson JP. Genetic variants in the complement system predisposing to age-related macular degeneration: a review. Mol Immunol. 2014 Oct; 61(2): 118-125). The complement pathway is a major part of innate immunity in recognizing and killing foreign pathogens and can be activated by the classical, alternative, or lectin pathways. The terminal component of the complement pathway is the formation of the membrane attack complex (MAC), a complex of proteins on cell membranes that have cytolytic function. 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 the R95X nonsense mutation in C9, a key component of the MAC complex, have a 4.7-fold reduced risk of wet AMD (Nichiguchi KM, Yasuma TR, Tomida D, et al. C9-R95X polymorphism in patients with neovascular age-related macular degeneration. Invest Ophthalmol Vis Sci. 2012 Jan 31;53(1):508-512).

[0007] Therefore, preventing the formation of the MAC complex may serve as a therapeutic strategy for AMD. The natural inhibitor of the MAC complex is CD59, a glycosylphosphatidylinositol (GPI)-anchored membrane-bound protein that prevents the formation of the MAC complex. Preclinical studies have shown that intravitreal injection of AAV2 viral vectors expressing a soluble form of CD59 (sCD59) can reduce MAC deposition and show efficacy in rodent models of wet AMD (Cashman SM, Ramo K, Kumar-Singh RA non membrane-targeted human soluble CD59 attenuates choroidal neovascularization in a model of age related macular degeneration. PLoS One. 2011 Apr 28; 6(4): e19078), suggesting that expression of sCD59 can reduce MAC deposition in vivo and may have therapeutic efficacy in AMD.

[0008] (Summary of the invention) According to one aspect, the described invention provides a method for treating age-related macular degeneration (AMD) in a subject, the method comprising administering a pharmaceutical composition to an AMD affected eye of the subject by ocular injection, the composition comprising a nucleic acid encoding a soluble CD59 (sCD59) protein operably linked to a promoter, the nucleic acid encoding sCD59 being packaged into a delivery vector, the administration resulting in expression and secretion of the sCD59 protein by cells of the AMD affected eye, the expression resulting in treatment of AMD affected cells in the AMD affected eye.

[0009] According to one embodiment, the AMD is geographic atrophy (GA).

[0010] According to one embodiment, the ocular injection is an intravitreal injection. According to another embodiment, the intravitreal injection is a single injection.

[0011] According to one embodiment, the delivery vector is an adeno-associated virus (AAV) vector. According to another embodiment, the AAV vector is AAV2.

[0012] According to one embodiment, the promoter is a CAG promoter.

[0013] According to one embodiment, the pharmaceutical composition comprises about 3.56×10 10 DNAse-resistant particles (DRP), approximately 1.071 × 10 11 DRP, approx. 3.56×10 11 DRP and approx. 1.07×10 12 The dose of viral particles is selected from the group consisting of DRPs.

[0014] According to another aspect, the described invention provides a method of modulating impaired complement activity in a subject, the method comprising contacting diseased cells 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 recombinantly engineered human soluble CD59 (sCD59) protein in the diseased cells, wherein the sCD59 protein contains at least one mutation causing loss of function of a glycosylphosphatidylinositol (GPI) anchor domain causing loss of membrane targeting, and observing a physiological sign of impaired complement activity after contacting compared to an abnormal amount of the physiological sign observed before contacting, wherein a reduction after contacting compared to before contacting is a positive indication that the diseased cells are treated.

[0015] According to one embodiment, the complement activity disorder is GA.

[0016] According to one embodiment, the contacting is by intravitreal injection. According to another embodiment, the intravitreal injection is a single injection.

[0017] According to one embodiment, the diseased cell is a retinal cell.

[0018] According to one embodiment, the vector is AAV2.

[0019] According to one embodiment, the physiological indicia is best corrected visual acuity (BCVA). According to another embodiment, BCVA is measured as the mean change from baseline. According to another embodiment, the mean change from baseline is -7.100 letters.

[0020] According to one embodiment, the pharmaceutical composition comprises about 3.56×10 10 DNAse-resistant particles (DRP), approximately 1.071 × 10 11 DRP, approx. 3.56×10 11 DRP and approx. 1.07×10 12 The dose of viral particles is selected from the group consisting of DRPs.

[0021] According to another aspect, the described invention provides a method for treating a complement disorder comprising contacting a cell with a pharmaceutical composition having as an active agent a therapeutically effective amount of a nucleic acid encoding human sCD59 protein or a source of expression of human sCD59 protein, comprising administering the pharmaceutical composition to a subject in need thereof.

[0022] According to one embodiment, the complement disorder is GA.

[0023] According to one embodiment, the contacting is by intravitreal injection. According to another embodiment, the intravitreal injection is a single injection.

[0024] According to one embodiment, the diseased cell is a retinal cell.

[0025] According to one embodiment, the therapeutically effective amount is about 3.56×10 10 DNAse-resistant particles (DRP), approximately 1.071 × 1011 DRP, approx. 3.56×10 11 DRP and approx. 1.07×10 12 DRPs. [Brief description of the drawings]

[0026] [Figure 1] Figure 1 shows a schematic diagram illustrating a dose escalation study conducted to establish the safety of a single intravitreal injection of the gene therapy vector AAVCAGsCD59 for the treatment of patients with advanced dry age-related macular degeneration (AMD) with geographic atrophy (GA). DRP = DNAse resistant particle. [Diagram 2] Spaghetti plots showing square root converted changes in geographic atrophy (GA) lesion area (mm) from baseline to selected visits in study eyes labeled by cohort. sqrt = square root. [Diagram 3] Figure 1 shows plots showing least squares (LS) means with 95% confidence intervals (CI) for square root converted change from baseline in geographic atrophy (GA) lesion area (mm) measured by fundus autofluorescence (FAF) over time in study eyes labeled by cohort. CHG = change, sqrt = square root. [Figure 4] 1 shows plots showing least squares (LS) means with 95% confidence intervals (CI) for the change from baseline in square root converted geographic atrophy (GA) lesion area (mm) measured by fundus autofluorescence (FAF) over time in study eyes (pooled cohort). CHG=change, sqrt=square root. [Diagram 5] FIG. 1 shows plots showing least squares (LS) means with 95% confidence intervals (CI) for change from baseline in geographic atrophy (GA) lesion area (mm2) measured by fundus autofluorescence (FAF) over time in study eyes labeled by cohort. [Figure 6]FIG. 1 shows plots showing least squares (LS) means with 95% confidence intervals (CI) for change from baseline in geographic atrophy (GA) lesion area (mm2) measured by fundus autofluorescence (FAF) over time in study eyes (pooled cohort). [Figure 7] Spaghetti plots of change from baseline to selected visits in distance best corrected visual acuity (BCVA) (letters) in the study eyes labeled by cohort. Full analysis set. CHG=change. [Figure 8] Spaghetti plots of change from baseline to selected visits in best corrected visual acuity (BCVA) (letters) in distance in the fellow eye labeled by cohort. Full analysis set. CHG = change.

[0027] DETAILED DESCRIPTION OF THE PRESENT EMBODIMENT The invention as described can be better understood from the following description of exemplary embodiments in conjunction with the accompanying figures and drawings. It should be apparent to those skilled in the art that the described embodiments provided herein are merely exemplary and explanatory, and not limiting.

[0028] Definition: Various terms used throughout this specification shall have the definitions set forth herein.

[0029] The term "administering" as used herein includes in vivo administration and administration directly to tissues ex vivo. In general, the compositions may be administered systemically, either orally, bucally, parenterally, topically, by inhalation or insufflation (i.e., through the mouth or through the nose), or rectally, in unit dosage formulations containing conventional non-toxic pharma- ceutically acceptable carriers, adjuvants, and vehicles as desired, or may be administered locally by means such as, but not limited to, injection, implantation, conjugation, topical application, or parenterally.

[0030] The term "attenuate" as used herein means to reduce in power, effect, or value.

[0031] The term "CD59" as used herein refers to a membrane-bound glycoprotein found in association with the 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 salivary glands, bronchial epithelium, renal tubules and squamous epithelium. The CD59 protein inhibits the assembly of a functional membrane attack complex (MAC), thus protecting cells from complement-mediated activation and / or lysis. 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 specify a signal sequence for attachment of a glycosylphosphatidylinositol anchor (GPI anchor) at amino acid asparagine at position 77. The cDNA sequence of CD59 is shown in US Pat. No. 5,624,837 to Fodor et al., issued Apr. 29, 1997, and is incorporated herein by reference in its entirety.

[0032] The term "condition" as used herein refers to a variety of health conditions and is meant to include disorders or diseases caused by any underlying mechanism or insult.

[0033] The term "disease" or "disorder" as used herein refers to an impairment of health or a state of abnormal function.

[0034] The term "unit dosage form" as used herein refers to a physically discrete unit of active agent appropriate for the patient to be treated.

[0035] As used herein, the term "drug" refers to a therapeutic agent or any substance used in the prevention, diagnosis, mitigation, treatment, or cure of disease.

[0036] As used herein, the term "drusen" refers to yellow deposits under the retina.

[0037] The term "enhance" as used herein in its various grammatical forms refers to an increase or strengthening, or a bettering or increasing, in quality or quantity.

[0038] The terms "functional equivalent" or "functionally equivalent" are used interchangeably herein to refer to substances, molecules, polynucleotides, proteins, peptides, or polypeptides that have a similar or identical effect or use. A polypeptide that is functionally equivalent to SEQ ID NO:3 can have, for example, a biological activity, e.g., inhibitory activity, kinetic parameters, salt inhibition, cofactor-dependent activity, and / or functional unit size, that is substantially similar or identical to the expressed polypeptide of SEQ ID NO:3.

[0039] The term "inhibit" and its various grammatical forms, including but not limited to "inhibiting" or "inhibition," are used herein to refer to slowing down the amount or rate of a process, stopping a process entirely, or reducing, limiting, or blocking its action or function. Inhibition can include a decrease or reduction of 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% of the amount, rate, action function, or process of a substance.

[0040] The term "inhibitor" as used herein refers to a second molecule that binds to a first molecule, thereby decreasing the activity of the first molecule. Enzyme inhibitors are molecules that bind to an enzyme, thereby decreasing the enzyme activity. The binding of an inhibitor can stop a substrate from entering the active site of the enzyme and / or prevent the enzyme from catalyzing its reaction. The binding of an inhibitor can be either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and chemically alter it, for example, by modifying a key amino acid residue required for enzyme activity. In contrast, reversible inhibitors bind non-covalently and produce different types of inhibition depending on whether they bind to the enzyme, the enzyme-substrate complex, or both. Enzyme inhibitors are often evaluated by their specificity and potency.

[0041] The term "injury," as used herein, refers to damage or harm to a structure or function of the body caused by an external agent or force, which may be physical or chemical.

[0042] The terms "membrane attack complex" and "MAC" are used interchangeably herein to refer to an effector of the immune system that comprises a complex of proteins that typically forms on the surface of pathogen cell membranes as a result of activation of the host's complement system. Antibody-mediated complement activation leads to MAC deposition on the surface of infected cells, creating holes that disrupt the infected cell's plasma membrane, causing cell lysis and death. The MAC is composed of complement components C5b, C6, C7, C8 and some C9 molecules.

[0043] The term "modify" as used herein means to change, fluctuate, adjust, vary, change, affect, or regulate in a particular measure or proportion in one or more items.

[0044] The term "modulate" as used herein means to adjust, change, adapt, or regulate to a particular measure or proportion.

[0045] The term "nucleic acid" is used herein to refer to deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form, and, unless otherwise limited, encompasses known analogues which have the essential properties 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).

[0046] The term "nucleotide" is used herein to refer to a chemical compound consisting of a heterocyclic base, a sugar, and one or more phosphate groups. In the most common nucleotides, the base is a derivative of a purine or pyrimidine, and the sugar is the pentose deoxyribose or ribose. Nucleotides are monomers of nucleic acids, three or more of which are linked together to form nucleic acids. 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).

[0047] The following terms are used herein to describe sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence," (b) "comparison window," (c) "sequence identity," (d) "percentage of sequence identity," and (e) "substantial identity."

[0048] (a) The term "reference sequence" refers to a sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence, e.g., a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence.

[0049] (b) The term "comparison window" refers to a contiguous specific segment of a polynucleotide sequence, where the polynucleotide sequence may be compared to a reference sequence, and a portion of the polynucleotide sequence within the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides long, and may optionally be at least 30 contiguous nucleotides long, at least 40 contiguous nucleotides long, at least 50 contiguous nucleotides long, at least 100 contiguous nucleotides long, or longer. Those skilled in the art will understand that in order to avoid high similarity to the reference sequence due to the inclusion of gaps in the polynucleotide sequence, gap penalties are typically introduced and subtracted from the number of matches.

[0050] Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 (1988), or by computerized implementations of these algorithms, including but not limited to CLUSTAL in the PC / Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA. The CLUSTAL program is fully described in Higgins and Sharp, Gene 73:237-244 (1988), Higgins and Sharp, CABIOS 5:151-153 (1989), Corpet, et al., Nucleic Acids Research 16:10881-90 (1988), Huang, et al., Computer Applications in the Biosciences, 8:155-65 (1992), and Pearson, et al., Methods in Molecular Biology, 24:307-331 (1994).The BLAST family of programs that can be used for database similarity searching include 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. See Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).

[0051] Unless otherwise stated, the sequence identity / similarity values ​​provided herein refer to values ​​obtained using the BLAST 2.0 program suite using default parameters. Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Software for performing BLAST analysis is publicly available, for example, 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 that either match when aligned with words of the same length in database sequences or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. For nucleotide sequences, the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls off its maximum achieved value by an amount X, when the accumulation of one or more alignments of negative scoring residues causes the cumulative score to fall below zero, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0052] 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. Sci. 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 that a match between two nucleotide sequences or two amino acid sequences would occur by chance. BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins contain regions of non-random sequence that may be homopolymeric tracts, short-term repeats, or regions rich in one or more amino acids. Such low-complexity regions can be aligned between unrelated proteins even if other regions of the proteins are completely dissimilar. A number of low-complexity filter programs can be used to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993)) low complexity filters may be used alone or in combination.

[0053] (c) The term "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences is used herein to refer to residues in the two sequences that are the same when aligned for maximum correspondence over a particular comparison window. When percentage sequence identity is used with reference to proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, i.e., substitutions in which an amino acid residue is replaced with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), and thus does not alter the functional properties of the molecule. When sequences differ by conservative substitutions, the percentage 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 conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions are given a score between 0 and 1. Scoring of conservative substitutions is calculated, for example, according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988), for example as implemented in the program PC / GENE (Intelligenetics, Mountain View, Calif., USA).

[0054] (d) The term "percentage of sequence identity" as used herein means a value determined by comparing two optimally aligned sequences over a comparison window, where a portion of a polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. This percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0055] (e) The term "substantial identity" of a polynucleotide sequence means that the polynucleotide comprises a sequence having at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity, and at least 95% sequence identity, when compared to a reference sequence using one of the alignment programs described using standard parameters. Those skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by considering codon degeneracy, amino acid similarity, reading frame arrangement, and the like. Substantial identity of amino acid sequences for these purposes usually means at least 60%, or at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. Another indication that nucleotide sequences are substantially identical is whether 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 they encode are substantially identical. This can occur, for example, when a copy of a nucleic acid is made using the maximum codon degeneracy permitted by the genetic code. A further indication that two nucleic acid sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid. The term "substantial identity" of a protein sequence 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 a common functional activity. For example, amino acid sequences that contain a common structural domain with at least about 60% identity, or at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% identity.

[0056] The term "parenteral" as used herein refers to introduction into the body by injection (i.e., administration by injection), including, for example, intraocular (also known as intravitreal) (i.e., injection into the vitreous space of the eye), subretinal (i.e., injection into the subretinal space located between the photoreceptors of the retina and the retinal pigment epithelium (RPE) layer), subcutaneous (i.e., injection under the skin), intramuscular (i.e., injection into a muscle), intravenous (i.e., injection into a vein), intrathecal (i.e., injection into the space surrounding the spinal cord), intrasternal injection, or infusion techniques. Compositions of the described invention that are administered parenterally are delivered using a needle, for example, a surgical needle. The term "surgical needle" as used herein refers to any needle adapted for delivery of a fluid (i.e., flowable) composition of the described invention to a selected anatomical structure. Injectable preparations, for example, sterile injectable aqueous or oily suspensions, may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents.

[0057] As used herein, the term "pharmaceutical acceptable carrier" refers to any substantially non-toxic carrier that can be conventionally used for administration of pharmaceuticals, in which the isolated polypeptide of the present invention remains stable and bioavailable. A pharmaceutical acceptable carrier must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the mammal being treated. Furthermore, it should maintain the stability and bioavailability of the active agent. A pharmaceutical acceptable carrier may be liquid or solid, and is selected to provide the desired bulk, consistency, etc., when combined with the active agent and other components of a given composition, taking into account the planned mode of administration.

[0058] The term "pharmaceutically acceptable salt" means a salt that is suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and commensurate with a reasonable benefit / risk ratio.

[0059] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The essential property of such analogs of naturally occurring amino acids, when incorporated into a protein, is that the protein is specifically reactive with antibodies raised against the same protein, but entirely composed of naturally occurring amino acids.

[0060] The terms "polypeptide" and "protein" are also 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 can be chemically synthesized or recombinantly expressed. The polypeptides of the described invention can also be chemically synthesized. Synthetic polypeptides, prepared using well-known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can contain natural and unnatural amino acids. The amino acids used in peptide synthesis can be standard Boc (N-α-amino protected N-α-t-butyloxycarbonyl) amino acid resin, or base-labile N-α-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids, as originally described by Carpino and Han (1972, J. Org. Chem. 37:3403-3409), using standard deprotection, neutralization, coupling and washing protocols of the original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154). Both Fmoc and Boc N-α-amino protected amino acids can be obtained from Sigma, Cambridge Research Biochemical, or other chemical companies familiar to those skilled in the art. In addition, polypeptides can be synthesized using other N-α-protecting groups familiar to those skilled in the art. Solid phase peptide synthesis is well known to those of skill in the art and can be accomplished, for example, by the techniques provided 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 by using an automated synthesizer.The polypeptides of the invention may contain D-amino acids (which are resistant to L-amino acid specific proteases in vivo), combinations of D and L-amino acids, and various "designer" amino acids to impart special properties (e.g., β-methyl amino acids, C-α-methyl amino acids, and N-α-methyl amino acids, etc.). Synthetic amino acids include ornithine in place of lysine, and norleucine in place of leucine or isoleucine. In addition, the polypeptides may have peptidomimetic bonds (e.g., ester bonds) to prepare peptides with novel properties. For example, peptides incorporating reduced peptide bonds, i.e., R1-CH2-NH-R2 (where R1 and R2 are amino acid residues or sequences), may be generated. The reduced peptide bonds may be introduced as dipeptide subunits. Such polypeptides are resistant to protease activity and have extended half-lives in vivo. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The essential property of such analogs of naturally occurring amino acids is that, when incorporated into a protein, the protein is specifically reactive to antibodies raised against the same protein, but composed entirely of naturally occurring amino acids.

[0061] The terms "polypeptide", "peptide" and "protein" also include modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. As is well known and as described above, it will be understood that a polypeptide may not be completely linear. For example, polypeptides may be branched as a result of ubiquitination, and they may generally be cyclic, with or without branching, as a result of post-translational events, including natural processing events and events resulting from non-naturally occurring human manipulation. Cyclic, branched and branched cyclic polypeptides may also be synthesized by non-translational natural processes and entirely synthetic methods. In some embodiments, a peptide is of any length or size.

[0062] As used herein, the terms "preserve," "preserved," "preserving," or "preservation" refer to maintaining, keeping safe from harm or injury, protecting, preserving or maintaining functionality.

[0063] The terms "prevent," "prevented," "preventing," or "prevention," as used herein, refer to keeping, hindering, or avoiding an event, action, or operation from happening, occurring, or occurring.

[0064] The term "recombinant" refers to a cell or vector that has been modified by the introduction of a heterologous nucleic acid, or a cell derived from a cell so modified. Recombinant cells express genes that are not found in the same form within the native (non-recombinant) form of the cell, or express native genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all as a result of human intervention. As used herein, the term "recombinant" does not encompass modification of a cell or vector by naturally occurring events, such as events that occur without human intervention (e.g., spontaneous mutation, natural transformation, transduction / transposition).

[0065] The term "recombinant expression cassette" refers to a recombinantly or synthetically produced nucleic acid construct that has a set of specific nucleic acid elements that allow for transcription of a particular nucleic acid in a host cell. A recombinant expression cassette can be incorporated into a plasmid, a chromosome, mitochondrial DNA, a virus, or a nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, the nucleic acid to be transcribed, a promoter, and a transcription termination signal such as a polyA signal.

[0066] 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 prokaryotic or eukaryotic cells that have been genetically engineered to contain a cloned gene or gene of interest in the chromosome or genome of the host cell.

[0067] The term "recombinant protein" as used herein refers to a protein that is produced by genetic engineering, e.g., by the manipulation of a genetically modified organism, such as a microorganism.

[0068] As used herein, the term "reduce" or "reducing" refers to limiting the occurrence of a disorder in individuals at risk of developing the disorder.

[0069] The term "regulate" as used herein means to control or maintain a process, function or mechanism, such as a biological process.

[0070] The term "similar" is used interchangeably with the terms similar, comparable, or resembling and means having a common trait or characteristic.

[0071] As used herein, the term "solution" refers to a homogeneous mixture of two or more substances, often, but not necessarily, a liquid. In a solution, the molecules of the solute (or dissolved substance) are evenly distributed among the molecules of the solvent.

[0072] As used herein, the terms "soluble CD59," "sCD59," and "membrane-independent CD59" refer to a CD59 amino acid sequence that lacks a glycosylphosphatidylinositol (GPI) anchor or has a modified GPI anchor that lacks the function and ability to bind to cell membranes or cell membrane-associated structures, such as membrane-associated proteins.

[0073] The term "stimulate," as used herein, in any of its grammatical forms, refers to inducing activity or increasing activity.

[0074] The term "suspension" as used herein refers to a dispersion (mixture) in which a finely divided species is combined with another species such that the former is so finely divided and mixed that it does not settle rapidly. In everyday life, the most common suspensions are suspensions of solids in liquids.

[0075] As used herein, the terms "subject" or "individual" or "patient" or "participant" are used interchangeably to refer to members of animal species of mammalian origin, including humans. The term "subject in need thereof" is used to refer to a subject with heart failure or at risk of progressing to heart failure, including a subject with AMI causing disease manifestations of left ventricular remodeling.

[0076] The phrase "subject in need of such treatment" as used herein refers to a patient suffering from a disease, disorder, condition, or pathological process. In some embodiments, the term "subject in need of such treatment" is also used to refer to a patient who (i) may be administered at least one dose of an adenoviral vector construct expressing human soluble CD59 of the described invention, (ii) has received at least one dose of an adenoviral vector construct expressing human soluble CD59 of the described invention, or (iii) has received at least one dose of an adenoviral vector construct expressing human soluble CD59 of the described invention, unless the context and use of the phrase indicate otherwise.

[0077] As used herein, the term "substantially similar" means that a first value, aspect, trait, characteristic, number, or amount is 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, characteristic, number, or amount. For example, a polypeptide substantially similar to (SEQ ID NO:3) will 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 the amino acid sequence (SEQ ID NO:3).

[0078] The term "substitution" is used herein to refer to a situation in which one or more bases are exchanged for another one or more bases in a DNA sequence. The substitution may be a synonymous or non-synonymous substitution. As used herein, a "synonymous substitution" refers to the substitution of one base for another in an exon of a gene encoding a protein, such that the amino acid sequence produced is not altered. As used herein, the term "non-synonymous substitution" refers to the substitution of one base for another in an exon of a gene encoding a protein, such that the amino acid sequence produced is altered.

[0079] The term "symptom" as used herein refers to a phenomenon that arises from, accompanies, or serves as an indication of a particular disease or disorder.

[0080] As used herein, the term "syndrome" refers to a pattern of symptoms indicative of some disease or condition.

[0081] The term "therapeutic agent" as used herein refers to a drug, molecule, nucleic acid, protein, metabolite, composition or other substance that provides a therapeutic effect. The term "active" as used herein refers to an ingredient, component or constituent of the described inventive composition that is responsible for the intended therapeutic effect. The terms "therapeutic agent" and "active agent" are used interchangeably herein. The term "therapeutic component" as used herein 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 proportion of the population. One example of a commonly used therapeutic component is the ED50, which describes the dose at a particular dosage that is therapeutically effective against a particular disease manifestation in 50% of the population.

[0082] The terms "therapeutic amount," "therapeutically effective amount," "effective amount," or "pharmacologically effective amount" of an active agent are used interchangeably to refer to an amount sufficient to provide the intended benefit of treatment. Effective amounts of active agents that may be used in accordance with the described invention are generally about 1×10 per dose. 10 DNase resistant particles (DRP)~1×10 12DRP range. However, 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 used. Thus, dosage regimens can vary widely, but can be routinely determined by a physician using standard methods. In addition, the terms "therapeutic amount", "therapeutically effective amount" and "pharmaceutical effective amount" include preventative or prophylactic amounts of the compositions of the described invention. In preventative or prophylactic uses of the described invention, a pharmaceutical composition or medicament is administered to a patient susceptible to or otherwise at risk for a disease, disorder, or condition in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of the disease, disorder, or condition, including the biochemical, histological, and / or behavioral symptoms of the disease, disorder, or condition, its complications, and intermediate pathological phenotypes presented during the development of the disease, disorder, or condition. In general, it is preferred to use a maximum dose, i.e., the highest safe dose according to some medical judgment. The terms "dose" and "dosage" are used interchangeably herein.

[0083] The term "therapeutic effect" as used herein refers to an outcome of a treatment, the outcome of which is deemed desirable and beneficial. Therapeutic effect may include, directly or indirectly, the arrest, reduction, or elimination of a disease symptom. Therapeutic effect may also include, directly or indirectly, the arrest, reduction, or elimination of the progression of a disease symptom.

[0084] For any therapeutic agent described herein, the therapeutically effective amount can be initially determined from preliminary in vitro tests and / or animal models. The therapeutically effective dose can also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and efficacy of the administered compound. It is within the ability of a person skilled in the art to adjust the dose to achieve maximum efficacy based on the above methods and other well-known methods.

[0085] General principles for determining therapeutic efficacy are outlined below, which can be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference.

[0086] Pharmacokinetic principles provide a basis for modifying dosing regimens to obtain the desired degree of therapeutic efficacy while minimizing unacceptable adverse effects. In situations where the plasma concentration of a drug can be measured and related to a therapeutic window, additional guidance for dosage modifications can be obtained.

[0087] Drug products are considered to be pharmacologic equivalents if they contain the same active ingredient and are identical in strength or concentration, dosage form, and route of administration. Two pharmacologic equivalent drug products are considered to be bioequivalent if the rate and extent of bioavailability of the active ingredient in the two products do not differ significantly under appropriate test conditions.

[0088] The term "therapeutic window" refers to a concentration range that provides therapeutic efficacy without unacceptable toxicity. After administration of a dose of a drug, its effect usually shows a characteristic temporal pattern. There is a lag period before the drug concentration exceeds the minimum effective concentration ("minimum effective concentration, MEC") for the desired effect. After the onset of the response, the intensity of the effect increases as the drug continues to be absorbed and distributed. This peaks, after which drug elimination results in a decrease in the intensity of the effect that disappears when the drug concentration returns below the MEC. The duration of the drug's action is therefore determined by the period during which the concentration exceeds the MEC. The therapeutic goal is to obtain and maintain a concentration within the therapeutic window for the desired response with minimal toxicity. A drug response below the MEC for the desired effect is subtherapeutic, while for adverse effects the probability of toxicity increases above the MEC. Increasing or decreasing the drug dosage shifts the response curve up or down the intensity scale, which is used to modulate the effect of the drug. Increasing the dose also extends the duration of action of the drug, but at the risk of increasing the likelihood of side effects. Therefore, unless a drug is non-toxic, increasing the dose is not a useful strategy for extending the duration of action of a drug.

[0089] Instead, another dose of the drug should be given to maintain the concentration within the therapeutic window. In general, the lower limit of a drug's therapeutic range appears to be approximately equal to the drug concentration that produces about half of the possible therapeutic effect, and the upper limit of the therapeutic range is such that more than about 5% to about 10% of patients experience toxic effects. These numbers are highly variable, and some patients may benefit greatly from drug concentrations above the therapeutic range, while others may suffer significant toxicity at much lower values. The therapeutic goal is to maintain steady-state drug levels within the therapeutic window. For most drugs, the actual concentrations associated with this desired range are not known, and need not be known; it is sufficient to understand that efficacy and toxicity are generally concentration-dependent, and how drug dosage and frequency of administration affect drug levels. For the few drugs where the difference between the concentrations that produce efficacy and those that produce toxicity is small (2- to 3-fold), a plasma concentration range associated with effective therapy has been defined.

[0090] When a target level strategy is warranted, a desired target steady state concentration of the drug (usually in plasma) associated with efficacy and minimal toxicity is selected, and the dosage expected to achieve this value is calculated. The drug concentration is then measured, and the dosage adjusted as necessary to move closer to the target.

[0091] In most clinical situations, drugs are administered in a series of repeated doses or as a continuous infusion to maintain a steady-state concentration of the drug that is associated with a therapeutic window. To maintain a selected steady-state or target concentration ("maintenance dose"), the drug administration rate is adjusted so that the input rate is equal to the loss rate. When the clinician selects the desired concentration of the drug in plasma and knows the clearance and bioavailability for that drug in a particular patient, the appropriate dose and dosing interval can be calculated.

[0092] The term "treat" or "treating" includes preventing, substantially inhibiting, slowing, or reversing the progression of a disease, condition, or disorder, substantially ameliorating the clinical or aesthetic symptoms of a condition, substantially preventing the appearance of clinical or aesthetic symptoms of a disease, condition, or disorder, and protecting against adverse or unpleasant symptoms. Treating further refers to achieving one or more of the following: (a) reducing the severity of the disorder, (b) limiting the onset of symptoms characteristic of the disorder being treated, (c) limiting the worsening of symptoms characteristic of the disorder being treated, (d) limiting the recurrence of the disorder in patients who previously had the disorder, and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic for the disorder.

[0093] The terms "variant," "mutant," and "derivative" are used herein to refer to a nucleotide sequence or polypeptide sequence that has substantial identity to a reference nucleotide sequence or polypeptide sequence. Differences in sequence may be the result of natural or designed changes in sequence or structure. Natural changes may arise during the normal course of replication or duplication in the nature of a particular nucleic acid sequence. Designed changes may be specifically designed and introduced into a sequence for a particular purpose. Such specific changes may be made in vitro using various mutagenesis techniques. Such specifically generated sequence variants may be referred to as "mutants" or "derivatives" of the original sequence.

[0094] One skilled in the art can similarly generate polypeptide variants of polypeptide SEQ ID NO:3 that have single or multiple amino acid substitutions, deletions, additions or substitutions, but are functionally equivalent to SEQ ID NO:3. These variants can include, in particular: (a) variants in which one or more amino acid residues are replaced 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 contains a substituent, (d) variants in which an amino acid residue from one species is replaced with the corresponding residue in another species, either at a conserved or non-conserved position, and (d) variants in which the target protein is fused to another peptide or polypeptide, such as a fusion partner, protein tag or other chemical moiety, e.g., an epitope for an antibody, that can confer a useful property to the target protein. Techniques for obtaining such variants, including but not limited to genetic techniques (such as suppressions, deletions, mutations), chemical techniques, and enzymatic techniques, are known to those skilled in the art. As used herein, the term "mutation" refers to a change in the DNA sequence within an organism's genes or chromosomes that results in the creation of new characteristics or traits not found in the parental type, either through a change in the nucleotide sequence of the DNA encoding the gene or through a change in the physical arrangement of the chromosome, or the process by which such changes occur in the chromosome. Three mechanisms of mutation include substitutions (exchange of one base pair for another), additions (insertion of one or more bases into a sequence), and deletions (loss of one or more base pairs).

[0095] The term "vector" as used herein refers to a carrier genetically engineered to deliver genes to cells. The term "viral vector" as used herein refers to a virus used as a vector to deliver a gene of interest by infecting cells. Such viruses are modified so that 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 of interest) into chromosomes in cells, and adenoviruses, which introduce their DNA (including the gene of interest) into the nucleus of cells without integrating into chromosomes.

[0096] As used herein, the term "vehicle" refers to a substance that facilitates the use of a drug or other material with which it is mixed.

[0097] According to some embodiments, the described invention provides a nucleotide sequence encoding a human soluble CD59 (sCD59) protein. According to some embodiments, the nucleotide sequence is a complementary DNA (cDNA) sequence.

[0098] The cDNA sequence encoding human CD59 is known in the art. For example, the cDNA sequence is reported by Sawada, R. et al. 1989 Nucleic Acids Res 17(16):6728 and is available from the American Type Tissue Culture Collection (ATCC, Manassas, Va.). The 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, LA et al. 1990 Eur J. Immol 21(3):847-850).

[0099] According to some embodiments, human sCD59 lacks the primary amino acid sequence of a functional glycosylphosphatidylinositol (GPI) anchor. According to some embodiments, human sCD59 comprises a modified GPI anchor domain amino acid sequence that is functionally deficient and lacks the ability to target to a membrane. According to some embodiments, the modified GPI anchor domain amino acid sequence comprises a mutation. Such mutations include, but are not limited to, substitutions and deletions of nucleic acids encoding amino acids at the omega positions used to reduce or eliminate GPI anchor binding or to reduce or eliminate effective functionality of the GPI anchor. The omega amino acid is the amino acid to which the GPI is transferred. For example, such mutations include, but are not limited to, substitutions of nucleic acids encoding hydrophobic leucine (e.g., nucleic acid CTG) and alanine (e.g., nucleic acid GCA) with the less hydrophobic (i.e., more hydrophilic) amino acids glycine (e.g., nucleic acid CAG) and glutamic acid (e.g., nucleic acid GAA). Alternatively, the mutation may involve substituting the omega residue with another amino acid (eg, substituting a tyrosine with a glycine).

[0100] According to some embodiments, the human sCD59 protein of the described invention comprises conservative sequence modifications. Conservative sequence modifications are amino acid modifications that do not significantly affect or change the characteristics of the human sCD59 protein containing the amino acid sequence, i.e., amino acid sequences of sCD59 that present these side chains in the same relative positions function in a manner similar to human sCD59. Such conservative modifications include amino acid substitutions, additions, and deletions. Methods for modifying amino acid sequences are known in the art (e.g., site-directed mutagenesis or PCR-based mutagenesis). Such techniques are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989. According to some embodiments, the human sCD59 protein of the described invention comprises conservative amino acid substitutions. Conservative amino acid substitution is a substitution 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. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0101] According to some embodiments, the human sCD59 amino acid sequence is an amino acid sequence that is substantially identical to the amino acid sequence of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 70% identical to the amino acid sequence of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 75% identical to the amino acid sequence of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 80% identical to the amino acid sequence of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 90% identical to the amino acid sequence of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 95% identical to the amino acid sequence of the wild-type sequence.

[0102] According to some embodiments, the human sCD59 comprises SEQ ID NO: 3. According to some embodiments, the human sCD59 consists essentially of SEQ ID NO: 3. According to some embodiments, the human sCD59 is SEQ ID NO: 3.

[0103] According to some embodiments, the human sCD59 of the described invention is a recombinant protein.

[0104] A variety of commercially available expression vector / host systems are useful for containing and expressing sequences encoding CD59 proteins. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmids, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems contacted with viral expression vectors (e.g., baculovirus); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transfected with bacterial expression vectors (e.g., Ti, pBR322, or pET25b plasmids); or animal cell systems. See Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.

[0105] Techniques for altering nucleic acid sequences to produce recombinant proteins are well known in the fields of genetics and molecular biology. Conventional strategies for recombinant protein expression involve transfecting cells with a DNA vector containing a template for expressing the desired protein, then culturing the cells so that they transcribe and translate the desired protein. The cells are then lysed to extract the expressed protein for subsequent purification.

[0106] Cell types 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. Non-limiting examples of bacterial cells include Escherichia coli. Eukaryotic cells include, but are not limited to, mammalian, insect, yeast and algal 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., NS0) and mouse non-producing hybridoma cells (e.g., SP2 / O-Ag14). Non-limiting examples of insect cells include Spodoptera frugiperda pupa ovary cells (e.g., Sf9, Sf21). Non-limiting examples of yeast cells include Saccharomyces cerevisiae. Non-limiting examples of algal cells include Chlamydomonas reinhardtii.

[0107] Methods for expressing recombinant proteins also include cell-free systems. Cell-free protein expression involves the in vitro production of recombinant proteins in solution (i.e., cell lysates) using biomolecular translation machinery extracted from cells.

[0108] Various methods of protein purification may be used and are known in the art and are described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step or steps selected will depend, for example, on the production process used and the properties of the particular protein produced.

[0109] According to some embodiments, human sCD59 is a synthetic protein. Methods for preparing synthetic proteins are well known in the art. See, for example, Peptide Synthesis Protocols, Methods in Molecular Biology, vol. 35, Pennington, MW and Dunn, BM, 1995, XII, Humana Press, Inc. Totowa, New Jersey. Synthetic proteins, prepared using well-known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, may contain natural and unnatural amino acids. The amino acids used in peptide synthesis can be standard Boc amino acid resins, or base-labile N-α-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids as originally described by Carpino and Han (1972, J. Org. Chem. 37:3403-3409), using standard deprotection, neutralization, coupling and washing protocols of the original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154). Both Boc and Fmoc amino protected amino acids can be obtained from Sigma or other chemical companies familiar to those skilled in the art. Peptides can also be synthesized using other N-α protecting groups familiar to those skilled in the art.

[0110] According to some embodiments, a nucleotide sequence encoding human sCD59 is used to construct an expression vector. According to some embodiments, 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 an expression vector containing a nucleotide sequence encoding human sCD59 protein operably linked to appropriate transcriptional and translational control elements. These methods include, but are not limited to, for example, 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, NY, 1989.

[0111] In some embodiments, the nucleotide sequence encoding human sCD59 is operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a cell cycle specific promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the promoter is a tissue specific promoter. Examples of tissue specific promoters include, but are not limited to, human rhodopsin kinase (hRK) promoter and retinal pigment epithelium specific promoter (e.g., RPE65 promoter). In some embodiments, the promoter is a metabolically regulated promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promote is a hybrid promoter. A non-limiting example of a hybrid promoter is the cytomegalovirus (CMV) early enhancer element / first exon and first intron of chicken beta-actin gene / splice acceptor of rabbit beta-globin gene (CAG). Non-limiting examples of promoters are provided in U.S. Pat. No. 6,677,311 (B1) to Evans et al., issued on Jan. 13, 2004, U.S. Pat. No. 7,109,029 (B2) to Clark et al., issued on Sep. 19, 2006, and U.S. Pat. No. 5,998,205 to Hallenbeck et al., issued on Dec. 7, 1999, each of which is incorporated by reference in its entirety herein.

[0112] According to some embodiments, a nucleotide sequence encoding human sCD59 operably linked to a promoter is packaged into a delivery vector. According to some embodiments, a human sCD59 expression construct is packaged into a delivery vector.

[0113] According to some embodiments, the delivery vector is a viral vector, including but not limited to adenoviral vectors, lentiviral vectors, adeno-associated viral (AAV) vectors, and helper-dependent adenoviral vectors.

[0114] Adenoviral vectors are commercially available from the American Type Tissue Culture Collection (Manassas, Va.). Methods for constructing and using adenoviral vectors are described in Klein et al. 2007 Ophthalmology 114:253-262, and van Lecuwen et al. 2003 Eur. J. Epidemiol. 18:845-854. Adenoviral vectors have been used for 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, NJ, 109-128). Additionally, recombinant adenovirus vectors are used in gene therapy (US Pat. No. 7,235,391 to Wu et al., issued Jun. 26, 2007, which is incorporated by reference in its entirety).

[0115] Recombinant adenovirus vectors are generated, for example, from homologous recombination between shuttle vectors and provirus vectors (Wu et al., U.S. Patent No. 7,235,391, issued June 26, 2007). The adenovirus vectors used herein are replication-defective. For example, the adenovirus vectors are conditionally defective and lack the adenovirus E1 region. The polynucleotide encoding the protein of interest (e.g., human sCD59) is introduced into the position where the E1 coding sequence is removed. Alternatively, the polynucleotide encoding the protein of interest (e.g., human sCD59) can be inserted into the E3 region of adenovirus.

[0116] Defective adenoviral vectors can be generated and propagated using helper cell lines. Helper cell lines can 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. Alternatively, helper cells can be derived from cells of other mammalian species that are permissive for human adenoviruses (e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells). The generation and propagation of these replication-defective adenoviral vectors using helper cell lines is described in Graham et al 1977 J.Gen.Virol.36:59-72.

[0117] Lentiviral packaging vectors are commercially available from Invitrogen Corporation (Carlsbad Calif.). HIV-based packaging systems for the production of lentiviral vectors are 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. Numerous vector constructs are available that are packaged using a system based on the third generation lentiviral SIN vector backbone (Dull et al. 1998 J.Virol. 72:8463-8471). For example, 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, an HIV packaging signal, an RRE sequence linked to a marker gene cassette consisting of Aequora jellyfish green fluorescent protein (GFP) driven by the CMV promoter, and a woodchuck hepatitis virus PRE element that appears to enhance nuclear export. The GFP marker gene allows quantification of transfection or transduction efficiency by UV fluorescence microscopy or direct observation by flow cytometry (Kafri et al. 1997 Nature Genet. 17:314-317, and Sakoda et al. 1999 J. Mol. Cell. Cardiol. 31:2037-2047).

[0118] Manipulation of retroviral nucleic acid to construct retroviral vectors containing a gene of interest (e.g., a gene encoding the human sCD59 protein) and packaging cells is accomplished using techniques known in the art (e.g., see 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, NY; Miller, et al., Biotechniques. 7:981-990, 1989; Eglitis, et al., 1993, each of which is incorporated by reference in its entirety. (see, e.g., U.S. Pat. Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767, and 5,124,263, as well as WO 85 / 05629, WO 89 / 07150, WO 90 / 02797, WO 90 / 02806, WO 90 / 13641, WO 92 / 05266, WO 92 / 07943, WO 92 / 14829, and WO 93 / 14188).

[0119] Retroviral vectors can be constructed and packaged into non-infectious transducing viral particles (virions) using amphotropic packaging systems. 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. Patent Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767, and 5,124,263, as well as WO 85 / 05629, WO 89 / 07150, WO 90 / 02797, WO 90 / 02806, WO 90 / 13641, WO 92 / 05266, WO 92 / 07943, WO 92 / 14829, and WO 93 / 14188, each of which is incorporated by reference in its entirety. Generation of "producer cells" can be achieved by introducing a retroviral vector into a packaging cell. Examples of such retroviral vectors are found, for example, in Korman et al. 1987 Proc. Natl. Acad. Sci. USA. 84:2150-2154, Morgenstern et al. 1990 Nucleic Acids Res. 18:3587-3596, U.S. Pat. Nos. 4,405,712, 4,980,289, and 5,112,767, as well as WO 85 / 05629, WO 90 / 02797, and WO 92 / 07943.

[0120] Herpesvirus packaging vectors are commercially available from Invitrogen Corporation, (Carlsbad, Calif.). Exemplary herpesviruses include, but are not limited to, alpha-herpesviruses such as varicella zoster virus or pseudorabies virus; herpes simplex viruses such as HSV-1 or HSV-2; or herpesviruses such as Epstein-Barr virus. A method for preparing empty herpesvirus particles that can be packaged with a desired nucleotide segment (e.g., human sCD59 nucleotide or polynucleotide sequence) in the absence of a helper virus that is competent against most herpesviruses is described in Fraefel et al. (U.S. Patent No. 5,998,208, issued December 7, 1999, which is incorporated by reference in its entirety).

[0121] Herpesvirus DNA vectors can be constructed using techniques known to those skilled in the art. For example, DNA segments encoding the entire genome of herpesviruses are divided into 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, NY, 1989) or E. coli F element plasmids (O'Conner et al. 1989 Science 244:1307-1313), among many other vectors capable of carrying large DNA segments. For example, a set of cosmids containing overlapping clones representing the entire genome of various herpesviruses, including Epstein-Barr virus, varicella-zoster virus, pseudorabies virus and HSV-1, have been isolated. See M. van Ziji et al. 1988 J. Virol. 62:2191, Cohen et al. 1993 Proc. Nat'l Acad. Sci. USA90:7376, Tomkinson et al. 1993 J. Virol. 67:7298, and Cunningham et al. 1993 Virology 197:116.

[0122] Adeno-associated virus (AAV) is a dependent parvovirus in that it depends on co-infection with another virus (either an adenovirus or a member of the herpesvirus family) to undergo productive infection in cultured cells (Muzyczka 1992 Curr. Top. Microbiol. Immunol., 158:97 129). For example, recombinant AAV (rAAV) viruses can be generated by co-transfecting a plasmid containing a gene of interest (e.g., the human sCD59 gene) flanked by 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 a wild-type AAV coding sequence without the terminal repeats. The cells are also contacted with or transfected with adenovirus or plasmids carrying the adenoviral genes required for AAV helper function.

[0123] Unlike most viruses, AAV is essentially non-pathogenic, has low immunogenicity, and is broadly tropic, making it an attractive gene delivery candidate for virus-based gene therapy. Most naturally occurring AAV utilize glycan moieties for initial attachment to cell surfaces, and these interactions have been well characterized for several serotypes. Identified interacting glycan moieties include AAV serotype 2 (AAV2), AAV3, AAV6, and AAV8; N-terminal galactose in the case of AAV9; and specific N- or O-linked sialic acid moieties in the case of AAV1, -4, -5, and -6. Serotypes vary according to the cell type they infect, making AAV a highly useful system for preferentially transducing specific cell types.

[0124] 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).

[0125] Methods for constructing and using AAV vectors are known in the art. Such methods are described, for example, in U.S. Patent No. 5,139,941 (Wu et al.), issued on June 26, 2007, and U.S. Patent No. 4,797,368 (Carter et al.), issued on January 10, 1989. The use of AAV in gene delivery is further described in LaFace et al. 1988 Virology 162(2):483 486, Zhou et al. 1993 Exp. Hematol, 21:928-933, Flotte et al. 1992 Am. J. Respir. Cell Mol. Biol. 7(3):349-356, and Walsh et al. 1994 J. Clin. Invest 94:1440-1448.

[0126] Recombinant AAV vectors have been used for in vitro and in vivo transduction of marker genes (Kaplitt et al. 1994 Nat Genet.,8(2):148-154; Lebkowski et al. 1988 Mol. Cell. Biol.8(10):3988-3996; Samulski et al. 1991 EMBO J.10:3941-3950; Shelling and Smith 1994 Gene Therapy,1:165-169; Yoder et al. 1994 Blood,82(Supp.):1:347A; Zhou et al. 1993 Exp. Hematol 21:928-933; Tratschin et al. 1985 Mol. Cell. Biol.5:3258-3260; McLaughlin et al. 1988 J. Virol. 62(6):1963-1973) as well as for the transduction of genes involved in human disease (Flotte et al. 1992 Am. J. Respir. Cell Mol. Biol. 7(3):349-356; Ohi et al. 1990 Gene, 89(2):279-282; Walsh et al. 1994 J. Clin. Invest. 94:1440-1448; and Wei et al. 1994 Gene Therapy, 1:261 268).

[0127] According to some embodiments, the nucleotide sequence encoding human sCD59 operably linked to a promoter is packaged into an adeno-associated virus (AAV) vector. According to some embodiments, the AAV vector is AAV2. According to some embodiments, the AAV vector is AAV5. According to some embodiments, the AAV vector is AAV8.

[0128] According to some embodiments, the human sCD59 expression vector is packaged in an adeno-associated virus (AAV) vector. According to some embodiments, the AAV vector is AAV2. According to some embodiments, the AAV vector is AAV5. According to some embodiments, the AAV vector is AAV8.

[0129] According to some embodiments, the nucleotide sequence encoding human sCD59 is packaged between inverted terminal repeat (ITR) sequences in an AAV vector. According to some embodiments, the nucleotide sequence encoding human sCD59 operably linked to a promoter is packaged between inverted terminal repeat (ITR) sequences in an AAV vector. According to some embodiments, the human sCD59 expression vector is packaged between inverted terminal repeat (ITR) sequences in an AAV vector. According to some embodiments, the ITR sequences are AAV2 sequences. According to some embodiments, the ITR sequences are AAV5 sequences. According to some embodiments, the ITR sequences are AAV8 sequences.

[0130] According to some embodiments, the AAV vector is a hybrid vector. The hybrid vector contains an ITR sequence from one AAV serotype and a capsid protein from a different AAV serotype. According to some embodiments, the hybrid vector comprises an ITR sequence from AAV2 and a capsid protein from AAV5 (AAV2 / 5). According to some embodiments, the hybrid vector comprises an ITR sequence from AAV2 and a capsid protein from AAV8 (AAV2 / 8). According to some embodiments, the hybrid vector comprises an ITR sequence from AAV5 and a capsid protein from AAV2 (AAV5 / 2). According to some embodiments, the hybrid vector comprises an ITR sequence from AAV5 and a capsid protein from AAV8 (AAV5 / 8). According to some embodiments, the hybrid vector comprises an ITR sequence from AAV8 and a capsid protein from AAV2 (AAV8 / 2). According to some embodiments, the hybrid vector comprises ITR sequences from AAV8 and capsid proteins from AAV5 (AAV8 / 5).

[0131] According to some embodiments, the delivery vector is a non-viral vector. For example, the delivery vector is a synthetic gene delivery vehicle or vector that is not associated with a viral particle and specifically delivers genetic material to a target cell or tissue. Examples of non-viral vectors include, but are not limited to, liposomes, peptides, nanoparticles, emulsions, or encapsulated two or more phase systems or other suitable preparations. Thus, according to some embodiments, the described invention provides a non-viral vector with a nucleic acid that is loaded and contacted with a tissue or cell. As an example, naked DNA encoding a human sCD59 protein with a modified GPI anchor that is not membrane targeted, or a liposome containing a gene encoding a human sCD59 protein without a GPI anchor, is encapsulated within the liposome and the liposome is contacted with the tissue or cell so that the nucleic acid is effectively delivered to the tissue or cell.

[0132] According to some embodiments, the described invention provides a pharmaceutical composition. According to some embodiments, the pharmaceutical composition comprises a human sCD59 protein comprising a full length nucleic acid of CD59 modified to remove the signal sequence for attachment of the GPI anchor at the nucleotide encoding the amino acid asparagine at position 77. According to some embodiments, the nucleic acid sequence of the human sCD59 protein is modified by point mutation, substitution or deletion to obtain a nucleic acid sequence encoding an amino acid sequence having a modified amino acid sequence at the GPI anchor position, such that the protein cannot be attached to a cell membrane.

[0133] According to some embodiments, the described invention provides a pharmaceutical composition comprising a nucleic acid encoding CD59 or a human sCD59 protein expression source. In various embodiments, the CD59 protein comprises a membrane-independent (i.e., soluble) CD59 protein. According to some embodiments, the pharmaceutical composition is formulated as an ophthalmic formulation for administration to the eye. According to some embodiments, the pharmaceutical composition is formulated to enhance delivery to the fundus. According to some embodiments, the pharmaceutical composition is formulated to provide a localized sustained release to the retina. According to some embodiments, the pharmaceutical composition is formulated to provide an effective treatment of blood vessels and / or tissues involved in ocular diseases. According to some embodiments, the ocular disease is age-related macular degeneration (AMD). According to some embodiments, the AMD is wet or exudative AMD. According to some embodiments, the AMD is dry AMD or geographic atrophy (GA).

[0134] According to some embodiments, the pharmaceutical compositions of the described invention are formulated sufficiently pure for administration to a human subject, for example to the eye of a human subject. According to some embodiments, the pharmaceutical composition comprises one or more additional therapeutic agents. According to some embodiments, the one or more additional therapeutic agents are selected from the group consisting of growth factors, anti-inflammatory agents, and hypertensive agents, including but not limited to nitric oxide and calcium channel blockers, collagenase inhibitors, steroids (e.g., prednisolone), matrix metalloproteinase inhibitors, ascorbate, angiotensin H, angiotensin III, calreticulin, tetracycline, fibronectin, collagen, thrombospondin, transforming growth factor (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), IGF binding protein (IGFBP), epidermal growth factor (EGF), platelet derived growth factor (PDGF), neu differentiation factor (NDF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VAG), hepatocyte growth factor (HGF ... The one or more additional therapeutic agents include, but are not limited to, an anti-tumor agent, an anti-viral agent, an anti-bacterial agent, an anti-mycobacterial agent, an anti-fungal agent, an anti-proliferative agent, or an anti-apoptotic agent. Therapeutic agents that can be included in the pharmaceutical compositions of the described invention are well known in the art.See, e.g., Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman, et al., eds., McGraw-Hill, 1996, the contents of which are incorporated herein by reference in their entirety.

[0135] According to some embodiments, the one or more additional therapeutic agents are compounds, compositions, biologics, etc. According to some embodiments, the one or more additional therapeutic agents enhance, stabilize, synergize, or replace the ability of the human sCD59 protein to protect cells from MAC deposition. According to some embodiments, the one or more additional therapeutic agents are provided simultaneously with the pharmaceutical composition comprising the human sCD59 protein. According to some embodiments, the one or more additional therapeutic agents are provided after the pharmaceutical composition comprising the human sCD59 protein. According to some embodiments, the one or more additional therapeutic agents are provided before the pharmaceutical composition comprising the human sCD59 protein. According to some embodiments, the one or more additional therapeutic agents are used to treat the same, concurrent, or related symptoms, conditions, or diseases.

[0136] According to some embodiments, the pharmaceutical compositions of the described invention include a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, any and all solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, suitable for 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 their preparation. Some examples of materials that may serve as pharma- ceutically 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 as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, and 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 may also be present in the composition, according to the judgment of the formulator.

[0137] According to some embodiments, the described invention provides a method for treating a complement disorder (e.g., AMD). According to some embodiments, the method comprises contacting a cell or tissue with a pharmaceutical composition comprising a source of human sCD59 protein. According to some embodiments, the pharmaceutical composition comprises a nucleotide sequence encoding human sCD59 operably linked to a promoter. According to some embodiments, the pharmaceutical composition comprises a human sCD59 expression construct. According to some embodiments, the pharmaceutical composition comprises a nucleotide sequence encoding human sCD59 operably linked to a promoter packaged in a delivery vector. According to some embodiments, the pharmaceutical composition comprises a human sCD59 expression construct packaged in a delivery vector.

[0138] According to some embodiments, the human sCD59 protein is administered as a recombinant protein.

[0139] Without wishing to be bound by theory, it is understood that the plasma membrane of cells is normally protected from the effects of complement by cell surface proteins (e.g., CD59) that specifically inhibit activation of the C5b-9 pore upon binding of the C9 complement protein to membrane C5b-8 (Holguin et al. 1989 J. Clin. Invest. 84:7-17; Sims et al. 1989 J. Biol. Chem. 264:19228-19235; Davies et al. 1989 J. Exp. Med. 170:637-654; Rollins et al. 1990 J. Immunol. 144:3478-3483; and Hamilton et al. 1990 Blood 76:2572-2577). CD59 competes with the C9 complement protein for binding to the C8 complement protein in the C5b-8 complex, thereby reducing or preventing the formation of the C5b-9 membrane attack complex. Thus, CD59 acts to reduce both cell activation and cell lysis by the terminal complement MAC.

[0140] Theories have linked the cause of diseases such as age-related macular degeneration (AMD) to activation of the complement system and formation of MAC. Dinu (U.S. Patent Application No. 2007 / 0196367(A1), published August 23, 2007) has proposed preventing debris formation by inhibiting complement as a treatment for AMD.

[0141] Diseases associated with unregulated complement activity include bacterial infections such as those caused by Haemophilus influenza, Streptococcus pnemoniae, Neisseria meningitidis, etc.; angioedema; renal diseases, e.g., atypical hemolytic uremic syndrome; paroxysmal nocturnal hemoglobinuria; systemic lupus erythematosus; central nervous system diseases, including, but not limited to, Alzheimer's disease, Huntington's disease, and diseases of the retina, including age-related macular degeneration (AMD).

[0142] According to some embodiments, human soluble CD59 (sCD59) is effective in inhibiting MAC formation. According to some embodiments, MAC formation is inhibited by delivering a vector containing a nucleic acid encoding human sCD59 to a cell.

[0143] According to some embodiments, human sCD59 is effective in treating AMD. According to some embodiments, AMD is treated by delivering a vector containing a nucleic acid encoding human sCD59 to a cell. According to some embodiments, human sCD59. According to some embodiments, human sCD59 is effective in preventing the onset of AMD. According to some embodiments, the onset of AMD is prevented by delivering a vector containing a nucleic acid encoding human sCD59 to a cell. According to some embodiments, human sCD59 is effective in preventing the progression of AMD. According to some embodiments, the progression of AMD is prevented by delivering a vector containing a nucleic acid encoding human sCD59 to a cell. According to some embodiments, human sCD59 is effective in reversing the progression of AMD. According to some embodiments, the progression of AMD is reversed by delivering a vector containing a nucleic acid encoding human sCD59 to a cell. According to some embodiments, the AMD is wet or exudative AMD. According to some embodiments, the AMD is dry AMD or geographic atrophy (GA).

[0144] According to some embodiments, human sCD59 is effective in attenuating choroidal neovascularization (CNV). According to some embodiments, human sCD59 is delivered by an approach using a gene therapy method that is effective in attenuating choroidal neovascularization (CNV). According to some embodiments, human sCD59 is effective in reducing the degree of MAC deposition on CNV spots. According to some embodiments, the degree of MAC deposition on CNV spots is reduced by delivering a vector containing human sCD59-encoding nucleic acid into cells.

[0145] According to some embodiments, human sCD59 prevents lysis of retinal cells. According to some embodiments, lysis of retinal cells is prevented by delivering a vector containing a nucleic acid encoding human sCD59 to the cells.

[0146] According to some embodiments, human sCD59 is delivered by an adeno-associated virus (AAV) vector. According to some embodiments, the adeno-associated virus vector is AAV2. According to some embodiments, the adeno-associated virus is AAV5. According to some embodiments, the adeno-associated virus vector is AAV8. According to some embodiments, the AAV vector is a hybrid vector comprising an ITR sequence from AAV2 and a capsid protein from AAV5 (AAV2 / 5). According to some embodiments, the AAV vector is a hybrid vector comprising an ITR sequence from AAV2 and a capsid protein from AAV8 (AAV2 / 8). According to some embodiments, the AAV vector is a hybrid vector comprising an ITR sequence from AAV5 and a capsid protein from AAV2 (AAV5 / 2). According to some embodiments, the AAV vector is a hybrid vector comprising an ITR sequence from AAV5 and a capsid protein from AAV8 (AAV5 / 8). According to some embodiments, the AAV vector is a hybrid vector comprising ITR sequences from AAV8 and capsid proteins from AAV2 (AAV8 / 2). According to some embodiments, the AAV vector is a hybrid vector comprising ITR sequences from AAV8 and capsid proteins from AAV5 (AAV8 / 5).

[0147] According to some embodiments, the AAV vector is administered by injection. According to some embodiments, the injection is subretinal. According to some embodiments, the injection is intravitreal. According to some embodiments, the injection is a single injection. According to some embodiments, the injection is multiple injections.

[0148] Without wishing to be bound by theory, it is believed that contacting cells with a vector containing a nucleic acid encoding human sCD59 produces a subset of cells that are "factories" for the local production and secretion of sCD59 that can protect adjacent ocular cells, including retinal pigmented epithelial (RPE) cells and choroidal blood vessels.

[0149] Compositions and methods using nucleotide sequences encoding human sCD59 provide additional advantages over protein-based delivery methods. Peptides have a limited in vivo half-life and need to be readministered periodically. Current treatments for wet AMD include, for example, intraocular ranibizumab antibody injections every 4-6 weeks. This treatment method exposes patients to complications such as endophthalmitis and associated pathologies. The incidence of endophthalmitis is relatively low (0.16% per dose) in the presence of a strong immune system. However, the rate of endophthalmitis increases substantially due to a weakened complement system and the cumulative effect of continuous injections over many years, such as for the treatment of chronic diseases such as AMD. Thus, frequent injections of complement inhibitors into the eyes of AMD patients are undesirable or ineffective. The pharmaceutical compositions and methods described herein limit the frequency of injections, thus providing a safer and more effective treatment for subjects suffering from complement disorders such as AMD.

[0150] Viral vectors such as adenoviral vectors have been used to provide lifelong expression of transgenes in mice in vivo.For example, AAV vectors have promoted transgene expression in dogs for more than 7 years.In humans, AAV has been found to have therapeutic transgene expression for more than 3.7 years, the longest period studied.Adenovirus has been found to be an efficient vector for the delivery of transgenes to ocular tissues and has been found to be safe in some ocular gene therapy trials.Adenoviral vectors engineered for long-term transgene expression and techniques for large-scale 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 to be less immunogenic than adenoviral vectors.

[0151] According to some embodiments, delivery of human sCD59 to the eye of AMD patients using AAV vectors is effective for long-term transgene expression.

[0152] According to some embodiments, the described invention provides a pharmaceutical composition for treating AMD, comprising a vector carrying a nucleotide sequence encoding a recombinant engineered human sCD59 protein operably linked to a promoter sequence that causes expression of the protein in a cell, such that the nucleotide sequence carries at least one mutation that confers loss of glycosylphosphatidylinositol (GPI) anchor function, such that the protein is expressed as a recombinant membrane-independent (i.e., soluble) CD59 protein and is not targeted to the membrane. According to some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable buffer. According to some embodiments, the AMD is wet or exudative AMD. According to some embodiments, the AMD is dry or GA.

[0153] According to some embodiments, the pharmaceutical composition is formulated sterilely for ocular delivery. According to some embodiments, the pharmaceutical composition formulated for sterile ocular delivery is in a dose effective to treat AMD.

[0154] According to some embodiments, the pharmaceutical composition formulated for ocular delivery further comprises at least one of a pharma- ceutically acceptable buffer, a pharma- ceutically acceptable salt, and a pharma- ceutically acceptable emollient suitable for delivery by at least one route selected from intraocular injection, subconjunctival injection, sub-Tenon injection, eye drops, and ointments.

[0155] According to some embodiments, the vector is at least one of an engineered viral vector recombinantly linked to a nucleotide sequence encoding a sCD59 protein and a synthetic gene delivery vector for delivery of a nucleotide sequence encoding human sCD59. According to some embodiments, the viral vector is selected from the group consisting of an adenovirus, an adeno-associated virus, a herpes virus, a pox virus, and a lentivirus. According to some embodiments, 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.

[0156] According to some embodiments, the pharmaceutical composition further comprises a peptide for overall delivery (POD), the pharmaceutical composition being operatively linked to the compound such that the POD comprises a protein transduction domain (PTD) to obtain a conjugated compound. For example, the POD composition is as set forth in International Application PCT / US2008 / 010179, filed August 28, 2008, to Kumar-Singh et al., or U.S. Patent Application Publication No. 2010 / 0209447, published August 19, 2010, to Kumar-Singh et al., each of which is incorporated herein by reference in its entirety.

[0157] According to some embodiments, the pharmaceutical composition comprises a dose of viral vector particles administered to the affected eye. According to some embodiments, the dose of viral particles is about 1×10 7 ~Approx. 1×10 9 According to some embodiments, the dose of viral particles is in the range of about 1×10 8 ~Approx. 1×10 10 According to some embodiments, the dose of viral particles is in the range of about 1×10 9 ~Approx. 1×10 11 According to some embodiments, the dose of viral particles is in the range of about 1×1011 ~Approx. 1×10 12 According to some embodiments, the dose of viral particles is in the range of about 1×10 11 ~Approx. 1×10 13 According to some embodiments, the pharmaceutical composition further comprises at least one therapeutic agent selected from the group consisting of anti-inflammatory agents, anti-tumor agents, anti-viral agents, anti-bacterial agents, anti-mycobacterial agents, anti-fungal agents, anti-proliferative agents, and anti-apoptotic agents. According to some embodiments, the dose of viral particles is in the range of about 1×10 10 DNase resistant particles (DRP) ~ approx. 1 × 10 12 According to some embodiments, the dose of viral particles is about 3.56×10 10 According to some embodiments, the dose of viral particles is about 1.071 x 10 11 According to some embodiments, the dose of viral particles is about 3.56×10 11 According to some embodiments, the dose of viral particles is about 1.07×10 12 It is DRP.

[0158] According to some embodiments, the pharmaceutical composition comprises a promoter sequence. According to some embodiments, the promoter sequence is a universal ubiquitous promoter for expression in mammalian cells. According to some embodiments, the promoter is a promoter from a gene encoding actin, polyhedron, or hydroxyl-methylglutaryl CoA reductase (HMGCR). Such promoters include, but are not limited to, chicken beta-actin promoter or human beta-actin promoter. According to some embodiments, 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, rhodopsin promoter or eye or liver tissue-specific promoters.

[0159] According to some embodiments, 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 mutations that encode amino acids of the glycosylphosphatidylinositol (GPI) anchor domain of the protein, such that the resulting vector encodes an engineered recombinant membrane-independent (i.e., soluble) CD59 (sCD59) protein, the vector being a viral vector or a synthetic gene delivery vector; and contacting at least one ocular tissue of the subject with the composition, such that cells of the tissue locally express and secrete CD59, thereby treating the subject for AMD.

[0160] According to some embodiments, the viral vector is derived from a genetically engineered genome of at least one virus selected from the group consisting of adenovirus, adeno-associated virus, herpes virus, and lentivirus.

[0161] According to some embodiments, 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.

[0162] According to some embodiments, contacting at least one ocular tissue of the subject further comprises injecting by a route selected from the group consisting of intravitreal, subretinal, subconjunctival, subtenon's; subcutaneous and intravenous. According to some embodiments, the tissue contacted by the pharmaceutical composition comprises at least one tissue selected from the group consisting of the retinal pigment epithelium, retina, choroid, sclera, Bruch's membrane, and choroidal blood vessels.

[0163] According to some embodiments, the described invention provides a method of modulating complement activity or treating a complement activity disorder in a subject, the method comprising contacting a diseased tissue or organ of a subject at risk for or suffering from a complement activity disorder with a composition comprising a vector carrying a nucleotide sequence encoding a recombinant engineered human sCD59 protein operably linked to a promoter sequence causing expression of the protein in a cell, such that the protein comprises at least one mutation that results in loss of function of the glycosylphosphatidylinositol (GPI) anchor domain, such that the protein is recombinant membrane-independent (i.e., soluble) CD59 (sCD59) and is not targeted to a membrane, observing a physiological indicator of a complement activity disorder after the contact compared to an abnormal amount of the physiological indicator observed before the contact, such that a reduction after the contact compared to before the contact is a positive indication that the diseased tissue or organ is treated.

[0164] According to some embodiments, the diseased tissue is selected from the group consisting of epithelial tissue, endothelial tissue and vascular tissue. According to some embodiments, the diseased organ is selected from the group consisting of eye, heart, kidney, lung, liver, pancreas and vasculature. According to some embodiments, the subject is a tissue or organ donor or recipient. According to some embodiments, the subject is an immunocompromised patient who is an organ recipient.

[0165] According to some embodiments, the described method includes 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, paroxysmal nocturnal hemoglobinuria, systemic lupus erythematosus, atherosclerosis, and the like. According to some embodiments, the AMD is wet or exudative AMD. According to some embodiments, the AMD is dry AMD or geographic atrophy (GA). According to some embodiments, the disorder is AMD. According to some embodiments, the AMD is dry AMD (GA). According to some embodiments, observing further includes measuring an index selected from the group consisting of visual acuity, visual aberration, and amount of MAC deposits.

[0166] According to some embodiments, the described invention provides a method of treating a complement disorder, comprising contacting a tissue or cell with a pharmaceutical composition. According to some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition having as an active agent a nucleic acid encoding human sCD59 protein or a source of expression of human sCD59 protein, in an amount and for a time necessary to achieve a desired result. According to some embodiments, the method comprises treating AMD by contacting ocular tissue or cells with human sCD59 protein or a vector encoding human sCD59 protein.

[0167] According to some embodiments, the pharmaceutical composition is administered using any amount and any route of administration effective for treating AMD or other complement-related diseases and conditions. Thus, the phrase "an amount effective for treating AMD" as used herein refers to an amount of the pharmaceutical composition sufficient to beneficially prevent or ameliorate the symptoms of AMD.

[0168] The exact dosage of the pharmaceutical composition can be selected by the individual physician in consideration of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active agent or to maintain the desired effect. Additional factors that can be considered include the severity of the disease state (e.g., intermediate or advanced stages of AMD); the age, weight and sex 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 can be administered once, hourly, twice hourly, every 3-4 hours, once daily, twice daily, every 3-4 days, every week, or once every two weeks, depending on the half-life and clearance rate of the particular composition.

[0169] The active agents of the described invention may be formulated in unit dosage form for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. For any active agent, the therapeutically effective dose can be estimated first either in cell culture assays or animal models (usually mice, as provided herein, but potentially also rats, rabbits, dogs or pigs). Such information can then be used to determine doses and routes useful for administration to humans. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used to formulate a range of dosages for use in humans.

[0170] Therapeutic effective dose refers to the amount of active agent that improves symptoms or condition or prevents the progression of AMD.The therapeutic efficacy and toxicity of active agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal to 50% of the population).The dose ratio of toxic effect to therapeutic effect is therapeutic index, and can be expressed as LD50 / ED50 ratio.

[0171] The daily dosage of the pharmaceutical composition may vary over a wide range, such as from 0.001 to 100 mg per adult human per day. For ocular administration, the pharmaceutical composition 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 (μg) of the active ingredient, for symptomatic adjustment of the dosage to the patient being treated.

[0172] A unit dose typically contains about 0.001 micrograms to about 500 micrograms of active ingredient, about 0.1 micrograms to about 100 micrograms of active ingredient, or about 1.0 micrograms to about 10 micrograms of active ingredient. An effective amount of drug can be supplied at a dosage level of about 0.0001 mg / kg body weight to about 25 mg / kg body weight per day. For example, the range can be about 0.001 to 10 mg / kg body weight per day, or about 0.001 mg / kg body weight to 1 mg / kg body weight per day. The pharmaceutical composition can be administered on a regimen of, for example, 1 to 4 times per day, or more. The unit dose can be divided and administered, for example, in two or more divided doses.

[0173] According to some embodiments, the source of expression of human sCD59 protein is administered as a dose of viral or 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. The number of cells can be calculated from the area of ​​retina requiring treatment by methods known to those skilled in the art. According to some embodiments, the dose is about 1×10 10 DNase resistant particles (DRP) ~ approx. 1 × 10 12 According to some embodiments, the dose is about 3.56×10 10 According to some embodiments, the dose is about 1.071 x 10 11 According to some embodiments, the dose is about 3.56×10 11 According to some embodiments, the dose is about 1.07×1012 It is DRP.

[0174] According to some embodiments, the source of human sCD59 protein is administered by ocular injection, including but not limited to intraocular injection into the aqueous or vitreous humor, or into the outer layers of the eye, such as by subconjunctival or sub-Tenon injection.

[0175] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. According to some embodiments, the injectable preparations are sterile injectable preparations. The sterile injectable preparations may be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents (e.g., solutions in 1,3-butanediol). Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, USP standard, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. Any brand of fixed oil can be used, including but not limited to synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectable preparations. The injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0176] According to some embodiments, the sterile injectable preparations contain excipients, including, but not limited to, suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia), dispersing or wetting agents, including naturally occurring phosphatides (e.g., lecithin), or condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate).

[0177] A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). A solution is generally considered to be a homogeneous mixture of two or more substances. It is often, but not necessarily, liquid. In a solution, the molecules of the solute (or dissolved substance) are uniformly distributed among the molecules of the solvent. A suspension is a dispersion (mixture) in which a finely divided species is combined with another species, the former being so finely divided and mixed that it does not settle rapidly. In daily life, the most common suspension is the suspension of a solid in a liquid. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media. For parenteral application, particularly 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 that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound, allowing for the preparation of highly concentrated solutions. Alternatively, the active compound may be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0178] According to some embodiments, the described invention provides liquid dosage forms for ocular injection. Such liquid dosage forms include, but are not limited to, pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active agent, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing and emulsifying agents (e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide), oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluents, the ocular delivery pharmaceutical composition may also contain adjuvants, such as wetting agents and emulsifying and suspending agents.

[0179] According to some embodiments, the pharmaceutical compositions of the described invention may be in the form of a sterile injectable aqueous or oleaginous suspension. 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.

[0180] According to some embodiments, the described inventions include ophthalmic, surgical, audiological devices or products containing the disclosed compositions (e.g., gauze bandages or strips), and methods of making or using such devices or products. These devices may be coated, impregnated, associated with, or otherwise treated with the pharmaceutical compositions described herein.

[0181] According to some embodiments, the described invention provides administering a pharmaceutical composition to a subject. According to some embodiments, the administering step comprises oral administration, topical administration or parenteral administration. According to some embodiments, the parenteral administration is selected from the group consisting of intravitreal injection and subretinal injection.

[0182] According to some embodiments, the administering step comprises administering the pharmaceutical composition as a single dose or multiple doses. According to some embodiments, the administering step comprises administering the pharmaceutical composition as a single dose. According to some embodiments, the single dose is administered to the eye of the subject in need thereof. According to some embodiments, the subject in need thereof is afflicted with AMD. According to some embodiments, the subject in need thereof is afflicted with wet or exudative AMD. According to some embodiments, the subject in need thereof is afflicted with GA.

[0183] According to some embodiments, the compositions are administered in a pharma- ceutically acceptable solution that may routinely contain pharma- ceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic agents.

[0184] According to some embodiments, the pharmaceutical composition is an aqueous suspension or emulsion mixed with excipients suitable for the manufacture of aqueous suspensions and emulsions. Such excipients include, but are not limited to, suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia. The dispersing or wetting agent may be, for example, a naturally occurring phosphatide such as lecithin, or a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), or a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), or a condensation product of ethylene oxide with a fatty acid and a partial ester derived from a hexitol (e.g., polyoxyethylene sorbitol monooleate), or a condensation product of ethylene oxide with a fatty acid and a partial ester derived from a hexitol anhydride (e.g., polyethylene sorbitan monooleate).

[0185] Solutions or suspensions used for parenteral, intradermal, subcutaneous, intrathecal, or topical application may contain, but are not limited to, a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. A particular carrier for intravenous administration is saline or phosphate buffered saline (PBS).

[0186] Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents (e.g., solutions in 1,3-butanediol). Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, USP standard, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used or are used as solvents or suspending media. For this purpose, any brand of fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectable preparations.

[0187] Preparations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the preparation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents.The preparations may be presented in single-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carriers, for example saline, water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0188] Suspensions may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.

[0189] The pharmaceutical compositions of the described invention may further comprise conventional excipients, i.e., pharma- ceutically acceptable organic or inorganic carrier substances suitable for parenteral application that do not adversely react with the active compounds.Suitable pharma-ceutically acceptable carriers include, but are not limited to, water, saline, alcohol, vegetable oils, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, flavor oils; fatty acid mono- and diglycerides, petroleum fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0190] The pharmaceutical compositions of the described invention may be sterilized and, if desired, mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavorings and / or aromatic substances, which do not adversely react with the active compounds. For parenteral application, suitable vehicles include solutions (e.g., oily or aqueous solutions), as well as suspensions, emulsions, or implants. Aqueous suspensions may contain substances that increase the viscosity of the suspension, including, but not limited to, sodium carboxymethylcellulose, sorbitol and / or dextran. Optionally, suspensions may also contain stabilizers. These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, such as, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0191] According to some embodiments, pharmaceutical compositions of the described invention comprise a therapeutically effective amount of human sCD59 and optionally other therapeutic agents in a pharma- ceutically acceptable carrier. According to some embodiments, the components of the pharmaceutical compositions also are capable of being commingled in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

[0192] According to some embodiments, the pharmaceutical compositions of the described invention include pharma- ceutically acceptable salts. Pharmaceutically acceptable salts are salts that are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, PH Stahl, et al. describe pharma- ceutically acceptable salts in detail in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley VCH, Zurich, Switzerland: 2002).

[0193] When a range of values ​​is provided, each intervening value (to the tenth of the unit of the lower limit between the upper and lower limits of that range and any other stated ranges, or each value between that stated range, unless the context clearly indicates otherwise) is understood to be encompassed in the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also intended to be encompassed in the invention, as if any limit could be specifically excluded from a stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also intended to be included in the invention.

[0194] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, the preferred methods and materials are described below. All publications mentioned herein are incorporated herein for the purpose of disclosing and describing the methods and / or materials to which the publications are pertinent.

[0195] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. All technical and scientific terms used herein have the same meaning.

[0196] Publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the described invention(s) are not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may require independent confirmation. EXAMPLES

[0197] The following examples are provided so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only ones performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be allowed for. Unless otherwise indicated, parts are by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0198] Example 1: Adenoviral Vector Constructs Expressing CD59 An adenoviral vector construct expressing human soluble CD59 (sCD59) was prepared as described in U.S. Patents 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 encoding the C-terminal 26 amino acids, including the signal sequence for attachment of a glycosylphosphatidylinositol (GPI) anchor, was PCR amplified using a forward primer containing an XhoI site (5'ccccctcgagtggacaatcacaatggg3'; SEQ ID NO:1) and a reverse primer with an EcoRV site (5'taaggagatatcttaattttcaagctgttcgtta3'; SEQ ID NO:2). The reverse primer introduced a stop codon after asparagine 77, resulting in a sequence encoding the soluble form of human CD59 (sCD59) (MGIQGGSVLFGLLLVLAVFCHSGHSLQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNKCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLEN; SEQ ID NO: 3). 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 the 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. Epidemiol. 18:845-854).The GPI signal was thus recombinantly removed to obtain a construct expressing a soluble, secreted form of human CD59.

[0199] Example 2: A Phase 1 / 2a, Open-Label, Single-Site, Dose-Escalation Safety and Tolerability Study of a Single Intravitreal Injection of AAVCAGsCD59 in Patients With Advanced Nonexudative (Dry) Age-Related Macular Degeneration With Geographic Atrophy In this study, we conducted an open-label, non-randomized, phase I dose-escalation study to establish the safety of a single intravitreal injection of the gene therapy vector AAVCAGsCD59 (adeno-associated viral vector serotype 2) expressing soluble CD59 (sCD59), an inhibitor of the membrane attack complex (MAC), for the treatment of patients with advanced dry age-related macular degeneration (AMD) with geographic atrophy (GA). The study schema is shown in Figure 1. The planned total sample size is approximately 26 participants. Seventeen participants were ultimately enrolled. The objectives and endpoints of this study are listed in Table 1. The number of participants (planned and analyzed) is listed in Table 2.

[0200] [Table 1]

[0201] [Table 2]

[0202] Study population; inclusion / exclusion criteria The study population consisted of adult males or females aged 50 years 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 three participants, and then a BCVA Snellen equivalent of 20 / 80 or worse in the study eye after the first three participants. Total GA lesion size was 5 mm in the study eye. 2 (2 disc area (DA))~20mm 2 (8DA) and had a BCVA of 20 / 800 or better in the fellow eye.

[0203] Participants were excluded if they had GA secondary to non-AMD etiologies, previous or active choroidal neovascularization (CNV) in the study eye, active or uncontrolled glaucoma, were or were likely candidates for intraocular surgery in the study eye, or had acute or chronic infection in the study eye.

[0204] Participant trends Seventeen participants (100%) completed the study at week 26, and 16 participants (94.1%) completed the 2-year long-term follow-up study (Table 2). One participant (5.9%) discontinued the study early due to death unrelated to the study intervention (Table 2).

[0205] Demographic and other baseline characteristics A higher proportion of participants were women (64.7%), and all participants were white. The mean age was 81 years (range 69–95 years) (Table 3). The mean body mass index (BMI) was 28.5 kg / m 2 (20-39kg / m 2 The mean BMI was higher in cohort 1 than in cohort 2 or 3 (27.7 kg / m2, respectively). 2 and 27.5 kg / m 2 32.9kg / m 2 .

[0206] [Table 3-1]

[0207] [Table 3-2]

[0208] Baseline ocular characteristics Test Eye All participants had advanced dry AMD with GA, with a mean duration from first diagnosis of AMD of 12.13 years (range 2.25-42.67 years). Mean visual acuity was 37.26 ETDRS letters overall. Per protocol, participants in Cohort 1 had lower mean visual acuity than participants in either Cohort 2 or 3 (18.67 ETDRS letters [Cohort 1], 50.50 ETDRS letters [Cohort 2], and 38.73 ETDRS letters [Cohort 3]). Mean IOP was similar across all cohorts, with a mean IOP of 13.82 mmHg. Table 4 summarizes baseline ocular characteristics for the study eyes.

[0209] [Table 4]

[0210] Fellow Eye Fifteen of the 17 eyes had a history of advanced dry AMD with GA in the fellow eye. The mean duration of AMD from initial diagnosis was 13.02 years (range 2.25-42.67 years). The mean BCVA in the fellow eye was 44.00, 59.17, and 59.59 ETDRS letters in cohorts 1, 2, and 3, respectively, with an overall mean BCVA of 56.76 ETDRS letters. Intraocular pressure (IOP) was within the normal range (mean 13.88 mmHg) and similar across all cohorts.

[0211] GA lesions Test Eye The mean baseline GA lesion size in the study eyes was 11.12 mm 2 and were similar across the three cohorts. All participants had GA lesions involving the fovea. Fundus autofluorescence (FAF) patterns at the junctional zone included striped (11 participants [64.7%]), diffuse (4 participants [23.5%]), and focal (2 participants [11.8%]). No CNV was noted in the study eye by fluorescein angiography (FA).

[0212] [Table 5]

[0213] Fellow Eye Fourteen participants (82.4%) had GA in the fellow eye. Thirteen of these participants (92.9%) had lesions involving the fovea, and one participant (7.1%) had foveal-sparing GA. The mean GA area was 10.02 mm. 2 FAF patterns at the junctional zone included striped (8 participants [57.1%]), diffuse (5 participants [35.7%]), and focal (1 participant [7.1%]). Two participants (11.8%) had CNV by FA, ​​and 1 participant could not be graded.

[0214] Optical Coherence Tomography (OCT) Features Test Eye Overall, OCT features were consistent with participants with GA. Mean OCT central subfield thickness was 182.41 μm across all participants. Four participants had pigment epithelial detachment (PED) with a mean thickness of 143.25 μm. No participants had intraretinal or subretinal fluid or CNV by OCT. Mean drusen volume within a 5 mm circle was 0.05 mm across all participants. 3 It was.

[0215] Fellow Eye The mean OCT central subfield thickness was 211.96 μm across all participants. Eight participants had PED with a mean thickness of 150.63 μm. One participant had subretinal fluid and two participants had intraretinal fluid. Twelve (70.6%) participants did not have CNV on optical coherence tomography angiography (OCTA). Four participants had CNV type 1 on OCTA and one participant had CNV that could not be graded. The mean drusen volume by OCT in the central 5 mm circle was 0.11 mm across all participants. 3 It was.

[0216] Previous and Concomitant Therapies The most common previous therapy categories according to the WHO ATC classification included vitamins (94.1%), diuretics (58.8%), lipid-modifying agents (58.8%), and antithrombotic agents (52.9%). The most common previous ocular therapies were PT macrogol 400-based ophthalmic agents (29.4%) and propylene glycol-based ophthalmic agents (17.6%).

[0217] The most common concomitant therapy categories according to the WHO ATC classification included cardiovascular (58.8%) (e.g. diuretics, lipid modifying agents, and renin-angiotensin system antihypertensives), systemic anti-infectives (52.9%, e.g. systemic antibiotics), and ophthalmic (52.9%, with artificial tears [a catchall term] being the most common PT [23.5%]) treatments.

[0218] Medical history All participants had a diagnosis of AMD with GA in the study eye according to the inclusion criteria. The most common extraocular medical history was hypertension (76.5%) and arthritis (58.8%). The most common ocular procedure performed in both the study eye (82.4%) and fellow eye (82.4%) of participants was lens extraction with posterior chamber intraocular lens implantation.

[0219] Study intervention The study interventions administered to participants are outlined in Table 6. The study intervention was administered to the eye that met the I / E criteria and had the worse visual acuity. Three participants received dose 1 (3.56 × 10 10 DRP) and 3 participants received dose 2 (1.07 × 10 11 DRP), and 11 participants received dose 3 (3.56 × 10 11 received the DRP.

[0220] [Table 6] a The highest dose of AAVCAGsCD59 resulted in slow mobilization. After cohort 3, no doses were administered to any study participants as the responsible parties were enrolled. b The label contained information that met applicable regulatory requirements.

[0221] Distribution of AAVCAGsCD59 vector Quantitative polymerase chain reaction (PCR) was performed to detect the presence of AAVCAGsCD59 RNA in serum at baseline, day 7, week 4, week 12, and week 26. Of the 17 participants, three participants had quantifiable sCD59 in serum at any time point, and one additional participant had detectable AAVCAGsCD59 RNA below the lower limit of quantification (LLOQ) at a single time point. None of the participants had detectable sCD59 in serum at baseline. Two participants had quantifiable values ​​of sCD59 at day 7 that were below the LLOQ by week 4. One participant had quantifiable levels of sCD59 at week 4 that were undetectable by week 12. All participants were included in the high-dose cohort.

[0222] AAV2 serum neutralizing antibody (NAb) titers AAV2 neutralizing antibody titers were detected in all participants at baseline and were highly variable (ranging from 1:5.10 to 1:50819.74). Seven participants had baseline titers less than 1:100, five participants had titers between 1:100 and 1:10000, and five participants had titers greater than 1:10000. Nine of 17 participants had a baseline 4-fold increase in titers at any time point (termed "treatment-boosted NAb"). One participant had one time point greater than 4-fold from baseline, but subsequent phases were less than 4-fold above baseline (termed "transient positive"). There was no clear relationship between the occurrence of intraocular inflammation and either high baseline AAV2 neutralizing titers or changes from baseline AAV2 neutralizing titers after study intervention.

[0223] Anti-sCD59 serum antibody titers Participants did not have serum anti-sCD59 antibodies either at baseline or at any other measurement during the study.

[0224] Test evaluation / measurement Safety assessments included the following: Adverse events (AEs) and serious adverse events (SAEs), Physical examination and vital signs, Clinical laboratory measurements, including hematology, liver function tests, kidney function tests, blood chemistry, urinalysis, and pregnancy tests; Previous therapies and concomitant medications, Distance visual acuity test: Best corrected visual acuity (BCVA) using the Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chart; Intraocular pressure (IOP), Biomicroscopy, - Dilated examination of the lens, retina and fovea, Spectral domain optical coherence tomography (SD-OCT), Spectral domain optical coherence tomography angiography (SD-OCTA), Fundus autofluorescence (FAF) imaging, Color Fundus Photograph (CFP), Fluorescein angiography (FA), Serum anti-AAV2 antibody titers, Serum AAVCAGsCD59 vector distribution, Serum anti-sCD59 antibodies, and Aqueous sCD59 levels.

[0225] Assessment of response to study intervention Adverse events Table 7 summarizes overall systemic treatment-emergent adverse events (TEAEs) by dose level. Overall, 16 (94.1%) participants experienced one or more TEAEs, one participant (5.9%) died during the study, and nine (52.9%) participants experienced one or more SAEs. None of the systemic TEAEs, systemic SAEs, or deaths were considered related to treatment interventions.

[0226] [Table 7]

[0227] Incidence of systemic adverse events by organ system class The most frequently reported preferred terms (PTs) for systemic (non-ocular) TEAEs were urinary tract infection (five participants [29.4%]), falls (five participants [29.4%]), and bradycardia (three participants [17.6%]). The remaining PTs were reported by one or two participants. Table 8 provides an overview of systemic TEAEs reported in at least two participants. There was no clustering of AEs within one or more system organ classes (SOCs). None of these TEAEs were related to the study intervention.

[0228] [Table 8]

[0229] The majority of TEAEs were mild in severity. Nine TEAEs were severe and occurred in the following SOCs: cardiac disorders: 2 events; gastrointestinal disorders: 1 event; injury, toxic, and procedural complications: 1 event; benign, malignant, and unspecified neoplasms: 3 events; respiratory, thoracic, and mediastinal disorders: 1 event; vascular disorders: 1 event. One participant died on study day 244. This participant had an ongoing history of white blood cell disorders since 2016. The cause of death was reported as leukemia and was determined by the investigator to be unrelated to the study intervention.

[0230] Serious Adverse Events Nine participants (52.9%) experienced one or more serious adverse events (SAEs). None of the SAEs were considered related to the study intervention. SAEs were not clustered in a specific SOC. The SOCs with the most frequently reported SAEs were benign, malignant, and unspecified neoplasms, injury, toxic, and procedural complications, and cardiac disorders, with each SOC having three participants (17.6%). No TEAEs led to discontinuation of the study.

[0231] Ocular Adverse Events Test Eye Thirteen participants (76.5%) experienced ocular TEAEs in the study eye (Table 9). None of these TEAEs were considered SAEs. Nine of the AEs did not resolve by the end of the study. All ocular TEAEs in the study eye were mild in severity, except for one moderate AE (basal cell tumor [reported term: basal cell on lower right eyelid]). The number of TEAEs in the study eye was balanced between treatment groups. Twelve participants (70.6%) reported a study eye TEAE in the Eye Disorders SOC. Vitritis was reported in four participants (23.5%, all in cohort 3), and anterior chamber inflammation was reported in one participant (5.9%). Increased intraocular pressure was reported in two participants (11.8%). Retinal hemorrhage was reported in two participants (11.8%). The remaining PTs occurred in only one participant. Visual acuity loss was reported in three participants (17.6%, all in Cohort 3) (Table 10). None of the participants who reported a visual acuity loss AE had a clinically significant loss of 15 letters or more in the study eye at the two consecutive visits at the time of the AE.

[0232] Six participants (35.3%) reported ocular adverse events in the study eye that were deemed related to study treatment by the investigator. Five of the participants were included in cohort 3 and one participant was included in cohort 2. PTs for related events were anterior chamber inflammation (one participant), optic neuropathy (one participant), and vitritis (four participants) (Table 11). Anterior chamber inflammation (reported term: mild post-injection anterior chamber inflammation) and optic neuropathy (reported term: worsening cup-disc ratio bilaterally [oculus uterque, OU]) were in the same participant. All TEAEs in the study eye were mild, except for one moderate TEAE (basal cell carcinoma) in the study eyelid.

[0233] both eyes There were 5 TEAEs present in both eyes: 2 participants had a bilateral adverse event of optic neuropathy (reported term: worsening cup-to-disc ratio), 1 participant had allergic conjunctivitis, 1 participant had seasonal allergies, and 1 participant had an adverse event of cataract (reported term: worsening cataract).

[0234] [Table 9]

[0235] [Table 10]

[0236] [Table 11]

[0237] Fellow Eye Nine participants (52.9%) experienced ocular TEAEs in the fellow eye. None of the TEAEs were SAEs. One participant (Cohort 3) reported an optic neuropathy adverse event in the fellow eye that was deemed possibly related to study treatment by the investigator. Seven participants (41.2%) reported fellow eye TEAEs in the ocular disorder SOC. The most frequently reported PTs were dry eye (2 participants [11.8%]) and optic neuropathy OU (2 participants [11.8%]). Both terms reported for optic neuropathy were worsening cup-disc ratio. Both optic neuropathy adverse events were bilateral events. All remaining PTs were reported only once. There were no ocular inflammatory AEs in the fellow eye.

[0238] There was one severe TEAE in the fellow eye (PT: Skin disorder [reported term: superior scaly erythematous lesion of upper eyelid OS]) and one moderate TEAE in the fellow eye (PT: Retinal tear [reported term: Horseshoe tear of the retina]).

[0239] Ocular adverse events of interest The following AEs were considered of interest: ·Endophthalmitis, Intraocular inflammation, Increased intraocular pressure, ·Eye bleeding, Cataracts, and · Changes in retinal structure, deposits and degeneration.

[0240] These categories consisted of specific PTs as determined by the responsible party.

[0241] In addition to the above categories, preferred terms for ocular TEAEs were reviewed and the following adverse events of interest were included: Optic neuropathy Retinopathy ·Neovascular AMD, Retinal artery embolism, and -Vitreous floaters.

[0242] Test Eye Thirteen AEs of interest in nine participants were experienced in the study eye (Table 12). None of these events occurred in cohort 1. Five of these AEs were classified as intraocular inflammation (29.4%) with a PT of anterior chamber inflammation (cohort 2, 1 event, onset on day 27, duration 24 days) and vitritis (4 events, all in cohort 3). The 4 events of vitritis had onset dates of day 45 (duration 47 days), day 20 (duration 346 days), day 34 (duration 170 days), and day 29 (unresolved due to participant death during study). All intraocular inflammatory AEs were considered potentially related to study treatment by the investigator and were mild in severity. The anterior chamber inflammation event was treated with difluprednate from day 28 to day 70, and the 1 event of vitritis was treated with prednisolone ophthalmic from day 57 to day 85. The other 3 events of vitritis did not occur in treated participants.

[0243] Two AEs of transient intraocular pressure were reported in two participants, both of which occurred either on the same day or the day after the onset of the intraocular inflammatory adverse event. Both were mild in severity and were deemed by the investigator to be unrelated to the study intervention. The onset of these events occurred on days 28 and 34. One participant experienced an increase in IOP from 12 mmHg on day 15 to 21 mmHg on day 28. The pressure remained the same on day 21 but decreased to 14 mmHg by day 34. The participant did not receive any treatment for the increased intraocular pressure. Another participant experienced an increase in IOP from 17 mmHg on day 9 to 28 mmHg on day 34. The pressure decreased to 10 mmHg on day 43. The participant received brimonidine tartrate / timolol eye drops from days 34 to 42. Both participants recovered from the transient increase in intraocular pressure within 10 days, and both participants ultimately recovered from their intraocular inflammatory AEs.

[0244] Three AEs in two participants were classified as ocular hemorrhages. All that occurred in the study eye were mild in severity and were considered unrelated to the study intervention by the investigator. One of the ocular hemorrhages was a retinal hemorrhage that developed on day 727, and at the end of the study, the participant had not yet recovered. The other two ocular hemorrhages were in the same participant and were vitreous hemorrhages that developed on day 2. The reported terms for these two events were a small vitreous hemorrhage in the right eye (resolved on day 14) and loss of 16 VA letters secondary to a small vitreous hemorrhage (resolved on day 28), both coded to a PT of vitreous hemorrhage. Although the vitreous hemorrhage was considered unrelated to the study intervention, the relationship of the AE to the study procedure was not captured in this study. However, vitreous hemorrhage is an AE of intravitreous injections observed in clinical practice and clinical trials.

[0245] One AE was classified as cataract. This AE occurred on Day 120, was mild in severity, and was deemed unrelated to the study intervention by the investigator. The participant had a history of ongoing ocular cataract since 2009. The reported term for the event was worsening cataract, which remained unresolved at the end of the study.

[0246] There were two AEs in two participants: worsening of cup-to-distal diameter ratio (PT: optic neuropathy). Both participants experienced this adverse event in both the study and fellow eyes. One participant experienced an adverse event with onset on day 281, which was deemed by the investigator to be unrelated to the study intervention and was pending at the end of the study. The other participant experienced an adverse event with onset on day 244, which was deemed by the investigator to be possibly related to the study intervention and was pending at the end of the study. This participant did not experience any other concomitant ocular adverse events of interest. There were no cases of endophthalmitis in the study eye.

[0247] [Table 12]

[0248] Fellow Eye Seven AEs of interest in four participants were reported in the fellow eye. Six of the seven were mild in severity and one was moderate in severity (PT retinal tear). All AEs except one were considered by the investigator to be unrelated to the study intervention. The AE of worsening cup-disc ratio (PT: optic neuropathy) occurring on day 244 was considered by the investigator to be possibly related to the study intervention. This AE was still ongoing at the end of the study. This participant also had the same AE in the study eye. Another AE of worsening cup-disc ratio in both eyes was experienced by another participant. The PTs of the remaining AEs, occurring in one participant each, were retinal artery embolism, neovascular age-related macular degeneration, retinopathy, and cataract. One participant had an AE of retinal artery embolism (reported term: Hollenhorst plaque) occurring on day 457. The events were mild in severity, not considered related by the investigator, and the participants did not recover by the end of the study. No cases of endophthalmitis in the fellow eye were reported.

[0249] Clinical Laboratory Evaluation There were no clinically significant changes in the clinical data. The majority of laboratory values ​​were grade 0 or grade 1 (according to NCI-CTCAE grading). One participant (Cohort 3) had toxicity grade 4 neutropenia at week 4. This participant had toxicity grade 3 neutropenia at baseline, day 7, and week 12. Neutrophil levels returned to within normal range at the end of study (day 190). In addition, this participant had toxicity grade 2 or 3 lymphopenia from baseline to the end of study (day 190). This participant had a fatal AE of leukemia at day 244 (section 5.1.2.2).

[0250] Other safety assessments Vital signs / physical assessment There were no clinically significant changes in vital sign measurements over time from baseline in this study.There were no clinically significant changes in physical examinations related to the study intervention in this study.

[0251] Intraocular pressure (IOP) Baseline IOP ranged from 8 to 21.50 mmHg for the study eye and 9.5 to 21.50 mmHg for the fellow eye. The mean change in IOP from baseline was less than 2 mmHg for all cohorts in the study eye at weeks 26 and 104. The mean change in IOP from baseline was less than 2.5 mmHg for all cohorts in the fellow eye at weeks 26 and 104. The highest mean change in IOP from baseline was 3 mmHg from baseline for the study eye and 2.5 mmHg from baseline for the fellow eye.

[0252] Two participants experienced a transient increase in IOP AEs of interest. Both participants experienced intraocular inflammatory AEs of interest either on the same day as the increase in IOP adverse events or the day before. One participant received no treatment, while the other received intraocular brimonidine tartrate / timolol.

[0253] Slit lamp examination In general, slit lamp examinations were consistent with the demographics of the participant population. There were no clinically significant changes in slit lamp examinations related to the study intervention, except for those noted in the AEs of interest listed above, in either the study or fellow eye.

[0254] Indirect / Dilated Ophthalmoscopy Other than the above-mentioned AEs of interest, there were no clinically significant changes related to the study intervention on indirect / dilated ophthalmoscopy in either the study or fellow eye.

[0255] Secondary evaluation Growth rate of GA lesions Individual participant data for change in GA growth from baseline in square-root transformed values ​​in the study eye are shown in Figure 2. Baseline GA lesion area in square-root transformed values ​​(mm) was 3.4 mm (range 2.85; 3.79) in cohort 1, 3.373 mm (range 3.01; 3.58) in cohort 2, and 3.222 mm (range 2.34; 4.42) in cohort 3. The mean change from baseline at week 26 was 0.153 mm in cohort 1, 0.263 mm in cohort 2, and 0.338 mm in cohort 3. The mean change from baseline at week 104 was 0.517 mm in cohort 1, 0.56 mm in cohort 2, and 0.487 mm in cohort 3.

[0256] Mixed model repeated measures (MMRM) was used to analyze GA growth over time for each of the dosing arms, with a pooled analysis of all cohorts (Figures 3 and 4). The model incorporated baseline lesion size, dose level, selected visit, and dose level (cohort) by visit interaction as covariates. Given the limited sample size, formal analyses looking at differences in GA growth by dosing arm were not performed. There were no apparent differences in GA growth by dose.

[0257] The square-root transformed GA lesion area in the fellow eye was 2.473 mm (range 0.85; 3.54) in cohort 1, 3.52 mm (range 3.41; 3.63) in cohort 2, and 3.041 mm (range 1.25; 4.76) in cohort 3. The mean change from baseline at week 104 ranged from 0.527 to 1.124 mm across the three cohorts.

[0258] Change in area of ​​GA lesions over time Baseline GA lesion area was similar across dosing arms. The mean baseline GA lesion area was 11.72 mm in cohort 1. 2 (range 8.15; 14.39), and 11.467 mm in cohort 2. 2(9.08;12.84), and 10.865 mm in cohort 3. 2 The mean mean mean GA growth rate was 1.018 (5.47; 19.50). The percent change from baseline at week 26 was 9.235 in cohort 1, 16.037 in cohort 2, and 14.888 in cohort 3. The percent change from baseline at week 104 was 32.572 in cohort 1, 35.830 in cohort 2, and 34.406 in cohort 3. Due to small numbers, there was no formal analysis of dosing arm differences by GA growth rate. MMRMs estimating change in GA lesion size for each dosing cohort and the pooled cohort are shown in Figures 5 and 6.

[0259] Conversion of dry AMD to wet AMD No participants in any of the cohorts converted from dry to wet AMD (defined as new presence of CNV) in the study eye as assessed by FA, ​​OCT, and OCTA. One participant in cohort 2 converted from dry to wet AMD in the fellow eye on day 83. This was likely due to the natural history of the disease and was not related to the study intervention.

[0260] Drusen volume Across all participants, there were no clinically significant changes in drusen volume in the study or fellow eye.

[0261] eyesight Individual participant data for visual acuity scores over time for the study eye are shown in Figure 7. Of note, the mean baseline BCVA in ETDRS letters for cohort 1 was lower than for cohorts 2 and 3. The mean BCVA for cohort 1 was 18.667 letters (range 13-26), for cohort 2 50.5 letters (48-53), and for cohort 3 38.727 letters (22.5-56.50). The mean change from baseline at week 26 was +3.667 letters, -0.500 letters, and -1.636 letters in cohorts 1, 2, and 3, respectively. The change from baseline at week 104 was +4.33 letters, -3.833 letters, and -6.40 letters in cohorts 1, 2, and 3, respectively. No significant trends in visual acuity differences between the three dose cohorts were observed.

[0262] The mean baseline BCVA in the fellow eye was lower for Cohort 1 compared to Cohorts 2 and 3. The mean baseline BCVA in the fellow eye in Cohort 1 was 44.000 letters (28.50-71.00), in Cohort 2 it was 59.167 letters (50.50-70.00), and in Cohort 3 it was 59.591 letters (35.50-78.00). 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 are shown in (Figure 8).

[0263] The number and percentage of participants with at least one loss of 10 or more letters, 15 or more letters, 20 or more letters, and 30 or more letters in distance BCVA from baseline over time are listed in Table 13. With regard to clinically significant changes in vision, two participants lost 15 or more letters at least once in the study eye. No participants lost 30 or more letters in the study eye. In the fellow eye, six participants (two in each cohort) lost 15 or more letters and two lost 30 or more letters at least once.

[0264] Consistent with the natural history of the disease, no participant gained 15 or more letters of visual acuity in either the study or fellow eye.

[0265] [Table 13]

[0266] Exploratory evaluation Intraocular (aqueous humor) sCD59 protein levels Aqueous humor levels of sCD59 were determined using a custom Western blot assay using a commercially available anti-CD59 rabbit polyclonal antibody (Abcam, Cambridge UK, Catalogue no. ab124396). Aqueous humor was collected at baseline before injection and at week 8. No participant had detectable levels of aqueous humor sCD59 protein before administration of the study intervention. Aqueous humor sCD59 protein was detected in 5 of 17 participants at week 8. Protein levels were highly variable, ranging from 250ng / ml to 5719.4ng / ml. All participants with detectable sCD59 protein were included in the high-dose cohort. Western blot analysis was not available for one participant (could not be electrophoresed for unknown reasons).

[0267] In summary, the three doses of AAVCAGsCD59 tested in this study were safe and well tolerated without dose-limiting toxicity. The most clinically significant AE related to the study intervention was intraocular inflammation, which occurred in 29.4% of participants. However, intraocular inflammation in all participants was mild and either self-limited or resolved with topical steroids. There were no systemic TEAEs related to the study intervention, which is consistent with the low systemic exposure of AAV2CAGsCD59 observed in serum. Production of the gene therapy product, sCD59, could be detected in the aqueous humor of a subset of participants.

[0268] Although the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular condition, material, composition of matter, process, process step or steps to the intended spirit and scope of the invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

1. 1. A pharmaceutical composition for use in a method for treating age-related macular degeneration (AMD) in a subject, the pharmaceutical composition comprising a nucleic acid encoding a soluble CD59 (sCD59) protein operably linked to a promoter, the nucleic acid encoding sCD59 being packaged in a delivery vector, the method comprising administering the pharmaceutical composition to an AMD-affected eye of the subject by ocular injection, wherein the administration results in expression and secretion of the sCD59 protein by cells in the AMD-affected eye, and wherein the expression results in treatment of AMD-affected cells in the AMD-affected eye.

2. The pharmaceutical composition of claim 1, wherein the AMD is geographic atrophy (GA).

3. The pharmaceutical composition of claim 1 , wherein the ocular injection is an intravitreal injection.

4. The pharmaceutical composition of claim 2 , wherein the intravitreal injection is a single injection.

5. 2. The pharmaceutical composition of claim 1, wherein the delivery vector is an adeno-associated virus (AAV) vector.

6. The pharmaceutical composition of claim 4, wherein the AAV vector is AAV2.

7. The pharmaceutical composition of claim 1 , wherein the promoter is a CAG promoter.

8. The pharmaceutical composition comprises about 3.56 x 10 １０ DNAse-resistant particles (DRP), approximately 1.071 x 10 １１ DRP, approximately 3.56×10 １１ DRP and approximately 1.07 x 10 １２ 10. The pharmaceutical composition of claim 1, comprising a dose of viral particles selected from the group consisting of DRP.

9. 1. A pharmaceutical composition for use in a method for modulating impaired complement activity in a subject, the pharmaceutical composition comprising a vector carrying a nucleotide sequence encoding a recombinantly engineered human soluble CD59 (sCD59) protein operably linked to a promoter sequence that causes expression of the recombinantly engineered human soluble CD59 (sCD59) protein in diseased cells, the method comprising contacting diseased cells of the subject with the pharmaceutical composition, wherein the sCD59 protein contains at least one mutation that causes a loss of function of a glycosylphosphatidylinositol (GPI) anchor domain, resulting in loss of membrane targeting, and observing a physiological sign of impaired complement activity after the contacting, compared to an abnormal amount of the physiological sign observed before the contacting, wherein a decrease after the contacting compared to before the contacting is a positive indication that the diseased cells are treated.

10. The pharmaceutical composition of claim 9, wherein the complement activity disorder is GA.

11. The pharmaceutical composition of claim 9, wherein the contacting is by intravitreal injection.

12. The pharmaceutical composition of claim 11 , wherein the intravitreal injection is a single injection.

13. The pharmaceutical composition of claim 9, wherein the diseased cells are retinal cells.

14. The pharmaceutical composition of claim 9, wherein the vector is AAV2.

15. 10. The pharmaceutical composition of claim 9, wherein the physiological indicia is best corrected visual acuity (BCVA).

16. 16. The pharmaceutical composition of claim 15, wherein BCVA is measured as the mean change from baseline.

17. 17. The pharmaceutical composition of claim 16, wherein the mean change from baseline is -7.100 letters.

18. The pharmaceutical composition comprises about 3.56 x 10 １０ DNAse-resistant particles (DRP), approximately 1.071 x 10 １１ DRP, approximately 3.56×10 １１ DRP and approximately 1.07 x 10 １２ 10. The pharmaceutical composition of claim 9, comprising a dose of viral particles selected from the group consisting of DRP.

19. 1. A pharmaceutical composition for use in a method for treating a complement disorder, wherein the pharmaceutical composition has as an active agent a nucleic acid encoding a human sCD59 protein or a source of expression of a human sCD59 protein, the method comprising administering the pharmaceutical composition to a subject in need thereof, the method comprising contacting a cell with a therapeutically effective amount of the pharmaceutical composition.

20. 20. The pharmaceutical composition of claim 19, wherein the complement disorder is GA.

21. 20. The pharmaceutical composition of claim 19, wherein the contacting is by intravitreal injection.

22. 22. The pharmaceutical composition of claim 21, wherein the intravitreal injection is a single injection.

23. 20. The pharmaceutical composition of claim 19, wherein the diseased cells are retinal cells.

24. The therapeutically effective amount is about 3.56×10 １０ DNAse-resistant particles (DRP), approximately 1.071 x 10 １１ DRP, approximately 3.56×10 １１ DRP and approximately 1.07 x 10 １２ 20. The pharmaceutical composition of claim 19, wherein the viral particles are in a dose selected from the group consisting of DRP.

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