Methods for treating macular degeneration

Abstract

Disclosed herein are methods of treating geographic atrophy (GA) that is secondary to age-related macular degeneration (AMD).

Description

METHODS FOR TREATING MACULAR DEGENERATIONTECHNICAL FIELDThe present disclosure relates generally to methods of treating geographic atrophy (GA) that is secondary to age-related macular degeneration (AMD).BACKGROUND

[0001] Age-related macular degeneration (AMD) is the leading cause of blindness in patients over 60 years of age in developed countries, affecting approximately 1 in 4 people over 70 years of age. Globally, AMD accounts for approximately 9% of all blindness, affects approximately 196 million people and is projected to affect 288 million by 2040. Age-related macular degeneration is a slow and progressive disease of the macula. The dry (nonexudative) form of AMD comprises the majority (-90%) of AMD patients. The wet (exudative) form of AMD accounts for the other -10% of cases. The most significant loss of vision occurs in advanced forms of AMD such as wet AMD and GA. Geographic atrophy is characterized by progressive and irreversible loss of photoreceptors, RPE and choriocapillaris. It is typically bilateral, and enlargement of the atrophy leads to irreversible visual function loss. It can affect central visual acuity as well as reading, driving, and low- light vision. Globally, GA affects more than 5 million people. There is a need for improved approaches for treating patients with AMD and GA. The disclosed methods are directed to these and other important needs.SUMMARY

[0002] In some embodiments, the present disclosure is directed to methods of treating geographic atrophy (GA) in a subject, the methods comprising administering a pharmaceutical composition into a GA-affected eye of a subject by ocular injection, wherein the pharmaceutical composition comprises a nucleic acid encoding a soluble CD59 (sCD59) protein. In certain embodiments, the GA is secondary to age-related macular degeneration, i.e., the methods involve treating GA in a subject who also suffers from age-related macular degeneration. In further embodiments, the pharmaceutical composition comprises a dose of viral particles of about 8.2 x 1010vector genome (vg). In still further embodiments, the pharmaceutical composition comprises a dose of viral particles of about 4. 1 x 10nvg.

[0003] In certain embodiments, the GA covers an area of the affected eye measuring 2.5 mm2to 17.5 mm2. In some embodiments, the GA is non-subfoveal. In further embodiments, the GA is multifocal with at least one focal lesion of >1.25 mm2. In still further embodiments, the GA lesion is measured as the square root of GA lesion area by fundus autofluorescence.

[0004] In further embodiments, the subject is 60 years of age or older. In certain embodiments, the pharmaceutical composition is injected into only one eye. In some embodiments, the pharmaceutical composition is administered by a single intravitreal injection in the GA-affected eye.

[0005] In some embodiments, the treatment is assessed at about 18 months following administration of the pharmaceutical composition. In certain embodiments, the pharmaceutical composition is administered with prednisone. In further embodiments, the prednisone is administered orally for 20 days at a dose of about 40 mg / day. In still further embodiments, the dose of prednisone is tapered down by about 10 mg every 5 days.

[0006] In certain embodiments, the pharmaceutical composition is administered with a corticosteroid. In further embodiments, corticosteroid is administered by single periocular injection. In still further embodiments, the corticosteroid is triamcinolone acetonide (TA). In some embodiments, the TA is administered at a dose of about 40 mg.

[0007] In some embodiments, the sCD59 protein lacks a glycosyl phosphatidyl inositol (GPI) anchoring domain. In certain embodiments, the nucleic acid encoding the sCD59 protein comprises SEQ ID NO: 1. In some embodiments, the sCD59 protein comprises SEQ ID NO: 2. In further embodiments, the nucleic acid encoding the sCD59 protein is operably linked to a promoter sequence for expression and secretion of the sCD59 protein in the cells of the GA-affected eye. In still further embodiments, the nucleic acid encoding the sCD59 protein is packaged into a delivery vector.

[0008] In certain embodiments, the delivery vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is AAV2. In further embodiments, the promoter is a CAG promoter.

[0009] In some embodiments, the present disclosure is directed to methods of improving GA lesion growth in a subject, the methods comprising administering a pharmaceutical composition into a GA-affected eye of a subject by ocular injection, wherein the pharmaceutical composition comprises a nucleic acid encoding a sCD59 protein. In certain embodiments, the improvement a change in the square root transformed GA lesionarea (sqrt-GAA) from baseline at months 3, 6, 9, 12, 15, and 18 in the GA-affected eye. In further embodiments, the improvement is relative to a sham control. In still further embodiments, the improvement is measured at about 18 months after injection.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawing. For the purpose of illustrating the disclosed methods or uses, the drawing shows exemplary embodiments of the methods or uses; however, the methods or uses are not limited to the specific embodiments disclosed. In the drawing:

[0011] FIG. 1 is a schematic overview of the study described in Example 1. IVT = intravitreal.DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0001] In the present disclosure the singular forms “a”, “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a material” is a reference to at least one of such materials and equivalents thereof known to those skilled in the art, and so forth.

[0002] When a value is expressed as an approximation by use of the descriptor “about” or “substantially” it will be understood that the particular value forms another embodiment. In general, use of the term “about” or “substantially” indicates approximations that can vary depending on the desired properties sought to be obtained by the disclosed subject matter and is to be interpreted in the specific context in which it is used, based on its function. The person skilled in the art will be able to interpret this as a matter of routine. In some cases, the number of significant figures used for a particular value may be one nonlimiting method of determining the extent of the word “about” or “substantially”. In other cases, the gradations used in a series of values may be used to determine the intended range available to the term “about” or “substantially” for each value. Where present, all ranges are inclusive and combinable. That is, references to values stated in ranges include every value within that range.

[0003] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list and every combination of that list is to be interpreted as a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.”

[0004] It is to be appreciated that certain features of the disclosure which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. That is, unless obviously incompatible or excluded, each individual embodiment is deemed to be combinable with any other embodiments and such a combination is considered to be another embodiment. Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation. Finally, while an embodiment may be described as part of a series of steps or part of a more general structure, each said step may also be considered an independent embodiment in itself.Certain Terminology

[0005] Various terms used throughout this specification shall have the definitions set out herein.

[0006] The term “administering” as used herein includes in vivo administration, as well as administration directly to tissue ex vivo. Generally, compositions can be administered systemically either orally, buccally, parenterally, topically, by inhalation or insufflation (i.e., through the mouth or through the nose), or rectally in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired, or can be locally administered by means such as, but not limited to, injection, implantation, grafting, topical application, or parenterally.

[0007] The term “CD59” as used herein refers to a membrane-bound glycoprotein found associated with membranes of cells including both human hematopoietic and non- hematopoietic cells, for example on endothelial cells, peripheral nerve fibers, neurons, microglia, oligodendrocytes, astrocytes, ependymal cells, epithelial cells, acinar cells of the salivary glands, bronchial epithelium, renal tubules and squamous epithelium. CD59 proteininhibits assembly of functional membrane attack complexes (MACs) and thus protects 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 specifies a signal sequence for attachment of a glycosyl phosphatidyl inositol anchor (GPI anchor) at amino acid asparagine at position 77. A cDNA sequence of CD59 is shown in Fodor et al., U.S. Pat. No. 5,624,837 issued Apr. 29, 1997, which is incorporated herein by reference in its entirety.

[0008] The term "dosage unit form" as used herein refers to a physically discrete unit of active agent appropriate for the patient to be treated.

[0009] The term “drusen” as used herein refers to yellow deposits under the retina.

[0010] The term “enhance,” “improve” and the like as used herein in their various grammatical forms refer to an increase or to intensify in quality or quantity, or to make better or augment.

[0011] The terms “functional equivalent” or “functionally equivalent” are used interchangeably herein to refer to substances, molecules, polynucleotides, proteins, peptides, or polypeptides having similar or identical effects or use. A polypeptide functionally equivalent to SEQ ID NO: 2, for example, may have a biologic activity, e.g., an inhibitory activity, kinetic parameters, salt inhibition, a cofactor-dependent activity, and / or a functional unit size that is substantially similar or identical to the expressed polypeptide of SEQ ID NO: 2.

[0012] The term “fundus autofluorescence (FAF)” refers to a non-invasive retinal imaging modality that produces a density map of the ocular flurophore lipofuscin in the retinal pigment epithelium. FAF images may be obtained through the use of confocal laser scanning ophthalmoscopy (cSLO).

[0013] The term “inhibit” and its various grammatical forms, including, but not limited to, “inhibiting” or “inhibition”, are used herein to refer to reducing the amount or rate of a process, to stopping the process entirely, or to decreasing, limiting, or blocking the action or function thereof. Inhibition can include a reduction or decrease of the amount, rate, action function, or process of a substance by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%.

[0014] The terms “membrane attack complex” and “MAC” are used interchangeably herein to refer to an effector of the immune system comprising a complex of proteins typically formed on the surface of pathogen cell membranes as a result of activation of a host's complement system. Antibody-mediated complement activation leads to MAC deposition on the surface of infected cells, leading to pores that disrupt the cell membrane of the infected cells, resulting in cell lysis and death. The MAC is composed of complement components C5b, C6, C7, C8 and several C9 molecules.

[0015] The term “modify” as used herein means to change, vary, adjust, temper, alter, affect or regulate to a certain measure or proportion in one or more particulars.

[0016] The term “multifocal” when used in reference to GA means two or more atrophic lesions in the eye.

[0017] The term “nucleic acid” is used herein to refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).

[0018] The term “nucleotide” is used herein to refer to a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. In the most common nucleotides, the base is a derivative of purine or pyrimidine, and the sugar is the pentose deoxyribose or ribose. Nucleotides are the monomers of nucleic acids, with three or more bonding together in order to form a nucleic acid. Nucleotides are the structural units of RNA, DNA, and several cofactors, including, but not limited to, CoA, FAD, DMN, NAD, and NADP. Purines include adenine (A), and guanine (G); pyrimidines include cytosine (C), thymine (T), and uracil (U).

[0019] The following terms are used herein to describe the 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.”

[0020] (a) The term “reference sequence” refers to a sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

[0021] (b) The term “comparison window” refers to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be at least 30 contiguous nucleotides in length, at least 40 contiguous nucleotides in length, at least 50 contiguous nucleotides in length, at least 100 contiguous nucleotides in length, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence, a gap penalty typically is introduced and is subtracted from the number of matches.

[0022] Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted 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); 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 well described by 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, SASSOS (1992), and Pearson, et al., Methods in Molecular Biology, 24:307-331 (1994). The BLAST family of programs, which can be used for database similarity searches, includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley -Interscience, New York (1995).

[0023] Unless otherwise stated, sequence identity / similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters.Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology- Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits then are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, 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). Lor amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 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 word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=- 4, and a comparison of both strands. Lor amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Set. USA 89: 10915).

[0024] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Set. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. BLAST searches assume that proteins may be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs may be employed to reduce such low-complexity alignments. Lor example, the SEG (Wooten and Lederhen, Comput. Chem., 17: 149-163 (1993)) and XNU (Claverie andStates, Comput. Chem., 17: 191-201 (1993)) low-complexity filters may be employed alone or in combination.

[0025] (c) The term “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences is used herein to refer to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, i.e., where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Set., 4: 11-17 (1988) e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, Calif, USA).

[0026] (d) The term “percentage of sequence identity” is used herein mean the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0027] (e) The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity and at least 95% sequence identity,compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values may be adjusted appropriately to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, or at least 70%, at least 80%, at least 90%, or at least 95%. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. However, nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide that the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. The term "substantial identity" of protein sequences refers to a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are identical to aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and / or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60% identity, or at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% identity.

[0028] The term "parenteral" as used herein refers to introduction into the body by way of an injection (i.e., administration by injection), including, for example, intraocularly (also known as intravitreally) (i.e., an injection into the vitreous of the eye), subretinally (i.e., an injection into the subretinal space which exists between the photoreceptors of the retina and the retinal pigment epithelium (RPE) layer), subcutaneously (i.e., an injection beneath the skin), intramuscularly (i.e., an injection into a muscle); intravenously (i.e., an injection into a vein), intrathecally (i.e., an injection into the space around the spinal cord), intrastemal injection, or infusion techniques. A parenterally administered composition of the described invention is delivered using a needle, e.g., a surgical needle. The term "surgical needle" as used herein, refers to any needle adapted for delivery of fluid (i.e., capable of flow) compositions of the described invention into a selected anatomical structure. Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.

[0029] As used herein the term “pharmaceutically acceptable carrier” refers to any substantially non-toxic carrier conventionally useable for administration of pharmaceuticals in which the isolated polypeptide of the present invention will remain stable and bioavailable. The pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the mammal being treated. It further should maintain the stability and bioavailability of an active agent. The pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with an active agent and other components of a given composition.

[0030] The term “pharmaceutically acceptable salt” means those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit / risk ratio.

[0031] The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids.

[0032] The terms “polypeptide” and “protein” also are used herein in their broadest sense to refer to a sequence of subunit amino acids, amino acid analogs, or peptidomimetics. The subunits are linked by peptide bonds, except where noted. The polypeptides described herein may be chemically synthesized or recombinantly expressed. Polypeptides of the described invention also can be synthesized chemically. Synthetic polypeptides, prepared using the well-known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc (N-a-amino protected N-a-t- butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or the base-labile N-a-amino protected 9-fluorenylmethoxy carbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J. Org. Chem. 37:3403-3409). Both Fmoc and Boc N-a-amino protected amino acids can be obtained from Sigma,Cambridge Research Biochemical, or other chemical companies familiar to those skilled in the art. In addition, the polypeptides can be synthesized with other N-a-protecting groups that are familiar to those skilled in this art. Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, for example, in Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, Ill.; Fields and Noble, 1990, Int. J. Pept. Protein Res. 35: 161-214, or using automated synthesizers. The polypeptides of the invention may comprise D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), a combination of D- and L-amino acids, and various “designer” amino acids (e.g., P-methyl amino acids, C-a- methyl amino acids, and N-a- methyl amino acids, etc.) to convey special properties. Synthetic amino acids include ornithine for lysine, and norleucine for leucine or isoleucine. In addition, the polypeptides can have peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties. For example, a peptide may be generated that incorporates a reduced peptide bond, i.e., RI- CH2-NH- R2, where R1 and R2 are amino acid residues or sequences. A reduced peptide bond may be introduced as a dipeptide subunit. Such a polypeptide would be resistant to protease activity, and would possess an extended half-live in vivo. Accordingly, these terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, the protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids.

[0033] The terms “polypeptide”, “peptide” and “protein” also are inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gammacarboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. It will be appreciated, as is well known and as noted above, that polypeptides may not be entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslational events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well. In some embodiments, the peptide is of any length or size.

[0034] The terms “prevent”, “prevented”, “preventing” or “prevention” as used herein refer to the keeping, hindering or averting of an event, act, or action from happening, occurring or arising.

[0035] The term "recombinant" refers to a cell or vector that has been modified by the introduction of a heterologous nucleic acid or the cell that is derived from a cell so modified. Recombinant cells express genes that are not found in identical form within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of deliberate human intervention. The term "recombinant" as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation transduction / transposition) such as those occurring without deliberate human intervention.

[0036] The term "recombinant expression cassette" refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a host cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, a promoter, and a transcription termination signal such as a poly -A signal.

[0037] The term "recombinant host" refers to any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned genes, or gene of interest, in the chromosome or genome of the host cell.

[0038] The term “recombinant protein” as used herein refers to a protein produced by genetic engineering, for example, by manipulation of genetically modified organisms such as microorganisms.

[0039] The term “reduce” or “reducing” as used herein refers to the limiting of an occurrence of a disorder in individuals at risk of developing the disorder.

[0040] The term “sham” refers to a procedure performed using a syringe without a needle or peri / intraocular penetration.

[0041] The term “similar” is used interchangeably with the terms analogous, comparable, or resembling, meaning having traits or characteristics in common.

[0042] The term “solution” as used herein refers to a homogeneous mixture of two or more substances. It is frequently, though not necessarily, a liquid. In a solution, the molecules of the solute (or dissolved substance) are uniformly distributed among those of the solvent.

[0043] The terms “soluble CD59”, “sCD59” and "membrane independent CD59" as used herein refer to a CD59 amino acid sequence that lacks a GPI anchor or has a modified GPI anchor that lacks function and ability to bind to a cell membrane or a cell- membrane- associated structure such as a membrane-bound protein.

[0044] The term “suspension” as used herein refers to a dispersion (mixture) in which a finely- divided species is combined with another species, with the former being so finely divided and mixed that it doesn't rapidly settle out. In everyday life, the most common suspensions are those of solids in liquid.

[0045] The term “subfoveal” refers to as involving the center point of the fovea whereas the term “non-subfoveal” refers to not involving the center point of the fovea.

[0046] As used herein, the terms “subject” or “individual” or “patient” or “participant” are used interchangeably to refer to a member of an animal species of mammalian origin, including humans. The term “a subject in need thereof is used to refer to a subject having, or at risk of progression to heart failure, including a subject having an AMI that leads to a disease manifestation of left ventricular remodeling.

[0047] The phrase “subject in need of such treatment” as used herein refers to a patient who suffers from a disease, disorder, condition, or pathological process. In some embodiments, the term “subject in need of such treatment” also is used to refer to a patient who (i) will be administered at least one dose of the adenovirus vector construct expressing human sCD59 of the described invention; (ii) is receiving at least one dose of the adenovirus vector construct expressing human sCD59 of the described invention; or (iii) has received at least one dose of the adenovirus vector construct expressing human sCD59 of the described invention, unless the context and usage of the phrase indicates otherwise.

[0048] The term “substantially similar” as used herein means that a first value, aspect, trait, feature, number, or amount is of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of a second value, aspect, trait, feature, number, or amount. For example, a polypeptide substantially similar to (SEQ ID NO: 2) would have at least 70% amino acid sequence identity, at least 75% amino acid sequence identity, at least80% amino acid sequence identity, at least 90% sequence identity, or at least 95% amino acid sequence identity to amino acid sequence (SEQ ID NO: 2).

[0049] The term “substitution” is used herein to refer to a situation in which a base or bases are exchanged for another base or bases in a DNA sequence. Substitutions may be synonymous substitutions or nonsynonymous substitutions. As used herein, “synonymous substitutions” refer to substitutions of one base for another in an exon of a gene coding for a protein, such that the amino acid sequence produced is not modified. The term “nonsynonymous substitutions” as used herein refer to substitutions of one base for another in an exon of a gene coding for a protein, such that the amino acid sequence produced is modified.

[0050] The term “symptom” as used herein refers to a phenomenon that arises from and accompanies a particular disease or disorder and serves as an indication of it.

[0051] The term “syndrome,” as used herein, refers to a pattern of symptoms indicative of some disease or condition.

[0052] 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 the ingredient, component or constituent of the compositions of the described invention 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 percentage of a population. An example of a commonly used therapeutic component is the ED50 which describes the dose in a particular dosage that is therapeutically effective for a particular disease manifestation in 50% of a population.

[0053] The terms “therapeutic amount”, "therapeutically effective amount", an "amount effective", or “pharmaceutically effective amount” of an active agent is used interchangeably to refer to an amount that is sufficient to provide the intended benefit of treatment. An effective amount of the active agent(s) that can be employed according to the described invention generally ranges from about 8.2 x 1010vector genome (vg) to 4. 1 x 1011vg per dose. 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 employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods.Additionally, the terms “therapeutic amount”, “therapeutically effective amount” and “pharmaceutically effective amount” includes prophylactic or preventative amounts of the compositions of the described invention. In prophylactic or preventative applications of the described invention, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and / or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes presenting during development of the disease, disorder or condition. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to some medical judgment. The terms “dose” and “dosage” are used interchangeably herein.

[0054] The term “therapeutic effect” as used herein refers to a consequence of treatment, the results of which are judged to be desirable and beneficial. A therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation. A therapeutic effect can also include, directly or indirectly, the arrest, reduction or elimination of the progression of a disease manifestation.

[0055] For any therapeutic agent described herein the therapeutically effective amount may be initially determined from preliminary in vitro studies and / or animal models. A therapeutically effective dose may also be determined from human data. The applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan.

[0056] General principles for determining therapeutic effectiveness, which may 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, are summarized below.

[0057] Pharmacokinetic principles provide a basis for modifying a dosage regimen to obtain a desired degree of therapeutic efficacy with a minimum of unacceptable adverse effects. In situations where the drug's plasma concentration can be measured and related to the therapeutic window, additional guidance for dosage modification can be obtained.

[0058] Drug products are considered to be pharmaceutical equivalents if they contain the same active ingredients and are identical in strength or concentration, dosageform, and route of administration. Two pharmaceutically equivalent drug products are considered to be bioequivalent when the rates and extents of bioavailability of the active ingredient in the two products are not significantly different under suitable test conditions.

[0059] The term “treat” or “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).

[0060] The terms “variants”, “mutants”, and “derivatives” are used herein to refer to nucleotide or polypeptide sequences with substantial identity to a reference nucleotide or polypeptide sequence. The differences in the sequences may be the result of changes, either naturally or by design, in sequence or structure. Natural changes may arise during the course of normal replication or duplication in nature of the particular nucleic acid sequence. Designed changes may be specifically designed and introduced into the sequence for specific purposes. Such specific changes may be made in vitro using a variety of mutagenesis techniques. Such sequence variants generated specifically may be referred to as “mutants” or “derivatives” of the original sequence.

[0061] A skilled artisan likewise can produce polypeptide variants of polypeptide SEQ ID NO: 2 having single or multiple amino acid substitutions, deletions, additions or replacements, but functionally equivalent to SEQ ID NO: 2. These variants may include inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids; (b) variants in which one or more amino acids are added; (c) variants in which at least one amino acid includes a substituent group; (d) variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at conserved or non-conserved positions; and (d) variants in which a target protein is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the target protein, for example, an epitope for an antibody. The techniques for obtaining such variants, including,but not limited to, genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to the skilled artisan. As used herein, the term “mutation” refers to a change of the DNA sequence within a gene or chromosome of an organism resulting in the creation of a new character or trait not found in the parental type, or the process by which such a change occurs in a chromosome, either through an alteration in the nucleotide sequence of the DNA coding for a gene or through a change in the physical arrangement of a chromosome. Three mechanisms of mutation include substitution (exchange of one base pair for another), addition (the insertion of one or more bases into a sequence), and deletion (loss of one or more base pairs).

[0062] The term “vector” as used herein refers to a carrier that is genetically engineered to deliver a gene to a cell. The term “viral vector” as used herein refers to a virus used as a vector to deliver a gene of interest by infecting a cell. Such viruses are modified so they cannot cause disease when used in humans. Types of viruses include, but are not limited to, retroviruses, which integrate their genetic material (including the gene interest) into a chromosome in a cell, and adenoviruses, which introduce their DNA (including the gene of interest) into the nucleus of a cell without integrating the genetic material into a chromosome.

[0063] The term “vehicle” as used herein refers to a substance that facilitates the use of a drug or other material that is mixed with it.Methods of Treatment and Uses

[0064] In some embodiments, the present disclosure is directed to methods of treating geographic atrophy (GA) in a subject. According to some embodiments, the method comprises contacting cells 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 that is packaged into a delivery vector. According to some embodiments, the pharmaceutical composition comprises a human sCD59 expression construct that is packaged into a delivery vector. In certain embodiment, the methods comprising, consisting of, or consisting essentially of administering a pharmaceutical composition into a GA-affected eye of asubject, wherein the pharmaceutical composition comprises a nucleic acid encoding a soluble CD59 (sCD59) protein, thereby treating the GA.

[0065] In certain embodiments, the GA is secondary to age-related macular degeneration. Age-related Macular Degeneration (AMD) refers to a slow, progressive disease of the macula that impairs the ability of individuals to read, drive a car, recognize faces, and see objects in fine detail. The appearance of AMD begins with lipoproteinaceous yellow deposits (drusen) in the region of the macula and can progress towards advanced AMD where the most significant vision loss occurs. Advanced AMD is generally divided into 2 categories, which are not mutually exclusive: “wet” or “dry” AMD, also referred to as “exudative” or “non-exudative,” respectively. Wet AMD is associated with choroidal neovascularization (CNV) formation. Dry AMD is associated with drusen and pigment changes in the macula. The majority of vision loss occurs in advanced cases of dry AMD with the formation and enlargement of GA that is associated with loss of photoreceptors, retinal pigment epithelium (RPE), and choriocapillaris. According to some embodiments, the AMD is wet or exudative AMD. According to some embodiments, the AMD is dry AMD.

[0066] In some embodiments, the present disclosure is directed to methods of treating wet AMD in a subject. According to some embodiments, the method comprises contacting cells 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 that is packaged into a delivery vector. According to some embodiments, the pharmaceutical composition comprises a human sCD59 expression construct that is packaged into a delivery vector. In certain embodiment, the methods comprising, consisting of, or consisting essentially of administering a pharmaceutical composition into a AMD-affected eye of a subject, wherein the pharmaceutical composition comprises a nucleic acid encoding a sCD59 protein, thereby treating the wet AMD.

[0067] Without being bound by any theory, it is understood that plasma membranes of cells are normally protected from the effects of complement by cell-surface proteins, e.g., CD59, that specifically inhibit activation of the C5b-9 pore upon C9 complement protein binding to membrane C5b-8 (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 C9 complement protein for binding to C8 complement protein in the C5b-8 complex, thereby decreasing or preventing the formation of the C5b-9 membrane attack complex. CD59 thus acts to reduce both cell activation and cell lysis by terminal complement MACs.

[0068] Theories have connected causation of disease such as age-related macular degeneration (AMD) and activation of the complement system and formation of MAC. Dinu (U.S. patent application number 2007 / 0196367 Al published Aug. 23, 2007) proposes preventing debris formation by inhibiting complement as a therapeutic for AMD.

[0069] Diseases associated with uncontrolled complement activity include bacterial infection such as with Haemophilus influenza, Streptococcus pnemoniae, Neisseria meningitidis; angiodema; renal disease for example atypical haemolytic uremic syndrome; paroxysmal nocturnal hemoglobinuria; systemic lupus erythematosus; central nervous system diseases including Alzheimer's disease, Huntington's disease and diseases of the retina including, but not limited to, AMD.

[0070] According to some embodiments, human sCD59 is effective to inhibit MAC formation. According to some embodiments, MAC formation is inhibited by delivering into a cell a vector containing a human sCD59-encoding nucleic acid.

[0071] According to some embodiments, human sCD59 is effective to treat GA that is secondary to AMD. According to some embodiments, GA is treated by delivering into a cell a vector containing a human sCD59-encoding nucleic acid. According to some embodiments, human sCD59 is effective to prevent the onset of GA. According to some embodiments, the onset of GA is prevented by delivering into a cell a vector containing a human sCD59-encoding nucleic acid. According to some embodiments, human sCD59 is effective to prevent the progression of GA. According to some embodiments, the progression of GA is prevented by delivering into a cell a vector containing a human sCD59-encoding nucleic acid. According to some embodiments, human sCD59 is effective to reverse the progression of GA. According to some embodiments, the progression of GA is reversed by delivering into a cell a vector containing a human sCD59-encoding nucleic acid.

[0072] In some embodiments, the present disclosure is directed to methods of improving GA lesion growth in a subject, the methods comprising, consisting of, or consisting essentially of administering a pharmaceutical composition into a GA-affected eyeof a subject by ocular injection, wherein the pharmaceutical composition comprises a nucleic acid encoding sCD59 protein.

[0073] In some embodiments, the present disclosure is directed to methods of slowing the progression of GA lesion growth in a subject, the methods comprising, consisting of, or consisting essentially of administering a pharmaceutical composition into a GA- affected eye of a subject by ocular injection, wherein the pharmaceutical composition comprises a nucleic acid encoding a sCD59 protein.

[0074] In some embodiments, the present disclosure is directed to methods of improving low luminance visual acuity (LLVA) in a subject suffering from GA, the methods comprising, consisting of, or consisting essentially of administering a pharmaceutical composition into a GA-affected eye of a subject by ocular injection, wherein the pharmaceutical composition comprises a nucleic acid encoding a sCD59 protein. In certain embodiments, the improvement in LLVA is determined by an improvement in an Early Treatment Diabetic Retinopathy Study (ETDRS) letter score. According to some embodiment, the methods of improving LLVA in a subject suffering from GA comprise contacting the GA-affected eye with a composition including a vector carrying a nucleotide sequence encoding a recombinantly engineered human sCD59 protein operably linked to a promoter sequence causing expression of the protein in a cell, such that the protein includes at least one mutation resulting in loss of function of GIP anchoring domain, such that the protein is recombinant membrane-independent sCD59 and is not membrane targeting; and, observing an improvement in ETDRS after contacting, in comparison to the ETDRS observed prior to contacting

[0075] In some embodiments, the present disclosure is directed to methods of improving visual function and retinal function in a subject suffering from GA, the methods comprising, consisting of, or consisting essentially of administering a pharmaceutical composition into a GA-affected eye of a subject by ocular injection, wherein the pharmaceutical composition comprises a nucleic acid encoding a sCD59 protein. In certain embodiments, the improvement in visual function and retinal function is determined by an improvement in reading speed (Radner Chart). In certain embodiments, the improvement in visual function and retinal function is determined by an improvement in retinal sensitivity by mesopic microperimetry (macular integrity assessment (MAIA)). According to some embodiment, the methods of improving visual function and retinal function in a subject suffering from GA comprise contacting the GA-affected eye with a composition including avector carrying a nucleotide sequence encoding a recombinantly engineered human sCD59 protein operably linked to a promoter sequence causing expression of the protein in a cell, such that the protein includes at least one mutation resulting in loss of function of GPI anchoring domain, such that the protein is recombinant membrane-independent CD59 (sCD59) and is not membrane targeting; and, observing an improvement in reading speed after contacting, in comparison to the reading speed observed prior to contacting.

[0076] In some embodiments, the present disclosure is directed to methods of improving visual acuity in a subject suffering from GA, the methods comprising, consisting of, or consisting essentially of administering a pharmaceutical composition into a GA- affected eye of a subject by ocular injection, wherein the pharmaceutical composition comprises a nucleic acid encoding a sCD59 protein. In certain embodiments, the visual acuity is best corrected visual acuity (BCVA). In certain embodiments, the improvement in visual acuity is determined by an improvement in Early Treatment Diabetic Retinopathy Study (ETDRS) letter score. According to some embodiment, the methods of improving visual acuity in a subject suffering from GA comprise contacting the GA-affected eye with a composition including a vector carrying a nucleotide sequence encoding a recombinantly engineered human sCD59 protein operably linked to a promoter sequence causing expression of the protein in a cell, such that the protein includes at least one mutation resulting in loss of function of GIP anchoring domain, such that the protein is recombinant membraneindependent sCD59 and is not membrane targeting; and, observing an improvement in BCVA or ETDRS after contacting, in comparison to the BCVA or ETDRS observed prior to contacting.

[0077] In some embodiments, the present disclosure is directed to methods of improving functional reading independence and vision-related quality of life in a subject suffering from GA, the methods comprising, consisting of, or consisting essentially of administering a pharmaceutical composition into a GA-affected eye of a subject by ocular injection, wherein the pharmaceutical composition comprises a nucleic acid encoding a sCD59 protein. In certain embodiments, the improvement in functional reading independence and vision-related quality of life is determined by an improvement in the Functional Reading Independence (FRI) Index. In certain embodiments, the improvement in functional reading independence and vision-related quality of life is determined by an improvement in the National Eye Institute Visual Functioning Questionnaire-25 (NEI-VFQ- 25) composite score. According to some embodiment, the methods of improving functionalreading independence and vision-related quality of life in a subject suffering from GA comprise contacting the GA-affected eye with a composition including a vector carrying a nucleotide sequence encoding a recombinantly engineered human sCD59 protein operably linked to a promoter sequence causing expression of the protein in a cell, such that the protein includes at least one mutation resulting in loss of function of GIP anchoring domain, such that the protein is recombinant membrane-independent sCD59 and is not membrane targeting; and, observing an improvement in FRI Index or NEI-VFQ-25 composite score after contacting, in comparison to the FRI Index or NEI-VFQ-25 observed prior to contacting

[0078] In certain of the foregoing embodiments, the improvement is relative to a subject or a subject population at substantially the same level of disease progression receiving a standard of care treatment that does not comprise sCD59. In certain embodiments the improvement is relative to a subject or a subject population at substantially the same level of disease progression receiving a sham treatment that does not comprise sCD59. In certain embodiments the improvement is relative to a subject or a subject population at substantially the same level of disease progression receiving no treatment.

[0079] In certain of the foregoing embodiments, the improvement a change in the square root transformed GA lesion area (sqrt-GAA) from baseline at months 3, 6, 9, 12, 15, and 18 in the GA-affected eye. In certain of the foregoing embodiments, the improvement is relative to a subject or a subject population who is not suffering from GA.

[0080] In further embodiments, the improvement is relative to a sham control injection. In certain embodiments, the sham control injection is performed using a syringe without a needle or peri / intraocular penetration.

[0081] In certain embodiments, the GA covers an area of the affected eye measuring 2.5 mm2to 17.5 mm2. In some embodiments, the GA is non-subfoveal. In further embodiments, the GA is multifocal with at least one focal lesion of >1.25 mm2. In still further embodiments, the GA lesion is measured as the square root of GA lesion area by fundus autofluorescence.

[0082] In some embodiments, the subject is 80 years of age or older. In certain embodiments, the subject is 70 years of age or older. In further embodiments, the subject is 60 years of age or older. In certain embodiments, the subject is 50 years of age or older.

[0083] In still further embodiments, the treatment or the improvement is determined at about 18 months after administration of the pharmaceutical composition comprising a nucleic acid encoding the sCD59 protein into a GA-affected eye of a subject.

[0084] According to some embodiments, the described invention provides a nucleotide sequence encoding human sCD59 protein. According to some embodiments, the nucleotide sequence is a complementary DNA (cDNA) sequence.

[0085] cDNA sequences encoding human CD59 are known in the art. For example, cDNA sequences have been reported by Sawada, R. et al. 1989 Nucleic Acids Res 17(16): 6728 and are available from the American Type Tissue Culture Collection (ATCC, Manassas, Va.). A cDNA encoding CD59 has also been cloned from human T-cell leukemia (YT) and human erythroleukemia (K562) cell lines, and CD59 has been transiently expressed in COS cells (Walsh, L. A. et al. 1990 Eur J. Immol 21(3): 847-850).

[0086] In some embodiments, the sCD59 protein lacks a GPI anchoring domain. According to some embodiments, the human sCD59 comprises a modified GPI anchor domain amino acid sequence that is functionally defective and lacks the ability to target a membrane. According to some embodiments, the modified GPI anchor domain amino acid sequence comprises a variation. Such variations include, but are not limited to, substitution and deletion of nucleic acids encoding amino acids at omega positions used to reduce or eliminate the attachment of the GPI anchor or reduce or eliminate the effective functionality of the GPI anchor. Omega amino acids are amino acids to which GPI is transferred. For example, such a variation includes, but is not limited to, substituting the nucleic acids encoding hydrophobic leucine (e.g., nucleic acids CTG) and alanine (e.g., nucleic acids GCA) with nucleic acids encoding glycine (e.g., nucleic acids CAG) and glutamate (e.g., nucleic acids GAA), which are less hydrophobic (i.e., more hydrophilic) amino acids. Alternatively, a variation may include substituting the omega residue with another amino acid, such as a glycine for a tyrosine.

[0087] According to some embodiments, the human sCD59 protein of the described invention includes conservative sequence modifications. Conservative sequence modifications are amino acid modifications that do not significantly affect or alter the characteristics of the human sCD59 protein containing the amino acid sequence, i.e., amino acid sequences of sCD59 that present these side chains at the same relative positions will function in a manner similar to human sCD59. Such conservative modifications include amino acid substitutions, additions and deletions. Methods of modifying amino acid sequences are known in the art e.g., site-directed mutagenesis or PCR based mutagenesis. Such techniques are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 and Ausubel et al., Current Protocols inMolecular 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 substitutions are ones in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0088] According to some embodiments, the human sCD59 amino acid sequence is an amino acid sequence that is substantially identical to that of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 70% identical to that of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 75% identical to that of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 80% identical to that of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 90% identical to that of the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 95% identical to that of the wild-type sequence.

[0089] In certain embodiments, the nucleic acid encoding the sCD59 protein comprises or consists essentially of SEQ ID NO: 1. In certain embodiments, the nucleic acid encoding the sCD59 protein comprises or consists essentially of a sequence that is at least 90% identical to SEQ ID NO: 1. In certain embodiments, the nucleic acid encoding the sCD59 protein comprises or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 1.

[0090] In some embodiments, the sCD59 protein comprises or consists essentially of SEQ ID NO: 2. In certain embodiments, the sCD59 protein comprises or consists essentially of a sequence that is at least 90% identical to SEQ ID NO: 2. In certain embodiments, the sCD59 protein comprises or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 2.

[0091] According to some embodiments, the human sCD59 of the described invention is a recombinant protein.

[0092] In still further embodiments, the nucleic acid encoding the sCD59 protein is packaged into a delivery vector.

[0093] A variety of commercially available expression vector / host systems are useful to contain and express a CD59 protein encoding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems contacted with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti, pBR322, or pET25b plasmid); or animal cell systems. See Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.

[0094] Techniques for altering a nucleic acid sequence to produce a recombinant protein are well known in the art of genetics and molecular biology. Traditional strategies for recombinant protein expression involve transfecting cells with a DNA vector that contains a template for expressing a desired protein and 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.

[0095] Types of cells used in recombinant protein expression include, but are not limited to, prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacterial cells. A non -limiting example of a bacterial cell includes Escherichia coli. Eukaryotic cells include, but are not limited to, mammalian, insect, yeast and algae cells. Non-limiting examples of mammalian cells include human embryonic kidney cells (e.g., HEK293, HEK293T), baby hamster kidney cells (e.g., BHK21), Chinese hamster ovary (CHO) cells, mouse myeloma cells (e.g., NSO) and murine non-producing hybridoma cells (e.g., SP2 / O-Agl4). Non-limiting examples of insect cells include, Spodoptera frugiperda pupa ovarian cells (e.g., Sf9, Sf21). A non-limiting example of a yeast cell includes Saccharomyces cerevisiae. A non-limiting example of an algae cell includes Chlamydomonas reinhardtii.

[0096] Methods for expressing recombinant proteins also include cell -free systems. Cell-free protein expression includes the in vitro production of recombinant proteins in solution (i.e., cell lysate) using biomolecular translation machinery extracted from cells.

[0097] Various methods of protein purification may be employed and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer- Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular protein produced.

[0098] According to some embodiments, the human sCD59 is a synthetic protein. Methods of preparing synthetic proteins are well-known in the art. See, for example, Peptide Synthesis Protocols, Methods in Molecular Biology, vol. 35, Pennington, M. W. and Dunn, B. M., 1995, XII, Humana Press, Inc. Totowa, New Jersey. Synthetic proteins, prepared using well-known techniques such as solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), orthe base-labile N-a-amino protected 9- Fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J. Org. Chem. 37:3403-3409). Both Boc and Fmoc amino protected amino acids can be obtained from Sigma or other chemical companies familiar to those skilled in art. The peptides can also be synthesized with other N-a protecting groups familiar to those skilled in the art.

[0099] According to some embodiments, the 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 expression vectors containing the nucleotide sequence encoding the human sCD59 protein operably linked to appropriate transcriptional and translational control elements. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination or genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989.

[0100] In further embodiments, the nucleic acid encoding the sCD59 protein is operably linked to a promoter sequence for expression and secretion of the sCD59 protein in the cells of the GA-affected eye. According to some embodiments, the promoter is a constitutive promoter. According to some embodiments, the promoter is a cell cycle-specificpromoter. According to some embodiments, the promoter is a ubiquitous promoter. According to some embodiments, the promoter is a tissue-specific promoters. Examples of tissue-specific promoters include, but are not limited to, human rhodopsin kinase (hRK) promoter and retinal pigmemt epithelium specific promoter (e.g., RPE65 promoter). According to some embodiments, the promoter is a metabolically regulated promoter. According to some embodiments, the promoter is an inducible promoter. According to some embodiments, the promoter is a hybrid promoter. A non-limiting example of a hybrid promoter is cytomegalovirus (CMV) early enhancer element / the first exon and the first intron of chicken beta-actin gene / the splice acceptor of the rabbit beta-globin gene (CAG). Nonlimiting examples of promoters are shown in Evans et al. U.S. Pat. No. 6,677,311 Bl issued Jan. 13, 2004; Clark et al. U.S. Pat. No. 7,109,029 B2 issued Sep. 19, 2006; and Hallenbeck et al. U.S. Pat. No. 5,998,205 issued Dec. 7, 1999, each of which is incorporated herein by reference in its entirety.

[0101] According to some embodiments, the delivery vector is a viral vector.

[0102] According to some embodiments, the vector is at least one of: an engineered viral vector recombinantly linked to the nucleotide sequence encoding the sCD59 protein; and a synthetic gene delivery vector for delivery of the nucleotide sequence encoding human sCD59. According to some embodiments, the viral vector is selected from the group consisting of adenovirus, adeno- associated virus (AAV), a herpesvirus, a poxvirus, a lentivirus, and helper-dependent adenovirus vectors. 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.

[0103] Adenovirus vectors are commercially available from American Type Tissue Culture Collection (Manassas, Va.). Methods of constructing adenovirus vectors and using adenovirus vectors are described in Klein et al. 2007 Ophthalmology 114: 253-262, and van Lecuwen et al. 2003 Eur. J. Epidemiol. 18: 845-854. Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al. 1991 Gene, 101: 195-202) and vaccine development (Graham et al. 1991 Methods in Molecular Biology: Gene Transfer and Expression Protocols 7, (Murray, Ed.), Humana Press, Clifton, N.J., 109-128). Further, recombinant adenovirus vectors are used for gene therapy (Wu et al. U.S. Pat. No. 7,235,391 issued Jun. 26, 2007 which is incorporated herein by reference in its entirety).

[0104] Recombinant adenovirus vectors are generated, for example, from homologous recombination between a shuttle vector and a pro virus vector (Wu et al., U.S.Pat. No. 7,235,391 issued Jun. 26, 2007). The adenovirus vectors used herein are replication defective. For example, the adenovirus vectors are conditionally defective, lacking adenovirus El region. A polynucleotide encoding a protein of interest, human sCD59 for example, is introduced at the position from which the El -coding sequences have been removed. Alternatively, the polynucleotide encoding the protein of interest (e.g., human sCD59) may be inserted in the E3 region of the adenovirus.

[0105] Defective adenovirus vectors can be generated and propagated using a helper cell line. Helper cell lines may be derived from human cells such as, 293 human embryonic kidney cells (HEK293), muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. Generation and propagation of these replication defective adenovirus vectors using a helper cell line is described in Graham et al 1977 J. Gen. Virol. 36: 59-72.

[0106] Lentiviral packaging vectors are commercially available from Invitrogen Corporation (Carlsbad Calif). An HIV-based packaging system for the production of lentiviral vectors is prepared using constructs described in Naldini et al. 1996 Science 272: 263-267; Zufferey et al. 1997 Nature Biotechnol. 15: 871-875; and Dull et al. 1998 J. Virol. 72: 8463-8471. A number of vector constructs are available to be packaged using a system, based on third-generation lentiviral SIN vector backbone (Dull et al. 1998 J. Virol. 72: 8463- 8471). 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 selfinactivating 3' LTR containing a deletion in the U3 promoter region, the HIV packaging signal, RRE sequences linked to a marker gene cassette consisting of the Aequora jellyfish green fluorescent protein (GFP) driven by the CMV promoter, and the woodchuck hepatitis virus PRE element, which appears to enhance nuclear export. The GFP marker gene allows quantitation of transfection or transduction efficiency by direct observation of UV fluorescence microscopy or flow cytometry (Kafri et al. 1997 Nature Genet. 17: 314-317; and Sakoda et al. 1999 J. Mol. Cell. Cardiol. 31 : 2037-2047).

[0107] Manipulation of retroviral nucleic acids to construct a retroviral vector containing a gene of interest (e.g., gene that encodes for human sCD59 protein) and packaging cells is accomplished using techniques known in the art (See, e.g., Ausubel, et al., 1992, Volume 1, Section III (units 9.10.1-9.14.3); Sambrook, et al., 1989. Molecular Cloning:A Laboratory Manual. Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Miller, et al., Biotechniques. 7:981-990, 1989; Eglitis, et al., Biotechniques. 6:608-614, 1988; U.S. Pat. Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767, and 5,124,263; and PCT patent publications numbers 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).

[0108] A retroviral vector can be constructed and packaged into non-infectious transducing viral particles (virions) using an amphotropic packaging system. Examples of such packaging systems are described in Miller et al. 1986 Mol. Cell Biol. 6 : 2895-2902; Markowitz et al. 1988 J. Virol. 62: 1120-1124; Cosset et al. 1990 J. Virol. 64: 1070-1078; U.S. Pat. Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767, and 5,124,263, and PCT patent publications numbers 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 accomplished by introducing retroviral vectors into the packaging cells. Examples of such retroviral vectors are found in, for example, Korman et al. 1987 Proc. Natl. Acad. Sci. USA. 84: 2150-2154; Morgenstem et al. 1990 Nucleic Acids Res. 18: 3587-3596; U.S. Pat. Nos. 4,405,712, 4,980,289, and 5,112,767; and PCT patent publications numbers WO 85 / 05629, WO 90 / 02797, and WO 92 / 07943.

[0109] Herpesvirus packaging vectors are commercially available from Invitrogen Corporation, (Carlsbad, Calif). Exemplary herpesviruses include, but are not limited to, an a- herpesvirus, such as Varicella-Zoster vims or pseudorabies vims; a herpes simplex vims such as HSV-1 or HSV-2; or a herpesvirus such as Epstein-Barr vims. A method for preparing empty herpesvirus particles that can be packaged with a desired nucleotide segment, for example a human sCD59 nucleotide or polynucleotide sequence, in the absence of a helper vims that is capable to most herpesviruses is described in Fraefel et al. (U.S. Pat. No. 5,998,208, issued Dec. 7, 1999 which is incorporated by reference in its entirety).

[0110] The herpesvirus DNA vector can be constmcted using techniques known to the skilled artisan. For example, DNA segments encoding the entire genome of a herpesvirus is divided among a number of vectors capable of carrying large DNA segments, e.g., cosmids (Evans, et al., Gene 79, 9-20, 1989), yeast artificial chromosomes (YACS) (Sambrook, J. et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989) or E. coli F element plasmids (O'Conner et al.1989 Science 244: 1307- 1313). For example, sets of cosmids have been isolated which contain overlapping clones that represent the entire genomes of a variety of herpesviruses including Epstein-Barr virus, Varicella-Zoster virus, pseudorabies virus and HSV-1. See M. van Ziji et al. 1988 J. Virol. 62: 2191; Cohen et al. 1993 Proc. Nafl Acad. Sci. U.S.A. 90: 7376; Tomkinson et al. 1993 J. Virol. 67: 7298; and Cunningham et al. 1993 Virology 197: 116.

[0111] Adeno-associated virus (AAV) is a dependent parvovirus in that it depends on coinfection with another virus (either adenovirus or a member of the herpes virus family) to undergo a productive infection in cultured cells (Muzyczka 1992 Curr. Top. Microbiol. Immunol., 158:97 129). For example, recombinant AAV (rAAV) virus can be made by cotransfecting a plasmid containing a gene of interest (e.g., human sCD59 gene), flanked by the two AAV terminal repeats (McLaughlin et al. 1988 J. Virol., 62(6): 1963-1973; Samulski et al. 1989 J. Virol, 63: 3822-3828) and an expression plasmid containing wild-type AAV coding sequences without terminal repeats. Cells are also contacted or transfected with adenovirus or plasmids carrying the adenovirus genes required for AAV helper function.

[0112] Unlike most viruses, AAVs are innately nonpathogenic, poorly immunogenic, and broadly tropic, making them attractive gene delivery candidates for virusbased gene therapies. Most naturally occurring AAVs utilize glycan moieties for initial attachment to a cell surface, and these interactions have been well characterized for a number of serotypes. The interacting glycan moieties identified include AAV serotype 2 (AAV2), AAV3, AAV6 and AAV8; N- terminal galactose for AAV9; and specific N- or O-linked sialic acid moieties for AAV1, -4, -5, and -6. Serotypes differ by the types of cells they infect, making AAV a very useful system for preferentially transducing specific cell types.

[0113] 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).

[0114] Methods of constructing AAV vectors and using AAV vectors are known in the art. Such methods are described, for example, in U.S. Pat. No. 5,139,941 (Wu et al.) issued Jun. 26, 2007 and U.S. Pat. No. 4,797,368 (Carter et al.) issued Jan. 10, 1989. 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.

[0115] Recombinant AAV vectors have been used successfully 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) and transduction of genes involved in human diseases (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).

[0116] 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.

[0117] According to some embodiments, the nucleotide sequence encoding human sCD59 is packaged between inverted terminal repeat (ITR) sequences within 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 within an AAV vector. According to some embodiments, the human sCD59 expression vector is packaged between inverted terminal repeat (ITR) sequences within 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.

[0118] According to some embodiments, the AAV vector is a hybrid vector. Hybrid vectors contain ITR sequences from one AAV serotype and a capsid protein from a different AAV serotype. According to some embodiments, the hybrid vector comprises ITR sequences from AAV2 and a capsid protein from AAV5 (AAV2 / 5). According to some embodiments, the hybrid vector comprises ITR sequences from AAV2 and a capsid protein from AAV8 (AAV2 / 8). According to some embodiments, the hybrid vector comprises ITR sequences from AAV5 and a capsid protein from AAV2 (AAV5 / 2). According to some embodiments, the hybrid vector comprises ITR sequences from AAV5 and a capsid proteinfrom AAV8 (AAV5 / 8). According to some embodiments, the hybrid vector comprises ITR sequences from AAV8 and a capsid protein from AAV2 (AAV8 / 2). According to some embodiments, the hybrid vector comprises ITR sequences from AAV8 and a capsid protein from AAV5 (AAV8 / 5).

[0119] According to some embodiments, the delivery vectors are non-viral vectors. For example, the delivery vectors are synthetic gene delivery vehicles or vectors that are not related to a virus particle and that specifically deliver the gene material to the target cells or tissue. 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 preparation. Thus, according to some embodiments, the described invention provides a non- viral vector with nucleic acid that is loaded and contacted to a tissue or cell. By way of example, a liposome containing naked DNA encoding a human sCD59 protein having a modified GPI anchor that does not target a membrane, or a gene encoding a human sCD59 protein having no GPI anchor, is encapsulated in the liposome and the liposome is contacted to the tissue or cell such that the nucleic acid is effectively delivered to the tissue or cell.

[0120] According to some embodiments, delivery of human sCD59 to the RA- affected eyes of using an AAV vector is effective for long-term transgene expression.

[0121] According to some embodiments, the pharmaceutical composition is administered by a route selected from the group consisting of intravitreal, subretinal, subconjunctival, subtenon; subcutaneous and intravenous. According to some embodiments, the pharmaceutical composition is administered by injection. According to some embodiments, the tissues contacted by the pharmaceutical composition comprises at least one tissue selected from the group consisting of retinal pigment epithelium, retina, choroid, sclera, Bruch's membrane and choroidal blood vessels.

[0122] In certain embodiments, the pharmaceutical composition is injected into only one eye. In certain embodiments, the pharmaceutical composition is injected into both eyes. In some embodiments, the pharmaceutical composition is administered by a single intravitreal injection in the GA-affected eye.

[0123] According to some embodiments the administering step comprises administering the pharmaceutical composition as a single dose or as 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 a subject in need thereof. According to some embodiments,the subject in need thereof is suffering from AMD. According to some embodiments, the subject in need thereof is suffering from wet or exudative AMD. According to some embodiments, the subject in need thereof is suffering from GA.

[0124] Without being bound by theory, it is believed that contacting cells with a vector containing a human sCD59-encoding nucleic acid produces a subset of cells that are 'factories' for local production and secretion of sCD59, which may protect adjacent ocular cells including retinal pigment epithelium (RPE) cells and choroidal blood vessels.

[0125] The exact dosage of the pharmaceutical composition may be chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, e.g., intermediate or advanced stage of AMD; age, weight and gender of the patient; diet, time and frequency of administration; route of administration; drug combinations; reaction sensitivities; and tolerance / response to therapy. Long-acting pharmaceutical compositions may be administered one time, hourly, twice hourly, every 3 to four hours, once daily, twice daily, every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition.

[0126] The active agents of the described invention can be formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. For any active agent, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, as provided herein, usually mice, but also potentially from rats, rabbits, dogs, or pigs. Such information can then be used to determine useful doses and routes of administration for humans. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.

[0127] A therapeutically effective dose refers to that amount of active agent that ameliorates the symptoms or condition or prevents progression of GA. Therapeutic efficacy and toxicity of active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population). The dose ratio oftoxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50 / ED50.

[0128] According to some embodiments, a source of expression of a human sCD59 protein is administered as a dose of a viral vector or a nucleic acid vector. In further embodiments, the pharmaceutical composition comprises a dose of viral particles of about 8.2 x 1010vector genome (vg). In still further embodiments, the pharmaceutical composition comprises a dose of viral particles of about 4. 1 x 1011vg. In certain embodiments, the dose of viral particles is alternatively expressed as DNAse-resistant particles (DRP).

[0129] In certain embodiments, the pharmaceutical composition is administered with prednisone or prednisolone. In further embodiments, the prednisone or prednisolone is administered orally. In still further embodiments, the prednisone or prednisolone is administered orally for 20 days at a dose of about 40 mg / day. In still further embodiments, the dose of prednisone or prednisolone is tapered down by about 10 mg every 5 days. For example, at day 1 the prednisone or prednisolone is administered orally at a dose of 40 mg / day, at day 5 the prednisone or prednisolone is administered orally at a dose of 30 mg / day, at day 10 the prednisone or prednisolone is administered orally at a dose of 20 mg / day, and at day 15 the prednisone or prednisolone is administered orally at a dose of 15 mg / day. In certain embodiments, the prednisone or prednisolone is administered starting on the same day that the pharmaceutical composition comprising a nucleotide sequence encoding human sCD59 is administered. In certain embodiments, the prednisone or prednisolone is taken in the morning with or after a meal.

[0130] In certain embodiments, the pharmaceutical composition comprising a nucleotide sequence encoding human sCD59 is administered with a corticosteroid. In further embodiments, corticosteroid is administered by single periocular injection. In still further embodiments, the corticosteroid is triamcinolone acetonide (TA). In some embodiments, the TA is administered at a dose of about 40 mg. In certain embodiments, the prednisone or prednisolone is administered starting four days after the pharmaceutical composition comprising a nucleotide sequence encoding human sCD59 is administered.

[0131] In certain embodiments, the pharmaceutical composition comprising a nucleotide sequence encoding human sCD59 is administered with a VEGF treatment. In certain embodiments, the VEGF treatment is initiated if the subject has suspected onset or presence of new exudative MNV (also referred to as active CNV or wet AMD). In certain embodiments, the VEGF treatment is ranibizumab or aflibercept.

[0132] In certain embodiments, the pharmaceutical composition comprising a nucleotide sequence encoding human sCD59 is not administered with a complement inhibitor.Pharmaceutical Compositions

[0133] 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 that was modified to remove the signal sequence for attachment of the GPI anchor at the nucleotides encoding amino acid asparagine at position 77. According to some embodiments, the nucleic acid sequence of human sCD59 protein is modified by point mutations, substitutions or deletions to obtain a nucleic acid sequence that encodes an amino acid sequence that has a modified amino acid sequence at the GPI anchor location, such that the protein is unable to attach to a membrane of a cell.

[0134] According to some embodiments, the pharmaceutical composition is formulated sterile for ocular delivery. According to some embodiments, the pharmaceutical composition formulated for sterile ocular delivery is in a dose effective to treat GA.

[0135] According to some embodiments, the pharmaceutical composition formulated for ocular delivery further includes at least one of a pharmaceutically acceptable buffer, a pharmaceutically acceptable salt and a pharmaceutically acceptable emollient suitable for delivery by at least one route selected from: intra-ocular injection, subconjunctival injection, subtenon injection, eye drop, and ointment.

[0136] According to some embodiments, the described invention provides a pharmaceutical composition that comprises a CD59-encoding nucleic acid or a source of human sCD59 protein expression. In various embodiments, the CD59 protein includes a membrane -independent (i.e., soluble) CD59 protein. According to some embodiments, the pharmaceutical composition is compounded as an ophthalmologic formulation for administration to the eye. According to some embodiments, the pharmaceutical composition is compounded to enhance delivery to the fundus. According to some embodiments, the pharmaceutical composition is compounded to provide sustained release locally to the retina. According to some embodiments, the pharmaceutical composition is formulated to provide effective treatment of vessels and / or tissue involved in ocular diseases. According to some embodiments, the ocular disease is age-related macular degeneration (AMD). According tosome embodiments, the AMD is wet or exudative AMD. According to some embodiments, the AMD is dry AMD or GA.

[0137] According to some embodiments, the pharmaceutical composition of the described invention is formulated sufficiently pure for administration to a human subject, e.g., to the eye of a human subject. According to some embodiments, the pharmaceutical composition includes one or more additional therapeutic agent(s). According to some embodiments, the additional therapeutic agent or agents are selected from the group consisting of growth factors, anti-inflammatory agents, vasopressor agents, including, but not limited to, nitric oxide and calcium channel blockers, collagenase inhibitors, steroids (e.g., prednisolone), matrix metalloproteinase inhibitors, ascorbates, angiotensin H, angiotensin III, calreticulin, tetracyclines, fibronectin, collagen, thrombospondin, transforming growth factors (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), epidermal growth factor (EGF), platelet derived growth factor (PDGF), neu differentiation factor (NDF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), heparin-binding EGF (HBEGF), thrombospondins, von Willebrand Factor-C, heparin and heparin sulfates, and hyaluronic acid. According to some embodiments, the additional therapeutic agent or agents include, without limitation, anti-tumor, antiviral, antibacterial, anti- mycobacterial, anti-fungal, antiproliferative or anti-apoptotic agents. Therapeutic agents that are included in the pharmaceutical composition of the described invention are well known in the art. See for example, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman, et al., eds., McGraw-Hill, 1996, the contents of which are herein incorporated by reference herein.

[0138] According to some embodiments, the additional therapeutic agent or agents is a compound, composition, biologic or the like. According to some embodiments, the additional therapeutic agent or agents potentiate, stabilize, synergize or substitute for the ability of human sCD59 protein to protect cells from MAC deposition. According to some embodiments, the additional therapeutic agent or agents are provided at the same time as the pharmaceutical composition that comprises the human sCD59 protein. According to some embodiments, the additional therapeutic agent or agents are provided after the pharmaceutical composition that comprises the human sCD59 protein. According to some embodiments, the additional therapeutic agent or agents are provided before the pharmaceutical composition that comprises the human sCD59 protein. According to some embodiments, the additionaltherapeutic agent or agents are used to treat the same, a concurrent or a related symptom, condition or disease.

[0139] According to some embodiments, the pharmaceutical composition of the described invention comprises a pharmaceutically acceptable carrier. Pharmaceutical acceptable carriers include, but are not limited to, any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995 provides various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as glucose and sucrose; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil, and soybean oil; glycols such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0140] According to some embodiments, the pharmaceutical composition further includes a peptide for overall delivery (POD), the pharmaceutical composition operably linked to the compound to obtain a conjugated compound, such that the POD includes a protein transduction domain (PTD). For example, the POD composition is one shown in Kumar-Singh et al. PCT7US2008 / 010179 filed Aug. 28, 2008 or Kumar-Singh et al. U.S. publication 2010 / 0209447 published Aug. 19, 2010, each of which is incorporated herein by reference in its entirety.

[0141] According to some embodiments, the pharmaceutical composition comprises a dose of viral vector particles administered to an affected eye. In further embodiments, the pharmaceutical composition comprises a dose of viral particles of about 8.2 x 1010vector genome (vg). In still further embodiments, the pharmaceutical composition comprises a dose of viral particles of about 4. 1 x 1011vg.

[0142] According to some embodiments, the pharmaceutical composition further includes at least one therapeutic agent selected from the group consisting of an antiinflammatory, an antitumor, an antiviral, an antibacterial, an anti-mycobacterial, an antifungal, an anti-proliferative and an anti-apoptotic.

[0143] According to some embodiments, the described invention provides a method for formulating a composition for treating GA in a subject, the method comprising engineering a vector to deliver and express a human sCD59 nucleotide sequence encoding an amino acid sequence corresponding to human sCD59, such that the nucleotide sequence includes a mutation encoding for amino acids of a GPI anchoring domain of the protein, such that the resulting vector encodes an engineered recombinant membrane- independent (i.e., soluble) CD59 (sCD59) protein, and the vector is a viral vector or a synthetic gene delivery vector; and, contacting at least one ocular tissue of the subject with the composition, such that the cells of the tissue express and secrete the CD59 locally, thereby treating the subject for GA.

[0144] According to some embodiments, the source of expression of human sCD59 protein is administered by ocular injections. Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. According to some embodiments, the injectable preparation is a sterile injectable preparation. The sterile injectable preparation may be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3- butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any bland, fixed oil can be employed including, but not limited to, synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0145] According to some embodiments, the sterile injectable preparation includes excipients. Such excipients include, without limitation, suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia), dispersing or wetting agentsincluding, a naturally-occurring phosphatide (e.g., lecithin), or condensation products of an alkylene oxide with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyl-eneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate).

[0146] The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable or diluent or solvent, for example, as a solution in 1, 3- butanediol. A solution generally is considered as a homogeneous mixture of two or more substances; it is frequently, though not necessarily, a liquid. In a solution, the molecules of the solute (or dissolved substance) are uniformly distributed among those of the solvent. A suspension is a dispersion (mixture) in which a finely- divided species is combined with another species, with the former being so finely divided and mixed that it does not rapidly settle out. In everyday life, the most common suspensions are those of solids in liquid water. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. 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 which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension also may contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0147] According to some embodiments, the described invention provides liquid dosage forms for ocular injection. Such liquid dosage forms include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active agent(s), the liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, ocular- delivered pharmaceutical compositions can also include adjuvants such as wetting agents and emulsifying and suspending agents.

[0148] According to some embodiments, the pharmaceutical composition of the described invention may be in the form of a sterile injectable aqueous or oleaginous suspension. Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.

[0149] According to some embodiments, the described invention comprises ophthalmological devices, surgical devices, audiological devices or products which contain disclosed compositions (e.g., gauze bandages or strips), and methods of making or using such devices or products. These devices may be coated with, impregnated with, bonded to, or otherwise treated with the pharmaceutical composition described herein.

[0150] According to some embodiments, the composition is administered in a pharmaceutically acceptable solution, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic agents.

[0151] According to some embodiments, the pharmaceutical composition is an aqueous suspension or emulsion in admixture 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; dispersing or wetting agents may be a naturally-occurring phosphatide such as lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.

[0152] Solutions or suspensions used for parenteral, intradermal, subcutaneous, intrathecal, or topical application may include, but are not limited to, a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Administered intravenously, particular carriers are physiological saline or phosphate buffered saline (PBS).

[0153] The injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that may be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution, suspension or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils conventionally are employed or as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0154] Formulations for parenteral administration include aqueous and nonaqueous sterile injection solutions that may contain anti-oxidants, buffers, bacteriostats and solutes, which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi -dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline, water- for-inj ection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0155] Suspensions, in addition to the active compounds, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.

[0156] The pharmaceutical composition of the described invention may further include conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral application which do not deleteriously react with the active compounds. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil; fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0157] The pharmaceutical composition of the described invention may be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and / or aromatic substances and the like which do not deleteriously react with the active compounds. For parenteral application, suitable vehicles include solutions, such as oily or aqueous solutions, as well as suspensions, emulsions, or implants. Aqueous suspensions may contain substances which increase the viscosity of the suspension and include, for example, but not limited to, sodium carboxymethyl cellulose, sorbitol and / or dextran. Optionally, the suspension also may contain stabilizers. These compositions also may contain adjuvants including preservative agents, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It also may be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0158] According to some embodiments, the pharmaceutical composition of the described invention comprises a therapeutically effective amount of human sCD59 and optionally other therapeutic agents included in a pharmaceutically-acceptable carrier. According to some embodiments, the components of the pharmaceutical composition also arecapable of being commingled in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

[0159] According to some embodiments, the pharmaceutical composition of the described invention includes a pharmaceutically acceptable salt. Pharmaceutically acceptable salts are those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well-known in the art. For example, P. H. Stahl, et al. describe pharmaceutically acceptable salts in detail in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley VCH, Zurich, Switzerland: 2002).SequencesEXAMPLES

[0160] The examples and embodiments described herein are for illustrative purposes only and various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.EXAMPLE 1: A Phase 2b Clinical Trial to Evaluate Intravitreal JNJ-81201887 (AAVCAGsCD59) Compared to Sham Procedure for the Treatment of Geographic Atrophy (GA) Secondary to Age-related Macular Degeneration (AMD)

[0161] An ongoing, non-limiting example of a Phase 2b, randomized, doublemasked, multicenter, dose-ranging, sham-controlled clinical trial to evaluate intravitreal JNJ- 81201887 (AAVCAGsCD59) compared to sham procedure for the treatment of geographic atrophy (GA) secondary to age-related macular degeneration (AMD)s, is provided herein. Description of the Compound

[0162] The investigational gene therapy, JNJ-81201887 (AAVCAGsCD59), is a recombinant adeno-associated virus serotype 2 (rAAV2) with a transgene encoding soluble CD59 (sCD59).Efficacy Objectives and EndpointsOverall Design

[0163] This is a Phase 2b, randomized, double-masked, multicenter, dose-ranging, sham-controlled study in participants 60 years of age or older with GA secondary to AMD conducted to evaluate the efficacy and safety of a single intravitreal injection in the study eye of the gene therapy JNJ-81201887 (AAV2CAGsCD59) after 18 months. The JNJ-81201887 gene therapy will be administered to a single eye for each participant.

[0164] Participants meeting all eligibility criteria will be randomized in a 1 : 1 : 1 ratio (stratified by lesion contiguity [multifocal vs non-multifocal] and baseline GA area [2.5 to 10 mm2 or >10 to 17.5 mm2]) to 1 of 2 dose levels of JNJ-81201887 (low dose: 8.2 x 1010vg dose; high dose: 4. 1 x 1011vg dose) or a sham arm. vg = vector genome.

[0165] Participants will receive a 20-day oral prednisone course starting on Day 1 and a single periocular corticosteroid injection on Day 4 for prophylaxis of intraocular inflammation (except those in the sham arm who will receive matching placebo and sham periocular injection). Triamcinolone acetonide (TA) 40 mg is recommended as the periocular corticosteroid injection.

[0166] Study participants will undergo an up to 28-day screening period during which all baseline procedures / tests will be performed on or prior to Day 1 / randomization (also start steroid / placebo course after randomization), after which dose administration will occur on Day 4. Participants will be followed for a period of 18 months. The study duration is approximately 19 months.Study PopulationNumber of Participants

[0167] A target of 300 participants will be enrolled in this study.Inclusion Criteria

[0168] Each potential participant must satisfy all of the following criteria to be enrolled in the study:Age1. 60 years of age or older.Type of Participant and Disease CharacteristicsStudy Eye2. Have non-subfoveal (i.e., not involving the center point of the fovea) GA secondary to AMD with an area measuring 2.5 mm2to 17.5 mm2(1 and 7 disc areas respectively), determined from screening images of FAF and SD-OCT.3. If GA is multifocal, at least one focal lesion must be >1.25 mm2(0.5 disc area).4. GA can be photographed in its entirety by FAF, using a 30-degree image centered on the fovea.5. GA can be measured separately from any areas of peripapillary atrophy, as assessed by the CRC.6. Presence of any pattern of hyper autofluorescence (hyper AF) in the junctional zone of GA (lack of hyper AF is exclusionary).7. Sufficiently clear ocular media, adequate fixation and pupillary dilation to permit fundus imaging of adequate quality.8. Best corrected distance visual acuity (BCVA) of 35 letters or better using the ETDRS chart. a. If both eyes are eligible, the eye with the worse visual acuity attributed to GA will be the study eye. b. If both eyes are eligible and have the same BCVA, then the right eye will be designated as the study eye.Fellow eye9. Fellow eye must be present with a BCVA of counting fingers or better. Systemic10. Medically stable on the basis of physical examination, including assessment of vital signs, medical history, and clinical laboratory tests performed at screening; if these results are outside the normal reference ranges, the participant may be included at the investigator’s discretion, (i.e., if the investigator judges the abnormalities or deviations from normal to be not clinically significant or to be appropriate and reasonable for the population under study), except for the following, for which participants must be excluded: alanine aminotransferase, aspartate aminotransferase or gamma-glutamyl transferase are >2 x the ULN (upper limit of normal), hemoglobin is <9 g / dL, white blood cell count is <2,500 / mm3, platelet count is <80,000 / mm3, or hematocrit is >54% (male) or >49% (female).Sex and Contraceptive / Barrier Requirements11. Man or woman (according to their reproductive organs and functions assigned by chromosomal complement).12. A woman of childbearing potential must have a negative highly sensitive serum (b-human chorionic gonadotropin) test for the sample collected at Screening and negative urine pregnancy test on Day 4 before receiving the study intervention.13. A woman must be (a) not of childbearing potential or (b) of childbearing potential and practicing a highly effective method of contraception (failure rate of <1% per year when used consistently and correctly) and agrees to remain on a highly effective method throughout the study.14. A woman must agree not to donate eggs (ova, oocytes) or freeze them for future use for the purposes of assisted reproduction for a period of 18 months from the dosing.15. A male participant must wear a condom when engaging in any activity that allows for passage of ejaculate to a woman of childbearing potential for a period of 18 months from dosing. Male participants should also be advised of the benefit for a female partner of childbearing potential to use a highly effective method of contraception, as a condom may break or leak.16. A male participant must agree not to father a child or donate sperm for the purpose of reproduction for a period of 18 months from dosing.Informed Consent17. Must sign an ICF (or their legally acceptable representative must sign) indicating that the participant understands the purpose of, and procedures required for, the study and is willing to participate in and able to complete all required assessments during the study.18. Must provide consent (or their legally acceptable representative must sign) if the participant agrees to provide an optional DNA sample for research (where local regulations permit). Refusal to give consent for the optional DNA research sample does not exclude a participant from participation in the study.19. Willing and able to adhere to specified lifestyle restrictions.Exclusion Criteria

[0169] Any potential participant who meets any of the following criteria will be excluded from participating in the study:Medical ConditionsStudy Eve1. History of or presence of retinal disease other than GA: diabetic retinopathy, central serous chorioretinopathy, inherited retinal degeneration, toxic maculopathies (i.e., hydroxychloroquine maculopathy), arterial and venous occlusive disease, macular hole that is present or has been previously repaired, or choroidal melanoma.2. Presence of macular fibrosis or retinal epithelial tear, clinically relevant myopic degeneration, or vitreous hemorrhage. Benign conditions of the vitreous (i.e., posterior vitreous detachment) or peripheral retina (i.e., paving stone degeneration, lattice degeneration, etc.) are permitted.3. History of transpupillary thermotherapy, photodynamic therapy or external -beam radiation therapy in the region of study eye.4. Any prior thermal laser in the macular region, regardless of indication.5. History of retinal detachment (with or without repair) .6. History of vitrectomy, comeal transplant, trabeculectomy, or tube-shunt surgery.7. Uncontrolled glaucoma (defined as IOP >22 despite treatment with more than 2 anti -glaucoma medications) or advanced glaucoma (cup to disc ratio >0.8).8. History of known steroid-induced ocular hypertension / glaucoma.9. Any previous intraocular or periocular surgery or intraocular laser procedure (i.e., Selective Laser Trabeculoplasty (SLT), Yttrium aluminum garnet (Y AG) within 3 months of administration of study intervention.10. Any concurrent ocular disease that, in the opinion of the investigator, would require surgery within 18 months or could affect the interpretation of test results during the course of the study.11. Aphakia or absence of the posterior capsule unless it occurred as a result of yttrium aluminum garnet (Y AG) laser posterior capsulotomy in association with prior posterior chamber intraocular lens implantation.Either Eye12. History or presence of uveitis and / or vitritis or inflammatory ocular disease a. Mild postop inflammation not exceeding 2 months in duration following a prior interventional ocular procedure is permitted.13. Active, infectious conjunctivitis, keratitis, scleritis, or endophthalmitis.14. Any sign of diabetic retinopathy or central serous chorioretinopathy.15. Presence or history of exudative MNV in either eye (also referred to as active CNV or wet AMD).Systemic16. Acute coronary syndrome, myocardial infarction or coronary artery revascularization, cerebrovascular accident, transient ischemic attack within 6 months of administration of study intervention.17. Uncontrolled hypertension defined as systolic blood pressure >170 mm Hg or diastolic blood pressure >100 mm Hg.18. Predisposition or history of increased risk of infection (e.g., history of splenectomy or chronic immunosuppression).19. History of known complement deficiency or current / past treatment with complement inhibitors.20. Active malignancy within past 12 months except for appropriately treated carcinoma in situ of the cervix, non-melanoma skin carcinoma, and prostate cancer with a Gleason score of <6 and a stable PSA (prostate-specific antigen) for >12 months.21. History of allergy to fluorescein that is not amenable to treatment.22. Pregnant, or breastfeeding, or planning to become pregnant within 18 months from dosing.23. Medical (e.g., uncontrolled diabetes, known congestive heart failure, clinically significant valvular heart disease, clinically significant cardiac rhythm or conduction abnormalities) or psychiatric conditions or any condition that may compromise the well-being of the participant or that could prevent, limit, or confound the protocol-specified assessments.24. Known allergies, hypersensitivity, or intolerance to JNJ-81201887 or its excipients.Prior / Concomitant TherapyStudy eye25. Previous intravitreal drug delivery, implant, anti-complement agents, or device implantation. A single intra- or peri -operative administration of a corticosteroid during cataract surgery for cystoid macular edema prophylaxis at least 6 months prior to enrollment is permitted.Either eye26. Prior treatment with any complement inhibitors (investigational or non- investigational) given by any route (e.g., ocular or systemic).27. Prior treatment of any kind for exudative AMD / MNV.Systemic28. Contraindications to the use of corticosteroids per local prescribing information.29. Taken any disallowed therapies before the planned dose of study intervention.30. Prior treatment with any gene therapy given by any route (e.g., ocular or systemic).31. Treatment for active systemic or localized infection.32. Received or plans to receive live, attenuated vaccine within 4 weeks of study enrollment or plans to receive live, attenuated vaccine anytime during the study. Non-live vaccines are permitted.Prior / Concurrent Clinical Study ExperienceStudy eye33. Previous participation (where the study participant was randomized to a study treatment or a sham / placebo assignment) in an ocular interventional clinical trial for AMD, except in cases where: (a) The intervention in the prior ocular interventional clinical trial was administration of vitamins or minerals; (b) It is documented (confirmed written documentation from the previous trial’s study site or sponsor) that the study participant 1) was randomized to and received sham / placebo treatment in the previous ocular interventional trial and 2) is no longer participating in that trial or follow-up from that trial.Systemic34. Received an investigational intervention within 6 weeks or 5 half-lives (whichever is longer) before the planned dose of study intervention or is currently enrolled in an investigational study.Other Exclusions35. Employee of the investigator or study site, with direct involvement in the proposed study or other studies under the direction of that investigator or study site, as well as family members of the employees or the investigator.Intervention Groups and DurationSingle liquid intravitreal injection in the study eye of 1 of 2 doses of JNJ-81201887 at the following strengths:

[0170] Group / Arm A (Low dose): 8.2 x 1010vg dose

[0171] Group / Arm B (High dose): 4.1 x 1011vg doseSham procedure / placebo control:

[0172] Group / Arm C: Single sham procedure (syringe with no needle) to mimic intravitreal injection in the study eye (placebo) Prophylaxis of intraocular inflammation:

[0173] Participants in Arms A (low dose) and B (high dose) will receive a 20-day oral prednisone course starting 3 days prior to the day of study intervention administration and a single periocular corticosteroid injection on the day of study intervention administration (Day 4) for prophylaxis of intraocular inflammation.

[0174] Participants in Arm C (single sham procedure) will receive a 20-day oral prednisone-matched placebo course and a sham corticosteroid periocular injection on the day of the sham procedure to maintain masking.Efficacy Evaluations

[0175] The following are the primary and secondary efficacy evaluations. Retinal Imaging• Blue-light FAF (Heidelberg)• Spectral Domain Optical Coherence Tomography (SD-OCT)Visual Acuity• Best Corrected Visual Acuity (BCVA, using the Early Treatment of Diabetic Retinopathy Study [ETDRS] Chart)• Best Corrected Low Luminance Visual Acuity (LEVA)• Low Luminance Visual Acuity Deficit (LLVD)Visual and Retinal Function• Reading Speed (Radner Chart)• Mesopic Microperimetry (MAIA; where available)• Patient Reported Outcomes (PROs): Functional Reading Independence (FRI) Index and National Eye Institute Visual Functioning Questionnaire-25 (NEI-VFQ-25)SAFETY EVALUATIONS

[0176] Safety evaluations include ocular adverse events of interest (including intraocular inflammation and new onset exudative macular neovascularization [MNV] among others as described in the body of the protocol), systemic adverse events, physical examination (including vital signs), ocular examination, ocular imaging parameters (for example, FAF, SD-OCT, fluorescein angiography, and color fundus photography), laboratory evaluations (clinical chemistry and hematologic measures), and pregnancy testing, if applicable.STATISTICAL METHODSSample Size Determination

[0177] Based on a meta-analysis of sham arms from completed GA clinical trials and natural history studies with comparable inclusion / exclusion criteria, the estimated mean and standard deviation for the change from baseline in GA area (square root transformed) at 18 months is 0.48 mm and 0.27 mm, respectively. Assuming 15% drop out by 18 months, it is estimated that 100 participants per arm will provide 87% power to detect at least one active treatment showing a between-group difference of 0.12 mm (corresponding to a 25% reduction in growth) at significance level of 0.025 (1-sided).Analysis Sets

[0178] For the purposes of analysis, the following analysis populations are defined:Efficacy Analyses

[0179] A mixed model for repeated measures (MMRM) will be used for the primary efficacy analysis of change in sqrt-GAA in the study eye, will include treatment, time (via a categorical variable for visit), treatment-by-time interaction, one indicator variable for stratification factor (GA lesion contiguity) as fixed effects and baseline sqrt-GAA as a co variate. An unstructured covariance will be used to model the within subject variability. The between-treatment differences in least-squares means and their 2-sided 95% confidence intervals at Month 18 will be presented. A Dunnett’s step-down procedure which controls the overall Type 1 error rate (two-sided alpha level of 0.0499 at the final analysis) will be used to test the primary endpoint.Safety Analyses

[0180] Safety will be summarized by change from baseline in physical examination (including vital signs), ocular examination, clinical chemistry, hematologic measures, and ocular and systemic treatment-emergent adverse events and SAEs, including ocular adverse events of interest (intraocular inflammation and new onset exudative MNV and others as outlined in the body of this report). In addition, certain imaging parameters will be assessed on a regular basis to monitor post-injection changes to the retina, including FAF, SD-OCT, fluorescein angiography and color fundus photography and changes in and abnormal ocular examination findings will be summarized descriptively. Observed values and change from baseline in clinical laboratory tests and vital signs will be summarized through Month 18 using descriptive statistics. The number and percentage of participants with intraocular pressure (IOP) >25 mm Hg at least one post-baseline visit and participants with an IOP rise by >10 mm Hg from baseline will be presented. The number and percentageof participants with a score >2+ in the anterior chamber cells and / or vitreous cells and vitreous haze (at least one of them) in the study eye at baseline, and at least one post-baseline visit will be presented. Shift tables from baseline to worst post-baseline value will be presented for intraocular inflammation scores (0, 0.5, 1+, 2+, 3+, 4+). The number and percentage of participants with treatment-emergent abnormal slit lamp biomicroscopy and indirect / dilated ophthalmoscopy findings over time will be presented.Immunogenicity

[0181] The incidence of anti-sCD59 transgene protein and anti-AAV2 viral capsid protein antibodies will be summarized for all participants who received an intravitreal injection of the study intervention (JNJ-81201887).

[0182] A listing of participants who are positive for antibodies to sCD59 transgene protein and anti-AAV2 viral capsid protein will be provided. The titers of antibodies to sCD59 transgene protein or anti-AAV2 viral capsid protein will be summarized for participants who are positive for antibodies to sCD59 transgene protein or anti-AAV2 viral capsid protein.

[0183] The incidence of cellular immune responses against the AAV2 viral capsids and the sCD59 transgene protein will be summarized for all participants who received an intravitreal injection of the study intervention (JNJ-81201887). A listing of participants who are positive for cellular immune responses to AAV2 viral capsids and the sCD59 transgene protein will be provided.

Claims

What is claimed:

1. A method of treating geographic atrophy (GA) in a subject, the method comprising administering a pharmaceutical composition into a GA-affected eye of a subject by ocular injection, wherein the pharmaceutical composition comprises a nucleic acid encoding a soluble CD59 (sCD59) protein.

2. The method of claim 1, wherein the GA is secondary to age-related macular degeneration.

3. The method of claim 1 or 2, wherein the pharmaceutical composition comprises a dose of viral particles of about 8.2 x IO10vector genome (vg).

4. The method of any one of the preceding claims, wherein the pharmaceutical composition comprises a dose of viral particles of about 4. 1 x 1011vector genome (vg).

5. The method of any one of the preceding claims, wherein the GA covers an area of the affected eye measuring 2.5 mm2to 17.5 mm2.

6. The method of any one of the preceding claims, wherein the GA is non-subfoveal.

7. The method of any one of the preceding claims, wherein the GA is multifocal with at least one focal lesion of >1.25 mm2.

8. The method of any one of the preceding claims, wherein the GA lesion is measured as the square root of GA lesion area by fundus autofluorescence.

9. The method of any one of the preceding claims, wherein the subject is 60 years of age or older.

10. The method of any one of the preceding claims, wherein the pharmaceutical composition is injected into only one eye.

11. The method of any one of the preceding claims, wherein the pharmaceutical composition is administered by a single intravitreal injection in the GA-affected eye.

12. The method of any one of the preceding claims, wherein the treatment is assessed at about 18 months following administration of the pharmaceutical composition.

13. The method of any one of the preceding claims, wherein the pharmaceutical composition is administered with prednisone.

14. The method of any one of the preceding claims, wherein the prednisone is administered orally for 20 days at a dose of about 40 mg / day.

15. The method of claim 14, wherein the dose of prednisone is tapered down by about 10 mg every 5 days.

16. The method of any one of the preceding claims, wherein the pharmaceutical composition is administered with a corticosteroid.

17. The method of claim 16, wherein the corticosteroid is administered by single periocular injection.

18. The method of claim 16, wherein the corticosteroid is triamcinolone acetonide (TA).

19. The method of claim 18, wherein the TA is administered at a dose of about 40 mg.

20. The method of any one of the preceding claims, wherein the sCD59 protein lacks a glycosyl phosphatidyl inositol (GPI) anchoring domain.

21. The method of any one of the preceding claims, wherein the nucleic acid encoding the sCD59 protein comprises SEQ ID NO: 1.

22. The method of any one of the preceding claims, wherein the sCD59 protein comprises SEQ ID NO: 2.

23. The method of any one of the preceding claims, wherein the nucleic acid encoding the sCD59 protein is operably linked to a promoter sequence for expression and secretion of the sCD59 protein in the cells of the GA-affected eye.

24. The method of any one of the preceding claims, wherein the nucleic acid encoding the sCD59 protein is packaged into a delivery vector.

25. The method of claim 24, wherein the delivery vector is an adeno-associated virus (AAV) vector.

26. The method of claim 25, wherein the AAV vector is AAV2.

27. The method of claim 23, wherein the promoter is a CAG promoter.

28. A method of improving geographic atrophy (GA) lesion growth in a subject, the method comprising administering a pharmaceutical composition into a GA-affected eye of a subject by ocular injection, wherein the pharmaceutical composition comprises a nucleic acid encoding a soluble CD59 (sCD59) protein.

29. The method of claim 28, wherein improvement a change in the square root transformed GA lesion area (sqrt-GAA) from baseline at months 3, 6, 9, 12, 15, and 18 in the GA-affected eye.

30. The method of claim 28 or 29 wherein the improvement is relative to a sham control.

31. The method of any one of claims 28 to 30, wherein the improvement is measured at about 18 months after injection.

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