Methods for treating macular degeneration

Abstract

This article discloses a method for treating geographic atrophy (GA) secondary to age-related macular degeneration (AMD).

Description

Technical Field

[0001] This disclosure relates in general to a method for treating geographic atrophy (GA) secondary to age-related macular degeneration (AMD). Background Technology

[0002] Age-related macular degeneration (AMD) is the leading cause of blindness in people aged 60 and older in developed countries, affecting approximately one-quarter of the population aged 70 and older. Globally, AMD accounts for about 9% of all blindness cases, affecting approximately 196 million people, and is projected to affect 288 million by 2040. AMD is a slow-progressing macular disease. The dry (non-exudative) form of AMD accounts for the majority (approximately 90%) of AMD patients. The wet (exudative) form of AMD accounts for the remaining approximately 10% of cases. The most significant vision loss occurs in late-stage forms of AMD, such as wet AMD and GA. Map-like atrophy is characterized by the progressive and irreversible loss of photoreceptor cells, RPE, and choroidal capillaries. It is usually bilateral, and the expansion of the atrophy leads to irreversible loss of visual function. It can affect central vision as well as reading, driving, and low-light vision. Globally, GA affects more than 5 million people. There is a need for improved methods for treating patients with AMD and GA. The methods disclosed in this invention address these and other important needs. Summary of the Invention

[0003] In some embodiments, this disclosure relates to methods of treating geographic atrophy (GA) in a subject, including administering a pharmaceutical composition to the subject's GA-affected eye via ocular injection, wherein the pharmaceutical composition contains nucleic acid encoding a soluble CD59 (sCD59) protein. In some embodiments, GA is secondary to age-related macular degeneration; that is, these methods involve treating GA in a subject who also suffers from age-related macular degeneration. In a further embodiment, the pharmaceutical composition contains about 8.2 × 10⁻⁶ ppm. 10 A dose of viral particles representing one vector genome (vg). In a further embodiment, the pharmaceutical composition comprises approximately 4.1 × 10^6 viral particles. 11 A dose of viral particles per vg.

[0004] In some implementations, the measurement of GA coverage of the affected eye is 2.5 mm. 2 Up to 17.5mm 2 The area of ​​the GA. In some embodiments, the GA is non-centrally depressed. In further embodiments, the GA is multifocal, with at least one lesion ≥1.25 mm. 2 The lesions are focal. In a further implementation, the GA lesion is measured as the square root of the GA lesion area by fundus autofluorescence.

[0005] In further embodiments, the subjects are 60 years of age or older. In some embodiments, the pharmaceutical composition is injected into only one eye. In some embodiments, the pharmaceutical composition is administered to the GA-affected eye via a single intravitreal injection.

[0006] In some embodiments, treatment is evaluated approximately 18 months after administration of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is administered in combination with prednisone. In a further embodiment, prednisone is administered orally for 20 days at a dose of approximately 40 mg / day. In an even further embodiment, the dose of prednisone is reduced by approximately 10 mg every 5 days.

[0007] In some embodiments, the pharmaceutical composition is administered together with a corticosteroid. In a further embodiment, the corticosteroid is administered via a single periorbital injection. In a still further embodiment, the corticosteroid is triamcinolone acetonide (TA). In some embodiments, TA is administered at a dose of approximately 40 mg.

[0008] In some embodiments, the sCD59 protein lacks a glycosylphosphatidylinositol (GPI) anchoring domain. In some 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 a further embodiment, the nucleic acid encoding the sCD59 protein is operatively linked to a promoter sequence to express and secrete the sCD59 protein in cells of the GA-affected eye. In a still further embodiment, the nucleic acid encoding the sCD59 protein is packaged into a delivery vector.

[0009] In some embodiments, the delivery vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is AAV2. In a further embodiment, the promoter is the CAG promoter.

[0010] In some embodiments, this disclosure relates to methods for improving the growth of GA lesions in a subject, methods comprising administering a pharmaceutical composition to the subject's GA-affected eye via ocular injection, wherein the pharmaceutical composition comprises a nucleic acid encoding the sCD59 protein. In some embodiments, improvement is the change in the square root-transformed GA lesion area (sqrt-GAA) of the GA-affected eye relative to baseline at months 3, 6, 9, 12, 15, and 18. In further embodiments, improvement is relative to a sham control. In still further embodiments, improvement is measured approximately 18 months after injection. Attached Figure Description

[0011] The invention and the detailed embodiments described below will be further understood when read in conjunction with the accompanying drawings. The drawings illustrate exemplary embodiments of the disclosed methods or uses for the purpose of illustrating them; however, these methods and uses are not limited to the specific embodiments disclosed. In the drawings: Figure 1 This is a schematic overview diagram of the study described in Example 1. IVT = Intravitreal space. Detailed Implementation

[0012] In this disclosure, unless the context clearly indicates otherwise, the singular forms “a,” “an,” “the,” and “the” include the plural meaning, and references to a specific numerical value include at least that specific value. Thus, for example, a reference to “material” means at least one of such materials, as well as equivalents of that material known to those skilled in the art.

[0013] When the descriptors “about” or “substantially” are used to represent values ​​as approximations, it should be understood that the specific value constitutes another embodiment. Generally, the use of the terms “about” or “substantially” indicates approximations that may vary depending on the desired characteristics the disclosed subject matter seeks to achieve, and will be interpreted based on their function in the specific context in which the approximation is used. Those skilled in the art will be able to interpret these approximations conventionally. In some cases, the number of significant figures used for a particular value can be a non-limiting method for determining the range of the term “about” or “substantially.” In other cases, the gradient used in a range of values ​​can be used to determine the expected range that can be used for each value with the term “about” or “substantially.” Where applicable, all ranges include end values ​​and are composable. That is, a reference to a value specified within a range includes every value within that range.

[0014] When a list is provided, unless otherwise indicated, it should be understood that each individual element in the list and each combination of the list is to be understood as a separate implementation. For example, a list of implementations presented as “A, B or C” will be understood to include implementations “A”, “B”, “C”, “A or B”, “A or C”, “B or C”, or “A, B or C”.

[0015] It should be understood that, for clarity, certain features of this disclosure are set forth in the context of individual embodiments, but may also be provided in combination in a single embodiment. That is, unless obviously incompatible or excluded, each individual embodiment is considered to be combinable with any other embodiment, and such combination is considered another embodiment. Conversely, for brevity, various features of this disclosure are set forth in the context of individual embodiments, but may also be provided individually or in any sub-combination. It should also be noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a premise for the use of exclusive terms such as “solely,” “only,” etc., or the use of “negative” to define the elements of the claims. Finally, although an embodiment may be described as part of a series of steps or a more general structure, each step may also be considered an independent embodiment in itself.

[0016] certain terms All terms used throughout this specification shall have the definitions set forth herein.

[0017] As used herein, the term “application” includes in vivo application as well as direct application to ex vivo tissues. Typically, a composition may contain a volumetric unit of a desired, conventionally nontoxic, pharmaceutically acceptable carrier, adjuvant, and solvent for systemic administration via oral, oral, enteric, topical, inhalation or blowing (i.e., through the mouth or nose), or rectal administration, or may be administered topically via, but not limited to, injection, implantation, transplantation, local application, or enteric administration.

[0018] As used herein, the term "CD59" refers to a membrane-bound glycoprotein associated with cell membranes, including both hematopoietic and non-hematopoietic cells, such as those found in endothelial cells, peripheral nerve fibers, neurons, microglia, oligodendrocytes, astrocytes, ependymal cells, epithelial cells, salivary gland acinar cells, bronchial epithelium, renal tubules, and squamous epithelium. The CD59 protein inhibits the assembly of the functional membrane attack complex (MAC), thus protecting cells from complement-mediated activation and / or lysis. The protein structure of CD59 comprises 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 comprises 26 amino acids at the C-terminus, which specifies a signal sequence for attaching a glycosylphosphatidylinositol anchor (GPI anchor) to asparagine at position 77. The cDNA sequence of CD59 is shown in U.S. Patent No. 5,624,837 to Fodor et al., issued on April 29, 1997, the full text of which is incorporated herein by reference.

[0019] As used herein, the term "dosage unit form" refers to the physical discrete unit of the active agent applicable to the patient to be treated.

[0020] As used in this article, the term "drusen" refers to yellow deposits under the retina.

[0021] As used in this article, the terms “enhancement”, “improvement”, etc., in various grammatical forms refer to an increase or strengthening in quality or quantity, or to something better or more enhanced.

[0022] The terms “functionally equivalent” or “functionally equivalent” are used interchangeably herein and refer to substances, molecules, polynucleotides, proteins, peptides, or polypeptides that have similar or identical effects or uses. For example, a polypeptide functionally equivalent to SEQ ID NO: 2 may have biological activity substantially similar to or the same as that of the expressed polypeptide of SEQ ID NO: 2, such as inhibitory activity, kinetic parameters, salt inhibition, cofactor-dependent activity, and / or functional unit size.

[0023] The term "fundus autofluorescence (FAF)" refers to a non-invasive retinal imaging modality that produces a density map of the ocular fluorophore lipofuscin in the retinal pigment epithelium. FAF images can be obtained using a confocal laser scanning ophthalmoscope (cSLO).

[0024] The term “inhibition” and its various grammatical forms, including but not limited to “inhibition”, are used herein to refer to reducing the amount or rate of a process, stopping the method entirely, or reducing, limiting, or blocking its action or function. Inhibition may include reducing or decreasing the amount, rate, action, or method 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%.

[0025] The terms “membrane attack complex” and “MAC” are used interchangeably in this document and refer to effectors of the immune system that consist of protein complexes that normally form on the surface of pathogen cell membranes due to activation of the host complement system. Antibody-mediated complement activation leads to MAC deposition on the surface of infected cells, causing pore disruption of the infected cell membrane, resulting in cell lysis and death. MAC is composed of complement components C5b, C6, C7, C8, and several C9 molecules.

[0026] As used in this article, the term “modification” means to alter, change, adjust, modify, influence, or regulate one or more details to a certain degree or proportion.

[0027] When used to describe GA, the term "multifocal" refers to two or more atrophic lesions in the eye.

[0028] The term “nucleotide” is used herein to refer to deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, includes known analogs that have the basic properties of natural nucleotides because they hybridize with single-stranded nucleotides in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).

[0029] The term "nucleotide" is used herein to refer to a compound consisting of a heterocyclic base, a sugar, and one or more phosphate ester groups. In the most common nucleotides, the base is a derivative of a purine or pyrimidine, and the sugar is a pentose deoxyribose or ribose. Nucleotides are monomers of nucleic acids, in which three or more are linked together to form nucleic acids. Nucleotides are the structural units of RNA, DNA, and several cofactors, including but not limited to CoA, FAD, DMN, NAD, and NADP. Purines include adenine (A) and guanine (G); pyrimidines include cytosine (C), thymine (T), and uracil (U).

[0030] The following terms are used in this document to describe sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.

[0031] (a) The term “reference sequence” refers to a sequence used as the basis for sequence comparison. A reference sequence may be a subset or the whole of a specified sequence; for example, as a fragment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

[0032] (b) The term “comparison window” refers to a contiguous and specific segment of a polynucleotide sequence that can be compared with a reference sequence, and wherein the polynucleotide sequence portion within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. Typically, the comparison window is at least 20 consecutive nucleotides in length, and optionally may be at least 30 consecutive nucleotides, at least 40 consecutive nucleotides, at least 50 consecutive nucleotides, at least 100 consecutive nucleotides, or longer. Those skilled in the art will understand that, in order to avoid high similarity to the reference sequence due to gaps in the polynucleotide sequence, a gap penalty is typically introduced and subtracted from the number of matches.

[0033] The sequence alignment methods used for comparison are well known in the art. The optimal alignment of sequences for comparison can be performed using the following: local homology algorithms, Smith and Waterman, Adv. Appl. Math. 2:482 (1981); homology alignment algorithms, Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); similarity search methods, Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); computerized implementations of these algorithms, including but not limited to: CLUSTAL in the PC / Gene program of 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 in Higgins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet et al., Nucleic Acids Research 16:10881-90 (1988); Huang et al., Computer Applications in the Biosciences, SASSOS (1992); and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). The family of BLAST programs that 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, edited by Ausubel et al., Greene Publishing and Wiley-Interscience, New York (1995).

[0034] Unless otherwise stated, the sequence identity / similarity values ​​provided herein refer to those obtained using the BLAST 2.0 program suite with default parameters. Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Software for performing BLAST analysis is publicly available, for example, from the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by recognizing short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighbor word score threshold (Altschul et al., ibid.). These initial neighbor word hits act as seeds for initiating a search to find the longer HSPs containing them. The word hits are then extended in both directions along each sequence, as long as it increases the cumulative alignment score. For nucleotide sequences, parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0) are used. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Word hits cease in each direction when the cumulative alignment score decreases by an amount X from its maximum realized value; the cumulative score becomes zero or lower due to the accumulation of one or more negative score 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) defaults to a word length (W) of 11, an expected value (E) of 10, a cutoff value of 100, M=5, N=-4, and a comparison of two strands. For amino acid sequences, the BLASTP program defaults to a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Set. USA 89:10915).

[0035] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Set. USA 90:5873-5787 (1993)). One similarity metric provided by the BLAST algorithm is the minimum sum probability (P(N)), which indicates the probability that a match will occur by chance between two nucleotide or amino acid sequences. BLAST search assumes that proteins can be modeled as random sequences. However, many real proteins contain regions of non-random sequence, which can be homopolymeric segments, short repeats, or regions rich in one or more amino acids. Such low-complexity regions can be aligned between unrelated proteins, even if the other regions of the protein are completely different. Multiple low-complexity filtering procedures can be used to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17: 149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993)) low-complexity filters can be used alone or in combination.

[0036] (c) In the context of two nucleic acid or polypeptide sequences, the terms “sequence identity” or “identity” are used herein to refer to identical residues in two sequences when compared against the largest corresponding sequence within a specified comparison window. When using a sequence identity percentage for a reference protein, it is recognized that dissimilar residue positions are often distinguished by conserved amino acid substitutions, i.e., where an amino acid residue is substituted by another amino acid residue with similar chemical properties (e.g., charge or hydrophobicity) and therefore does not alter the functional properties of the molecule. When sequences differ in terms of conserved substitutions, the sequence identity percentage can be adjusted upwards to correct for the conservatism of the substitution. Sequences that differ due to such conserved substitutions are referred to as having “sequence similarity” or “identity.” Methods for making such adjustments are well known to those skilled in the art. Typically, this involves scoring the conserved substitution as a partial rather than a complete mismatch, thereby increasing the percentage of sequence identity. Thus, for example, when identical amino acids are given 1 point and non-conservative substitutions are given 0 points, a conserved substitution is given 0–1 points. The scores for conservative replacements are calculated, for example, according to the algorithm of Meyers and Miller, Computer Applic. Biol. Set., 4:11-17 (1988), as implemented in program PC / GENE (Intelligenetics, Mountain View, Calif, USA).

[0037] (d) The term “sequence identity percentage” in this document refers to a value determined by comparing two best-aligned sequences in a comparison window, where the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence used for the best alignment of the two sequences (which does not contain additions or deletions). This percentage is calculated as follows: the number of positions in which the same nucleic acid base or amino acid residue appears in both sequences is determined to obtain the number of matching positions; this number of matching positions is divided by the total number of positions in the comparison window; and the result is then multiplied by 100 to obtain the sequence identity percentage.

[0038] (e) The term “substantially identical” for a polynucleotide sequence means that the polynucleotide contains a sequence having at least 70%, at least 80%, at least 90%, and at least 95% sequence identity compared to a reference sequence using one of the alignment procedures with standard parameters. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc. Substantially identical amino acid sequences for these purposes generally mean at least 60%, or at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. Another indication of substantially identical nucleotide sequences is that the two molecules hybridize under stringent conditions. However, if they encode substantially identical polypeptides, nucleic acids that do not hybridize under stringent conditions are still substantially identical. This can happen, for example, when nucleic acid copies are generated using the maximum codon degeneracy allowed by the genetic code. Another indication of substantially identical nucleic acid sequences is that the polypeptide encoded by the first nucleic acid is immunely cross-reactive with the peptide encoded by the second nucleic acid. The term "fundamental identity" of a protein sequence refers to the presence of a sufficient or minimum number of amino acid residues in the first amino acid sequence that are identical to those aligned to the second amino acid sequence, such that the first and second amino acid sequences may share a common domain and / or common functional activity. For example, amino acid sequences containing a shared domain have at least about 60% identity, or at least 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% identity.

[0039] As used herein, the term "extracorporeal" refers to introduction into the body via injection (i.e., administration by injection), including, for example, intraocular (also known as intravitreal) (i.e., injection into the vitreous humor of the eye), subretinal (i.e., injection into the subretinal space between the photoreceptors and the retinal pigment epithelium (RPE) layer of the retina), subcutaneous (i.e., injection under the skin), intramuscular (i.e., injection into muscles); intravenous (i.e., injection into veins), intrathecal (i.e., injection into the space surrounding the spinal cord), and intrasternal injection or infusion techniques. The non-enteral administration compositions of the present invention are delivered using a needle (e.g., a surgical needle). As used herein, the term "surgical needle" refers to any needle suitable for delivering (i.e., capable of flowing) the fluid compositions of the present invention to selected anatomical structures. Injectable formulations can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques, such as sterile injectable aqueous or oily suspensions.

[0040] As used herein, the term "pharmaceuticalally acceptable carrier" refers to any substantially nontoxic carrier generally suitable for drug administration, wherein the isolated polypeptides of the present invention will remain stable and bioavailable. A pharmaceutically acceptable carrier must have sufficiently high purity and sufficiently low toxicity to make it suitable for administration to the mammals to be treated. It should further maintain the stability and bioavailability of the active agent. A pharmaceutically acceptable carrier can be liquid or solid, and when combined with the active agent and other components of a given composition, it is selected to provide the desired volume, consistency, etc., taking into account the planned manner of administration.

[0041] The term "pharmaceutically acceptable salt" means a salt that is suitable for use in contact with human and lower animal tissues within the limits of reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, etc., and in proportion to a reasonable efficacy / risk ratio.

[0042] The terms “polypeptide,” “peptide,” or “protein” are used interchangeably herein and refer to polymers of amino acid residues. This term applies to polymers of amino acids in which one or more amino acid residues are artificial chemical analogs of naturally occurring amino acids, as well as polymers of naturally occurring amino acids. A fundamental property of such analogs of naturally occurring amino acids is that, when incorporated into a protein, the protein exhibits a specific reactivity to antibodies induced by the same protein but composed entirely of naturally occurring amino acids.

[0043] The terms “peptide” and “protein” are used herein in their broadest sense, referring to the sequence of subunit amino acids, amino acid analogs, or peptide mimics. Subunits are linked by peptide bonds unless otherwise stated. The peptides described herein can be chemically synthesized or recombinantly expressed. The peptides of the present invention can also be chemically synthesized. Synthetic peptides prepared using well-known solid-phase, liquid-phase, or peptide condensation techniques, or any combination thereof, can include natural and non-natural amino acids. The amino acids used for peptide synthesis can be standard Boc (Na-amino-protected Na-tert-butoxycarbonyl) amino acid resins with the standard deprotection, neutralization, coupling, and washing scheme of Merrifield’s original solid-phase method (1963, J. Am. Chem. Soc. 85:2149-2154), or base-labile Na-amino-protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J. Org. Chem. 37:3403-3409). Both Fmoc and Boc, whose Na-amino-protected amino acids are available from Sigma, Cambridge Research Biochemical, or other chemical companies familiar to those skilled in the art. Furthermore, other Na-protecting groups familiar to those skilled in the art can be used to synthesize peptides. Solid-phase peptide synthesis can be performed using techniques familiar to those skilled in the art and is provided, for example, in Stewart and Young, 1984, Solid Phase Synthesis, 2nd ed., Pierce Chemical Co., Rockford, Ill.; Fields and Noble, 1990, Int. J. Pept. Protein Res. 35: 161-214, or using an automated synthesizer. The peptides of the present invention may contain D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), combinations of D and L-amino acids, and various “designed” amino acids (e.g., P-methyl amino acids, Ca-methyl amino acids, and Na-methyl amino acids, etc.) to convey specific properties. Synthetic amino acids include ornithine for lysine and leucine for leucine or isoleucine. Furthermore, the peptides may have peptide-like bonds, such as ester bonds, to prepare peptides with novel properties. For example, a peptide incorporating a reduced peptide bond can be generated, namely RI-CH2-NH-R2, where R1 and R2 are amino acid residues or sequences. The reduced peptide bond can be introduced as a dipeptide subunit. This polypeptide will be resistant to protease activity and will have an extended in vivo half-life. Therefore, these terms also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of naturally occurring amino acids, as well as naturally occurring amino acid polymers.The fundamental property of such analogues of naturally occurring amino acids is that, when incorporated into a protein, the protein exhibits a specific responsiveness to antibodies induced by the same protein but composed entirely of naturally occurring amino acids.

[0044] The terms “polypeptide,” “peptide,” and “protein” also include modifications, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues. It should be understood that, as is well known and as stated above, polypeptides may not be perfectly linear. For example, as a result of pervasive proteination, polypeptides can be branched, and they can be cyclic, with or without branching, which is generally the result of post-translational events, including natural processing events and non-naturally occurring events induced by artificial manipulation. Cyclic, branched, and branched-cyclic polypeptides can be synthesized through non-translational natural processes as well as through fully synthetic methods. In some embodiments, peptides have any length or size.

[0045] As used in this article, the term "prevention" means maintaining, preventing, or avoiding the occurrence, presence, or effect of an event, behavior, or action.

[0046] The term "recombinant" refers to a cell or vector that has been modified by introducing a heterologous nucleic acid, or a cell derived from such a modified cell. Recombinant cells express genes not found in the same form in the native (non-recombinant) form of the cell, or express native genes that are abnormally, poorly, or not expressed at all due to intentional human intervention. As used herein, the term "recombinant" does not include alterations to cells or vectors through naturally occurring events (e.g., spontaneous mutation, natural transformation, transduction / transposition) (such as those events that occur without intentional human intervention).

[0047] The term "recombinant expression cassette" refers to a recombinant or synthetically produced nucleic acid construct that has a set of specific nucleic acid elements that allow a particular nucleic acid to be transcribed in a host cell. Recombinant expression cassettes are incorporated into plasmids, chromosomes, mitochondrial DNA, viruses, or nucleic acid fragments. Typically, in other sequences, the recombinant expression cassette portion of an expression vector includes the nucleic acid to be transcribed, a promoter, and transcription termination signals such as a poly-A signal.

[0048] The term "recombinant host" refers to any prokaryotic or eukaryotic cell containing a cloning vector or expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain a cloned gene or target gene in the host cell's chromosome or genome.

[0049] As used herein, the term "recombinant protein" refers to proteins produced through genetic engineering, such as by manipulating genetically modified organisms like microorganisms.

[0050] As used in this article, the term “reduction” refers to limiting the occurrence of a developmental disability in individuals at risk of developing such a disability.

[0051] The term "fake" refers to a procedure performed using a syringe, without a needle or periocular / intraocular puncture.

[0052] The term “similar” is used interchangeably with the terms similar, comparable, or similar, and refers to having common traits or characteristics.

[0053] As used herein, the term "solution" refers to a homogeneous mixture of two or more substances. It is usually a liquid, although not necessarily. In a solution, the molecules of the solute (or dissolved substance) are uniformly distributed among the molecules of the solvent.

[0054] As used herein, the terms “soluble CD59,” “sCD59,” and “membrane-independent CD59” refer to CD59 amino acid sequences that lack a GPI anchor or have a modified GPI anchor that lacks the function and ability to bind to the cell membrane or cell membrane-related structures such as membrane-binding proteins.

[0055] As used in this article, the term "suspension" refers to a dispersion (mixture) in which a finely dispersed class of substances is combined with another class of substances, wherein the former is so finely dispersed and mixed that it does not settle rapidly. In everyday life, the most common suspension is a suspension of solids in a liquid.

[0056] The term "central depression" refers to the point at which the central depression is located, while the term "non-central depression" refers to the point at which the central depression is not located.

[0057] As used herein, the terms “subject” or “individual” or “patient” or “participant” are used interchangeably to refer to a member of the animal class of mammalian origin, including humans. The term “subject in need of” is used to refer to a subject who has heart failure or is at risk of developing heart failure, including a subject with AMI that presents as a disease leading to left ventricular remodeling.

[0058] As used herein, the phrase “subject requiring such treatment” refers to a patient suffering from a disease, disorder, symptom, or pathological process. In some embodiments, the term “subject requiring such treatment” is also used to refer to a patient who (i) will be administered at least one dose of an adenovirus vector construct expressing human sCD59 of the present invention; (ii) has received at least one dose of an adenovirus vector construct expressing human sCD59 of the present invention; or (iii) has received at least one dose of an adenovirus vector construct expressing human sCD59 of the present invention, unless the context and usage of the phrase indicate otherwise.

[0059] As used herein, the term "substantially similar" means that the first value, aspect, trait, characteristic, quantity, or amount is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the second value, aspect, trait, characteristic, quantity, or amount. For example, a polypeptide substantially similar to (SEQ ID NO: 2) would have at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% amino acid sequence identity with the amino acid sequence (SEQ ID NO: 2).

[0060] The term "substitution" is used herein to refer to the exchange of one or more bases in a DNA sequence with one or more other bases. Substitution can be synonymous or non-synonymous. As used herein, "synonymous substitution" means that in an exon of a protein-coding gene, one base replaces another base, such that the resulting amino acid sequence is not modified. As used herein, the term "non-synonymous substitution" means that in an exon of a protein-coding gene, one base replaces another base, such that the resulting amino acid sequence is modified.

[0061] As used herein, the term “symptom” refers to a phenomenon that is caused by and accompanies a particular disease or disorder, and serves as an indication of it.

[0062] As used in this article, the term "syndrome" refers to a pattern of symptoms that indicates certain diseases or conditions.

[0063] As used herein, the term "therapeutic agent" refers to a drug, molecule, nucleic acid, protein, metabolite, composition, or other substance that provides a therapeutic effect. As used herein, the term "active" refers to an ingredient, component, or composition of the composition of the present invention responsible for the intended therapeutic effect. The terms "therapeutic agent" and "active agent" are used interchangeably herein. As used herein, the term "therapeutic component" refers to a therapeutically effective amount (i.e., dosage and frequency of administration) that eliminates, reduces, or prevents the progression of a particular disease manifestation in a certain percentage of a population. A commonly used example of a therapeutic component is ED50, which describes a dosage in a specific amount that shows therapeutic efficacy against a particular disease in 50% of a population.

[0064] The terms "therapeutic amount," "therapeutic effective amount," "effective amount," or "drug effective amount" for active agent are used interchangeably and refer to an amount sufficient to provide the intended therapeutic benefit. The effective amount of active agent used according to the present invention typically ranges from about 8.2 × 10⁻⁶ per dose. 10 One vector genome (vg) up to 4.1 × 10 11However, dosage levels are based on a variety of factors, including the type of injury, the patient's age, weight, sex, medical condition, severity of the condition, route of administration, and the specific active agent used. Therefore, dosage regimens can vary widely, but can be routinely determined by a physician using standard methods. Additionally, the terms "therapeutic dose," "therapeutic effective dose," and "pharmaceutical effective dose" include preventative doses of the compositions of the present invention. In the protective or preventative application of the present invention, the pharmaceutical composition or agent is administered to a patient who is susceptible to or at risk of a disease, disorder, or condition, in an amount sufficient to eliminate or reduce the risk of the disease, disorder, or condition, mitigate its severity, or delay its onset, including biochemical, histological, and / or behavioral symptoms, complications, and intermediate pathological phenotypes present during the development of the disease, disorder, or condition. Generally, the maximum dose is preferred, i.e., the highest safe dose based on certain medical judgments. The terms "dose" and "volume" are used interchangeably herein.

[0065] As used herein, the term "therapeutic effect" refers to the outcome of treatment that is judged to be desirable and beneficial. Therapeutic effects may include, directly or indirectly, the prevention, reduction, or elimination of disease manifestations. Therapeutic effects may also include, directly or indirectly, the prevention, reduction, or elimination of the progression of disease manifestations.

[0066] For any of the therapeutic agents described herein, the therapeutically effective dose may initially be determined by preliminary in vitro studies and / or animal models. The therapeutically effective dose may also be determined by human data. The applied dose may be adjusted based on the relative bioavailability and potency of the compound administered. Adjusting the dose to achieve maximum efficacy based on the methods described above and other well-known methods is within the capabilities of a person skilled in the art.

[0067] The general principles for determining treatment effectiveness, which can be found in Chapter 1 of Goodman and Gilman's *The Pharmacological Basis of Therapeutics*, 10th edition, McGraw-Hill (New York) (2001), are summarized below.

[0068] Pharmacokinetic principles provide the basis for modifying dosage regimens to achieve the desired level of therapeutic efficacy while minimizing unacceptable side effects. Additional guidance for dosage modifications can be obtained when the plasma concentration of the drug is measurable and correlated with the therapeutic window.

[0069] Pharmaceutical products are considered pharmaceutical equivalents if they contain the same active ingredient and are identical in strength or concentration, dosage form, and route of administration. Two pharmaceutically equivalent products are considered bioequivalent when the rate and extent of bioavailability of the active ingredient in the two products do not differ significantly under appropriate testing conditions.

[0070] The term “treatment” includes eliminating, substantially inhibiting, slowing or reversing the progression of a disease, symptom or disorder; substantially improving the clinical or aesthetic symptoms of a symptom; substantially preventing the occurrence of the clinical or aesthetic symptoms of a disease, symptom or disorder; and preventing harmful or bothersome symptoms. Treatment further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting the development of characteristic symptoms of the treated disorder; (c) limiting the worsening of the symptomatic features of the treated disorder; (d) limiting the recurrence of the disorder in patients who previously had the disorder; and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic about the disorder.

[0071] The terms “variant,” “mutant,” and “derivative” are used herein to refer to a nucleotide or polypeptide sequence that is substantially identical to a reference nucleotide or polypeptide sequence. Differences in the sequence can result from natural or designed alterations to the sequence or structure. Natural changes can occur during the normal or intrinsic replication of a particular nucleic acid sequence. Designed changes can be specifically engineered and introduced into the sequence for a particular purpose. Such specific changes can be performed in vitro using a variety of mutagenesis techniques. Such specifically generated sequence variants can be referred to as “mutants” or “derivatives” of the original sequence.

[0072] Those skilled in the art can also produce polypeptide variants of SEQ ID NO: 2, having one or more amino acid substitutions, deletions, additions, or replacements, but functionally equivalent to SEQ ID NO: 2. These variants may include, in particular: (a) variants in which one or more amino acid residues are substituted with conserved or non-conserved 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; (d) variants in which an amino acid residue from one class is substituted at a conserved or non-conserved position with a corresponding residue from another class; and (d) variants in which the target protein is fused with another peptide or polypeptide, such as a fusion chaperone, protein tag, or other chemical motif, which may confer useful properties to the target protein, such as an epitope of an antibody. Techniques for obtaining such variants, including but not limited to genetic (suppression, deletion, mutation, etc.), chemical, and enzymatic techniques, are known to those skilled in the art. As used herein, the term “mutation” refers to an alteration in the DNA sequence within a gene or chromosome of an organism, resulting in a new characteristic or trait not found in the parental type, or such an alteration occurring in a chromosome through a change in the nucleotide sequence of the DNA encoding the gene or through a change in the physical arrangement of the chromosome. The three mechanisms of mutation include substitution (exchanging one base pair for another), addition (insertion of one or more bases into the sequence), and deletion (loss of one or more base pairs).

[0073] As used herein, the term "vector" refers to a genetically engineered vector for delivering genes into cells. As used herein, the term "viral vector" refers to a virus used as a vector to deliver a target gene by infecting cells. These viruses are modified so that they do not cause disease when used in humans. Types of viruses include, but are not limited to, retroviruses and adenoviruses. Retroviruses integrate their genetic material, including the target gene, into the chromosomes of a cell, while adenoviruses introduce their DNA, including the target gene, into the cell nucleus without integrating the genetic material into the chromosomes.

[0074] As used in this article, the term "solvent" refers to a substance that facilitates the use of a drug or other material mixed with it.

[0075] Treatment methods and uses In some embodiments, this disclosure relates to methods of treating geographic atrophy (GA) in a subject. According to some embodiments, the method includes contacting cells or tissue with a pharmaceutical composition comprising a human sCD59 protein source. According to some embodiments, the pharmaceutical composition comprises a nucleotide sequence encoding human sCD59 operatively 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 operatively linked to a promoter and encapsulated in a delivery vector. According to some embodiments, the pharmaceutical composition comprises a human sCD59 expression construct encapsulated in a delivery vector. In some embodiments, these methods include, consist of, or substantially consist of the process of administering the pharmaceutical composition to the GA-affected eye of a subject, wherein the pharmaceutical composition comprises nucleic acid encoding a soluble CD59 (sCD59) protein, thereby treating GA.

[0076] In some implementations, GA is secondary to age-related macular degeneration (AMD). AMD is a slowly progressive disease of the macula that impairs an individual's ability to read, drive, recognize faces, and see details. AMD begins with lipoprotein yellow deposits (drusen) in the macular region and can progress to late-stage AMD, where the most significant vision loss occurs. Late-stage AMD is generally classified into two types, 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). Dry AMD is associated with drusen and pigmentary changes in the macula. Most vision loss occurs in late-stage cases of dry AMD, accompanied by the formation and expansion of GA, which is associated with the loss of photoreceptor cells, retinal pigment epithelium (RPE), and the choroidal capillary layer. According to some implementations, AMD is either wet or exudative. According to some implementations, AMD is dry AMD.

[0077] In some embodiments, this disclosure relates to methods of treating a subject with wet AMD. According to some embodiments, the method includes contacting cells or tissue with a pharmaceutical composition comprising a human sCD59 protein source. According to some embodiments, the pharmaceutical composition comprises a nucleotide sequence encoding human sCD59 operatively 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 operatively linked to a promoter and encapsulated in a delivery vector. According to some embodiments, the pharmaceutical composition comprises a human sCD59 expression construct encapsulated in a delivery vector. In some embodiments, these methods include, consist of, or substantially consist of the following steps: administering the pharmaceutical composition to the AMD-affected eye of a subject, wherein the pharmaceutical composition comprises nucleic acid encoding the sCD59 protein, thereby treating wet AMD.

[0078] Without being bound by any theory, it should be understood that the cell membrane is generally protected from complement by cell surface proteins (e.g., CD59), which specifically inhibit the activation of C5b-9 pores after C9 complement proteins bind 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 proteins for binding to C8 complement proteins in the C5b-8 complex, thereby reducing or preventing the formation of the C5b-9 membrane attack complex. Therefore, CD59 is used to reduce cellular activation and cell lysis by terminal complement MAC.

[0079] Theories have linked the etiology of diseases such as age-related macular degeneration (AMD) to the activation of the complement system and the formation of MAC. Dinu (US Patent Application No. 2007 / 0196367 A1, published August 23, 2007) proposed preventing fragment formation by inhibiting complement as a therapeutic agent for AMD.

[0080] Diseases associated with uncontrolled complement activity include: bacterial infections such as Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis; angioedema; kidney diseases such as atypical hemolytic uremic syndrome; paroxysmal nocturnal hemoglobinuria; systemic lupus erythematosus; and central nervous system diseases including Alzheimer's disease, Huntington's disease, and retinal diseases including but not limited to AMD.

[0081] According to some implementation schemes, human sCD59 effectively inhibits MAC formation. According to some implementation schemes, MAC formation is inhibited by delivering a vector containing nucleic acid encoding human sCD59 into cells.

[0082] According to some embodiments, human sCD59 is effective in treating GA secondary to AMD. According to some embodiments, GA is treated by delivering a vector containing nucleic acid encoding human sCD59 into cells. According to some embodiments, human sCD59 is effective in preventing the onset of GA. According to some embodiments, the occurrence of GA is prevented by delivering a vector containing nucleic acid encoding human sCD59 into cells. According to some embodiments, human sCD59 is effective in preventing the progression of GA. According to some embodiments, the progression of GA is prevented by delivering a vector containing nucleic acid encoding human sCD59 into cells. According to some embodiments, human sCD59 is effective in reversing the progression of GA. According to some embodiments, the progression of GA is reversed by delivering a vector containing nucleic acid encoding human sCD59 into cells.

[0083] In some embodiments, this disclosure relates to methods for improving the growth of GA lesions in a subject, methods comprising, consisting of, or substantially consisting of the following steps: administering a pharmaceutical composition to the GA-affected eye of the subject by ocular injection, wherein the pharmaceutical composition contains nucleic acid encoding the sCD59 protein.

[0084] In some embodiments, this disclosure relates to methods for slowing the growth and progression of GA lesions in a subject, methods comprising, consisting of, or substantially consisting of the following steps: administering a pharmaceutical composition to the GA-affected eye of the subject by ocular injection, wherein the pharmaceutical composition contains nucleic acid encoding the sCD59 protein.

[0085] In some embodiments, this disclosure relates to methods for improving low-light vision (LLVA) in subjects with GA, methods comprising, consisting of, or substantially consisting of the following steps: administering a pharmaceutical composition to the GA-affected eye of the subject by ocular injection, wherein the pharmaceutical composition comprises nucleic acid encoding the sCD59 protein. In some embodiments, improvement in LLVA is determined by improvement in the Early Treatment Diabetic Retinopathy Study (ETDRS) letter score. According to some embodiments, a method for improving LLVA in a subject with GA includes contacting the GA-affected eye with a composition comprising a carrier carrying a nucleotide sequence encoding the protein that causes expression of a recombinant engineered human sCD59 protein in cells, operatively linked to a promoter sequence, such that the protein includes at least one mutation resulting in loss of function of the GIP anchoring domain, such that the protein is a recombinant membrane-independent sCD59 and not membrane-targeted; and, improvement in ETDRS is observed after contact compared to ETDRS observed before contact.

[0086] In some embodiments, this disclosure relates to methods for improving visual and retinal function in subjects with GA, methods comprising, consisting of, or substantially consisting of the following steps: administering a pharmaceutical composition to the GA-affected eye of the subject by ocular injection, wherein the pharmaceutical composition contains a nucleic acid encoding the sCD59 protein. In some embodiments, the improvement in visual and retinal function is determined by an improvement in reading speed (Radner visual acuity chart). In some embodiments, the improvement in visual and retinal function is determined by an improvement in retinal sensitivity as measured by a mesoscopic microfield test (macular integrity assessment (MAIA)). According to some implementation schemes, a method for improving visual and retinal function in subjects with GA includes contacting the GA-affected eye with a composition comprising a carrier carrying a nucleotide sequence encoding a recombinant engineered human sCD59 protein operatively linked to a promoter sequence, causing expression of the protein in cells, such that the protein includes at least one mutation resulting in loss of function of the GPI anchoring domain, such that the protein is a recombinant membrane-independent CD59 (sCD59) and is not membrane-targeted; and, an improvement in reading speed is observed after contact compared to reading speed observed before contact.

[0087] In some embodiments, this disclosure relates to methods for improving the vision of a subject with GA, methods comprising, consisting of, or substantially consisting of the following steps: administering a pharmaceutical composition to the GA-affected eye of the subject by ocular injection, wherein the pharmaceutical composition comprises nucleic acid encoding the sCD59 protein. In some embodiments, the vision is best-corrected visual acuity (BCVA). In some embodiments, the improvement in vision is determined by an improvement in the Early Treatment Diabetic Retinopathy Study (ETDRS) letter score. According to some embodiments, a method for improving the vision of a subject with GA includes contacting the GA-affected eye with a composition comprising a carrier carrying a nucleotide sequence encoding the protein that causes expression of a recombinant engineered human sCD59 protein in cells, operatively linked to a promoter sequence, such that the protein includes at least one mutation resulting in loss of function of the GIP anchoring domain, such that the protein is a recombinant membrane-independent sCD59 and not membrane-targeted; and, an improvement in BCVA or ETDRS is observed after contact compared to BCVA or ETDRS observed before contact.

[0088] In some embodiments, this disclosure relates to methods for improving functional reading independence and vision-related quality of life in subjects with GA, methods comprising, consisting of, or substantially consisting of the following steps: administering a pharmaceutical composition to the GA-affected eye of the subject via ocular injection, wherein the pharmaceutical composition contains nucleic acid encoding the sCD59 protein. In some embodiments, improvements in functional reading independence and vision-related quality of life are determined by improvements in the Functional Reading Independence (FRI) index. In some embodiments, improvements in functional reading independence and vision-related quality of life are determined by improvements in the National Eye Institute Visual Function Questionnaire-25 (NEI-VFQ-25) composite score. According to some implementation schemes, methods for improving functional reading independence and vision-related quality of life in subjects with GA include exposing the GA-affected eye to a composition comprising a carrier carrying a nucleotide sequence encoding a recombinant engineered human sCD59 protein operatively linked to a promoter sequence, causing expression of the protein in cells, such that the protein includes at least one mutation resulting in loss of function of the GIP anchoring domain, such that the protein is a recombinant membrane-independent sCD59 and is not membrane-targeted; and that an improvement in the FRI index or NEI-VFQ-25 composite score is observed after exposure compared to the FRI index or NEI-VFQ-25 observed before exposure.

[0089] In some of the foregoing embodiments, the improvement is relative to subjects or groups of subjects receiving standard care excluding sCD59 who are at substantially the same level of disease progression. In some embodiments, the improvement is relative to subjects or groups of subjects receiving sham treatment excluding sCD59 who are at substantially the same level of disease progression. In some embodiments, the improvement is relative to subjects or groups of subjects who are not receiving treatment and are at substantially the same level of disease progression.

[0090] In some of the aforementioned embodiments, improvement is the change in the square root-transformed area of ​​GA lesions (sqrt-GAA) relative to baseline at months 3, 6, 9, 12, 15, and 18. In some of the aforementioned embodiments, improvement is relative to subjects or subject populations without GA.

[0091] In a further embodiment, the improvement is relative to a sham control injection. In some embodiments, the sham control injection is performed using a syringe without a needle or periocular / intraocular puncture.

[0092] In some implementations, the measurement of GA coverage of the affected eye is 2.5 mm. 2 Up to 17.5mm 2 The area of ​​the GA. In some embodiments, the GA is non-centrally depressed. In further embodiments, the GA is multifocal, with at least one lesion ≥1.25 mm. 2 The lesions are focal. In a further implementation, the GA lesion is measured as the square root of the GA lesion area by fundus autofluorescence.

[0093] In some embodiments, the subjects are 80 years of age or older. In some embodiments, the subjects are 70 years of age or older. In further embodiments, the subjects are 60 years of age or older. In some embodiments, the subjects are 50 years of age or older.

[0094] In a further embodiment, treatment or improvement is determined approximately 18 months after the administration of a pharmaceutical composition containing the sCD59 protein to the GA-affected eye of a subject.

[0095] According to some embodiments, the present invention provides a nucleotide sequence encoding the human sCD59 protein. According to some embodiments, the nucleotide sequence is a complementary DNA (cDNA) sequence.

[0096] The cDNA sequence encoding human CD59 is known in the art. For example, Sawada, R. et al. reported the cDNA sequence in NucleicAcids Res 17(16): 6728 in 1989, and these sequences were purchased from the American Type Culture Collection (ATCC, Manassas, Va.). The cDNA encoding CD59 has also been cloned from human T-cell leukemia (YT) and human erythroleukemia (K562) cell lines, and CD59 is transiently expressed in COS cells (Walsh, LA et al. 1990 Eur J. Immol21(3): 847-850).

[0097] In some embodiments, the sCD59 protein lacks a GPI anchoring domain. According to some embodiments, human sCD59 contains a modified GPI anchoring domain amino acid sequence that is functionally deficient and lacks the ability to target membranes. According to some embodiments, the modified GPI anchoring domain amino acid sequence contains variations. Such variations include, but are not limited to, substitutions and deletions of nucleic acids encoding amino acids at the ω position, used to reduce or eliminate the GPI anchor's linkage or to reduce or eliminate the GPI anchor's effective function. The ω amino acid is the amino acid for GPI transfer. For example, such variations include, but are not limited to, replacing nucleic acids encoding hydrophobic leucine (e.g., nucleic acid CAG) and alanine (e.g., nucleic acid GAA) with nucleic acids encoding glycine (e.g., nucleic acid CAG) and glutamic acid (e.g., nucleic acid GAA), which are less hydrophobic (i.e., more hydrophilic) amino acids. Alternatively, variations may include replacing the ω residue with another amino acid, such as replacing tyrosine with glycine.

[0098] According to some embodiments, the human sCD59 protein of the present invention includes conserved sequence modifications. Conserved sequence modifications are amino acid modifications that do not significantly affect or alter the characteristics of the human sCD59 protein containing an amino acid sequence, i.e., the amino acid sequence of sCD59 presenting these side chains at the same relative positions will function in a similar manner to human sCD59. Such conserved modifications include amino acid substitutions, additions, and deletions. Methods for modifying amino acid sequences are known in the art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described in: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989; and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989. According to some embodiments, the human sCD59 protein of the present invention includes conserved amino acid substitutions. A conserved amino acid substitution is a substitution in which an amino acid residue is replaced by 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0099] According to some embodiments, the human sCD59 amino acid sequence is substantially identical to the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 70% identical to the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 75% identical to the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 80% identical to the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 90% identical to the wild-type sequence. According to some embodiments, the human sCD59 amino acid sequence is at least 95% identical to the wild-type sequence.

[0100] In some embodiments, the nucleic acid encoding the sCD59 protein comprises or is substantially composed of SEQ ID NO: 1. In some embodiments, the nucleic acid encoding the sCD59 protein comprises or is substantially composed of at least 90% the same sequence as SEQ ID NO: 1. In some embodiments, the nucleic acid encoding the sCD59 protein comprises or is substantially composed of at least 95% the same sequence as SEQ ID NO: 1.

[0101] In some embodiments, the sCD59 protein comprises or is substantially composed of SEQ ID NO: 2. In some embodiments, the sCD59 protein comprises or is substantially composed of at least 90% the same sequence as SEQ ID NO: 2. In some embodiments, the sCD59 protein comprises or is substantially composed of at least 95% the same sequence as SEQ ID NO: 2.

[0102] According to some embodiments, the human sCD59 of the present invention is a recombinant protein.

[0103] In a further embodiment, the nucleic acid encoding the sCD59 protein is packaged into a delivery vector.

[0104] A variety of commercially available expression vector / host systems can be used to contain and express the CD59 protein-coding sequence. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant phage, plasmid, or copious DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems in contact with viral expression vectors (e.g., baculoviruses); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti, pBR322, or pET25b plasmids); or animal cell systems. See Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.

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

[0106] Cell types used for recombinant protein expression include, but are not limited to, prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacterial cells. Non-limiting examples of bacterial cells include *Escherichia coli*. Eukaryotic cells include, but are not limited to, mammalian, insect, yeast, and algal cells. Non-limiting examples of mammalian cells include human embryonic kidney cells (e.g., HEK293, HEK293T), juvenile hamster kidney cells (e.g., BHK21), Chinese hamster ovary (CHO) cells, mouse myeloma cells (e.g., NSO), and mouse non-hybridoma cells (e.g., SP2 / O-Agl4). Non-limiting examples of insect cells include fall armyworm (*Spodoptera frugiperda*) pupa ovary cells (e.g., Sf9, Sf21). Non-limiting examples of yeast cells include *Saccharomyces cerevisiae*. Non-limiting examples of algal cells include *Chlamydomonas reinhardtii*.

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

[0108] Various protein purification methods can 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 steps chosen will depend, for example, on the nature of the production method used and the specific protein being produced.

[0109] According to some embodiments, human sCD59 is a synthetic protein. Methods for preparing synthetic proteins are well known in the art. See, for example, Peptide Synthesis Protocols, Methods in Molecular Biology, Vol. 35, Pennington, MW and Dunn, BM, 1995, XII, Humana Press, Inc. Totowa, New Jersey. Synthetic proteins prepared using well-known techniques such as solid-phase, liquid-phase, or peptide condensation techniques, or any combination thereof, may include native and non-native amino acids. Amino acids used for peptide synthesis may be standard Boc amino acid resins with the standard deprotection, neutralization, coupling, and washing protocol of Merrifield's original solid-phase method (1963, J. Am. Chem. Soc. 85:2149-2154), or base-labile Na-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 are available from Sigma or other chemical companies familiar to those skilled in the art. These peptides can also be synthesized using other Na-protecting groups familiar to those skilled in the art.

[0110] According to some embodiments, the nucleotide sequence encoding human sCD59 is used to construct an expression vector. According to some embodiments, the present invention provides a human sCD59 expression construct. Methods for constructing expression vectors are well known to those skilled in the art. For example, such methods can be used to construct expression vectors containing a nucleotide sequence encoding a human sCD59 protein operatively linked to suitable 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. These techniques are described in: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989.

[0111] In a further embodiment, the nucleic acid encoding the sCD59 protein is operatively linked to a promoter sequence to express and secrete the sCD59 protein in 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-specific promoter. According to some embodiments, the promoter is a ubiquitous promoter. According to some embodiments, the promoter is a tissue-specific promoter. Examples of tissue-specific promoters include, but are not limited to, the human rhodopsin kinase (hRK) promoter and retinal pigment epithelium-specific promoters (e.g., the 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 heterozygous promoter. A non-limiting example of a heterozygous promoter is the cytomegalovirus (CMV) early enhancer element / first exon and first intron of the chicken β-actin gene / split acceptor (CAG) of the rabbit β-globulin gene. Non-limiting examples of promoters are shown in the following: U.S. Patent No. 6,677,311 B1, issued January 13, 2004, by Evans et al.; U.S. Patent No. 7,109,029 B2, issued September 19, 2006, by Clark et al.; and U.S. Patent No. 5,998,205, issued December 7, 1999, each of which is incorporated herein by reference in its entirety.

[0112] According to some implementation schemes, the delivery vector is a viral vector.

[0113] According to some embodiments, the vector is at least one of the following: an engineered viral vector recombinantly linked to a nucleotide sequence encoding the sCD59 protein; and a synthetic gene delivery vector for delivering the nucleotide sequence encoding human sCD59. According to some embodiments, the viral vector is selected from adenovirus, adeno-associated virus (AAV), herpesvirus, poxvirus, lentivirus, and helper-dependent adenovirus vectors. According to some embodiments, the synthetic gene delivery vector is selected from liposomes, lipid / polycationic (LPD), peptides, nanoparticles, gold particles, and polymers.

[0114] Adenoviral vectors are commercially available from the U.S. Type Culture Collection (Manassas, Va.). Methods for constructing and using adenoviral vectors are described in: Klein et al. 2007 Ophthalmology 114: 253-262; and van Lecuwen et al. 2003 Eur. J. Epidemiol. 18: 845-854. Adenoviral vectors have been used for eukaryotic gene expression (Levrero et al. 1991 Gene, 101: 195-202) and vaccine development (Graham et al. 1991 Methods in Molecular Biology: Gene Transfer and Expression Protocols 7, edited by Murray, Humana Press, Clifton, NJ, 109-128). Furthermore, recombinant adenoviral vectors are used in gene therapy (U.S. Patent No. 7,235,391, issued June 26, 2007 by Wu et al., the full text of which is incorporated herein by reference).

[0115] Recombinant adenovirus vectors are generated, for example, by homologous recombination between a shuttle vector and a proviral vector (U.S. Patent No. 7,235,391, issued June 26, 2007, by Wu et al.). The adenovirus vector used herein is replication-deficient. For example, the adenovirus vector is conditionally deficient, lacking the adenovirus E1 region. A polynucleotide encoding the target protein (e.g., human sCD59) is introduced at the location where the E1 coding sequence has been removed. Alternatively, a polynucleotide encoding the target protein (e.g., human sCD59) can be inserted into the E3 region of the adenovirus.

[0116] Helper cell lines can be used to generate and proliferate replication-deficient adenovirus vectors. Helper cell lines can be derived from human cells, such as 293 human embryonic kidney cells (HEK293), muscle cells, hematopoietic cells, or other embryonic mesenchymal or epithelial cells. Alternatively, helper cells can be derived from cells of other mammalian species that permit human adenoviruses, such as Vero cells or other monkey embryonic mesenchymal or epithelial cells. The use of helper cell lines to generate and proliferate these replication-deficient adenovirus vectors is described in: Graham et al. 1977 J. Gen. Virol. 36: 59-72.

[0117] Lentiviral encapsulation vectors are commercially available from Invitrogen Corporation (Carlsbad Calif). HIV-based encapsulation systems for producing lentiviral vectors were prepared using the constructs described in the following journals: 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. Many vector constructs can be encapsulated using systems based on third-generation lentiviral SIN vector backbones (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 by the Rous sarcoma virus (RSV) promoter sequence), a deleted self-inactivating 3' LTR in the U3 promoter region, an HIV encapsulation signal, and an RRE sequence linked to a marker gene cassette consisting of a jellyfish green fluorescent protein (GFP) driven by the CMV promoter and a groundhog hepatitis virus PRE element that appears to enhance extranuclear transfer. The GFP marker gene allows for the quantification 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).

[0118] Manipulating retroviral nucleic acids to construct retroviral vectors containing target genes (e.g., the gene encoding the human sCD59 protein) and encapsulating them in cells is accomplished using techniques known in the art (see, for example, Ausubel et al., 1992, Vol. 1, Chapter III (Units 9.10.1–9.14.3); Sambrook et al., 1989. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Miller et al., Biotechniques. 7:981–990, 1989; Eglitis et al., Biotechniques. 6:608–614, 1988; U.S. Patent Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767, and 5,124,263; and PCT Patent Publication Nos. 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 cited in full.

[0119] A bidirectional encapsulation system can be used to construct retroviral vectors and encapsulate them into non-infectious transduced viral particles (virions). Examples of such encapsulation systems are described in the following: Miller et al. 1986 Mol. Cell Biol. 6:2895-2902; Markowitz et al. 1988 J. Virol. 62: 1120-1124; Cosset et al. 1990 J. Virol. 64: 1070-1078; U.S. Patent Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767 and 5,124,263; and PCT Patent Publications WO 85 / 05629, WO 89 / 07150, WO 90 / 02797, WO 90 / 02806, WO 90 / 13641, WO92 / 05266, WO 92 / 07943, WO 92 / 14829 and WO All of these documents and publications, in their entirety, are incorporated herein by reference. The generation of “productive cells” can be accomplished by introducing a retroviral vector into encapsulated cells. Examples of such retroviral vectors are found, for example, in: Korman et al. 1987 Proc. Natl. Acad. Sci. USA, 84: 2150-2154; Morgenstern et al. 1990 Nucleic Acids Res. 18: 3587-3596; U.S. Patents 4,405,712, 4,980,289, and 5,112,767; and PCT Patent Publications WO 85 / 05629, WO 90 / 02797, and WO92 / 07943.

[0120] Herpesvirus encapsulation vectors are commercially available from Invitrogen Corporation (Carlsbad, Calif). Exemplary herpesviruses include, but are not limited to, alpha-herpesviruses such as varicella-zoster virus or pseudorabies virus; herpes simplex viruses such as HSV-1 or HSV-2; or herpesviruses such as EB virus. Fraefel et al. describe a method for preparing empty herpesvirus particles that can be encapsulated with desired nucleotide fragments (e.g., human sCD59 nucleotides or polynucleotide sequences) in the absence of helper viruses that can be used for most herpesviruses (U.S. Patent No. 5,998,208, issued December 7, 1999, the entire contents of which are incorporated herein by reference).

[0121] Herpesvirus DNA vectors can be constructed using techniques known to those skilled in the art. For example, DNA segments encoding the complete genome of a herpesvirus are divided into many vectors capable of carrying large DNA segments, such as granules (Evans et al., Gene 79, 9-20, 1989), yeast artificial chromosomes (YACS) (Sambrook, J. et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989), or E. coli F-element plasmids (O'Conner et al., 1989 Science 244: 1307-1313). For example, granule groups containing overlapping clones have been isolated, representing the complete genomes of various herpesviruses, including Epstein-Barr virus (EBV), varicella-zoster virus (VZV), pseudorabies virus (PRV), and HSV-1. See M. van Ziji et al. 1988 J. Virol. 62:2191; Cohen et al. 1993 Proc. Nat'l Acad. Sci. USA 90: 7376; Tomkinson et al. 1993 J. Virol. 67: 7298; and Cunningham et al. 1993 Virology 197: 116. Adeno-associated virus (AAV) is a parvovirus-dependent virus because it relies on co-infection with another virus (adenovirus or a member of the herpesvirus family) to undergo productive infection in cultured cells (Muzyczka 1992 Curr. Top. Microbiol. Immunol., 158:97 129). For example, recombinant AAV (rAAV) virus can be prepared by co-transfecting a plasmid containing a target gene flanked by two AAV terminal repeats (e.g., the human sCD59 gene) and an expression plasmid containing a wild-type AAV coding sequence without terminal repeats. Cells can also be contacted with or transfected with adenovirus or plasmids carrying adenovirus genes required for AAV helper function.

[0122] Unlike most viruses, AAVs are inherently nonpathogenic, weakly immunogenic, and broadly tropistic, making them attractive gene delivery candidates for virus-based gene therapy. Most naturally occurring AAVs utilize glycan moieties to initially attach to the cell surface, and these interactions have been well characterized for many serotypes. Identified interacting glycan moieties include AAV serotype 2 (AAV2), AAV3, AAV6, and AAV8; the N-terminal galactose of AAV9; and the specific N- or O-linked sialic acid moieties of AAV1, -4, -5, and -6. The serotypes differ depending on the cell types they infect, making AAVs a very useful system for preferentially transducing specific cell types.

[0123] Adeno-associated virus (AAV) encapsulation vectors are commercially available from GeneDetect (Auckland, New Zealand). AAV has a broad range of infectious hosts (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).

[0124] Methods for constructing and using AAV vectors are known in the art. Such methods are described, for example, in U.S. Patent No. 5,139,941 (Wu et al.), published June 26, 2007, and U.S. Patent No. 4,797,368 (Carter et al.), published January 10, 1989. The use of AAV in gene delivery is further described in the following: 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.

[0125] Recombinant AAV vectors have been successfully used for in vitro and in vivo transduction of marker genes (Kaplitt et al. 1994 Nat Genet., 8(2): 148-154; Lebkowski et al. 1988 Mol. Cell. Biol. 8(10): 3988-3996; Samulski et al. 1991 EMBO J. 10: 3941-3950; Shelling and Smith 1994 Gene Therapy, 1:165-169; Yoder et al. 1994 Blood, 82 (Supp.): 1: 347A; Zhou et al. 1993 Exp. Hematol 21: 928-933; Tratschin et al. 1985 Mol. Cell. Biol. 5: 3258-3260; McLaughlin et al. 1988 J. Virol. 62(6): (1963-1973) and the 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).

[0126] According to some embodiments, a nucleotide sequence encoding human sCD59, operatively linked to a promoter, is encapsulated 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.

[0127] According to some embodiments, the nucleotide sequence encoding human sCD59 is encapsulated between inverted terminal repeat (ITR) sequences within an AAV vector. According to some embodiments, the nucleotide sequence encoding human sCD59, operatively linked to a promoter, is encapsulated between inverted terminal repeat (ITR) sequences within an AAV vector. According to some embodiments, the human sCD59 expression vector is encapsulated between inverted terminal repeat (ITR) sequences within an AAV vector. According to some embodiments, the ITR sequence is an AAV2 sequence. According to some embodiments, the ITR sequence is an AAV5 sequence. According to some embodiments, the ITR sequence is an AAV8 sequence.

[0128] According to some embodiments, the AAV vector is a hybrid vector. The hybrid vector contains an ITR sequence from one AAV serotype and capsid proteins from different AAV serotypes. According to some embodiments, the hybrid vector contains an ITR sequence from AAV2 and a capsid protein from AAV5 (AAV2 / 5). According to some embodiments, the hybrid vector contains an ITR sequence from AAV2 and a capsid protein from AAV8 (AAV2 / 8). According to some embodiments, the hybrid vector contains an ITR sequence from AAV5 and a capsid protein from AAV2 (AAV5 / 2). According to some embodiments, the hybrid vector contains an ITR sequence from AAV5 and a capsid protein from AAV8 (AAV5 / 8). According to some embodiments, the hybrid vector contains an ITR sequence from AAV8 and a capsid protein from AAV2 (AAV8 / 2). According to some embodiments, the hybrid vector contains an ITR sequence from AAV8 and a capsid protein from AAV5 (AAV8 / 5).

[0129] According to some embodiments, the delivery vector is a non-viral vector. For example, the delivery vector is a synthetic gene delivery solvent or carrier that is independent of viral particles and specifically delivers genetic material to target cells or tissues. 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 formulations. Therefore, according to some embodiments, the present invention provides a non-viral vector containing nucleic acids, which is loaded and contacted with tissues or cells. For example, liposomes containing naked DNA or a gene encoding a human sCD59 protein with a modified GPI anchor having a non-target membrane, and the gene encoding a human sCD59 protein without a GPI anchor, are encapsulated in liposomes, thereby enabling efficient delivery of nucleic acids to the tissues or cells.

[0130] According to some implementation schemes, using AAV vectors to deliver human sCD59 to RA-affected eyes is effective for long-term transgene expression.

[0131] According to some embodiments, the pharmaceutical composition is administered via a route selected from the group consisting of intravitreal, subretinal, subconjunctival, subfascial, subcutaneous, and intravenous routes. According to some embodiments, the pharmaceutical composition is administered by injection. According to some embodiments, the tissue in contact with 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 vessels.

[0132] In some embodiments, the pharmaceutical composition is injected into only one eye. In some embodiments, the pharmaceutical composition is injected into both eyes. In some embodiments, the pharmaceutical composition is administered to the GA-affected eye via a single intravitreal injection.

[0133] According to some embodiments, the administration step includes administering the pharmaceutical composition in a single dose or multiple doses. According to some embodiments, the administration step includes administering the pharmaceutical composition in a single dose. According to some embodiments, a single dose is administered to the eye of a subject in need. According to some embodiments, the subject in need suffers from AMD. According to some embodiments, the subject in need suffers from wet or exudative AMD. According to some embodiments, the subject in need suffers from GA.

[0134] Unbound by theory, it is believed that contact between cells and a carrier containing nucleic acid encoding human sCD59 produces a subset of cells that act as “factories” for the local production and secretion of sCD59, protecting nearby eye cells, including retinal pigment epithelial (RPE) cells and choroidal vessels.

[0135] The precise dosage of the pharmaceutical composition can be selected by an individual physician considering the patient to be treated. The dosage and administration are adjusted to provide an adequate level of active agent or maintain the desired effect. Other factors to consider include the severity of the disease state, such as intermediate or advanced AMD; the patient's age, weight, and sex; diet, timing and frequency of administration; route of administration; drug combination; responsiveness; and tolerance / response to therapy. Depending on the half-life and clearance rate of the specific composition, long-acting pharmaceutical compositions can be administered once per hour, twice per hour, every 3 to 4 hours, once daily, twice daily, every 3 to 4 days, once weekly, or every two weeks.

[0136] The active agents of the present invention can be formulated in dosage units that facilitate administration and ensure uniform dosage. However, it should be understood that the total daily dosage of the compositions of the present invention will be determined by the attending physician within a reasonable medical judgment. For any active agent, the therapeutically effective dose can initially be estimated in cell culture assays or animal models, typically mice, but also rats, rabbits, dogs, or pigs, as provided herein. This information can then be used to determine the dosage and route of administration that is useful to humans. Pharmaceutical compositions exhibiting a large therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used to formulate dosage ranges for human use.

[0137] Therapeutic effective dose refers to the amount of an active agent that improves symptoms or disease or prevents the progression of GA. The therapeutic efficacy and toxicity of an active agent can be determined in cell cultures or laboratory animals using standard pharmaceutical procedures, such as ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal in 50% of the population). The dose ratio of toxicity to therapeutic effect is the therapeutic index, and it can be expressed as the LD50 / ED50 ratio.

[0138] According to some embodiments, the expression source of human sCD59 protein is administered in the form of a viral vector or nucleic acid vector. In a further embodiment, the pharmaceutical composition comprises approximately 8.2 × 10⁻⁶ ppm. 10 A dose of viral particles representing one vector genome (vg). In a further embodiment, the pharmaceutical composition comprises approximately 4.1 × 10^6 viral particles. 11 A dose of viral particles per vg. In some embodiments, the dose of viral particles may alternatively be expressed as anti-DNase particles (DRP).

[0139] In some embodiments, the pharmaceutical composition is administered together with prednisone or prednisolone. In further embodiments, prednisone or prednisolone is administered orally. In even further embodiments, prednisone or prednisolone is administered orally at a dose of about 40 mg / day for 20 days. In even further embodiments, the dose of prednisone or prednisolone is decreased by about 10 mg every 5 days. For example, on day 1, prednisone or prednisolone is administered orally at a dose of 40 mg / day; on day 5, prednisone or prednisolone is administered orally at a dose of 30 mg / day; on day 10, prednisone or prednisolone is administered orally at a dose of 20 mg / day; and on day 15, prednisone or prednisolone is administered orally at a dose of 15 mg / day. In some embodiments, prednisone or prednisolone is initiated on the same day as administration of the pharmaceutical composition containing the nucleotide sequence encoding human sCD59. In some embodiments, prednisone or prednisolone is taken in the morning with or after a meal.

[0140] In some embodiments, the pharmaceutical composition comprising the nucleotide sequence encoding human sCD59 is administered together with a corticosteroid. In a further embodiment, the corticosteroid is administered via a single periorbital injection. In a still further embodiment, the corticosteroid is triamcinolone acetonide (TA). In some embodiments, TA is administered at a dose of about 40 mg. In some embodiments, prednisone or prednisolone is initiated four days after administration of the pharmaceutical composition comprising the nucleotide sequence encoding human sCD59.

[0141] In some embodiments, a pharmaceutical composition comprising a nucleotide sequence encoding human sCD59 is administered in conjunction with VEGF treatment. In some embodiments, VEGF treatment is initiated if the subject is suspected of having or has a new exudative MNV (also known as active CNV or wet AMD). In some embodiments, VEGF treatment is ranibizumab or aflibercept.

[0142] In some implementations, the pharmaceutical composition containing the nucleotide sequence encoding human sCD59 is not administered with complement inhibitors.

[0143] Pharmaceutical Composition According to some embodiments, the present invention provides a pharmaceutical composition. According to some embodiments, the pharmaceutical composition comprises a human sCD59 protein comprising the full-length nucleic acid of CD59, which is modified to remove the signal sequence for linking a GPI anchor at the nucleotide encoding the amino acid asparagine at position 77. According to some embodiments, the nucleic acid sequence of the human sCD59 protein is modified by point mutation, substitution, or deletion to obtain a nucleic acid sequence encoding an amino acid sequence having a modified amino acid sequence at the GPI anchor position, such that the protein cannot bind to the cell membrane.

[0144] According to some embodiments, the pharmaceutical composition is formulated as sterile for ocular delivery. According to some embodiments, the pharmaceutical composition formulated for sterile ocular delivery is a dose effective for treating GA.

[0145] According to some embodiments, a pharmaceutical composition formulated for ocular delivery further comprises at least one of a pharmaceutically acceptable buffer, a pharmaceutically acceptable salt, and a pharmaceutically acceptable emollient suitable for delivery via at least one of the following routes: intraocular injection, subconjunctival injection, subfascial bursa injection, eye drops, and ointment.

[0146] According to some embodiments, the present invention provides a pharmaceutical composition comprising a nucleic acid encoding CD59 or a human sCD59 protein expression source. In various embodiments, the CD59 protein comprises a membrane-independent (i.e., soluble) CD59 protein. According to some embodiments, the pharmaceutical composition is formulated into an ophthalmic preparation for administration to the eye. According to some embodiments, the pharmaceutical composition is formulated to enhance delivery to the fundus. According to some embodiments, the pharmaceutical composition is formulated to provide sustained release locally to the retina. According to some embodiments, the pharmaceutical composition is formulated to provide effective treatment for blood vessels and / or tissues involved in an eye disease. According to some embodiments, the eye disease is age-related macular degeneration (AMD). According to some embodiments, AMD is wet or exudative AMD. According to some embodiments, AMD is dry AMD or GA.

[0147] According to some embodiments, the pharmaceutical composition of the present invention is formulated to be sufficiently pure for administration to a human subject, for example, for administration to the eye of a human subject. According to some embodiments, the pharmaceutical composition comprises one or more additional therapeutic agents. According to some implementation schemes, one or more additional therapeutic agents are selected from growth factors, anti-inflammatory agents, vasopressors, including but not limited to nitric oxide and calcium channel blockers, collagenase inhibitors, steroids (e.g., prednisolone), matrix metalloproteinase inhibitors, ascorbic acid, angiotensin H, angiotensin III, calreticulin, tetracycline, fibronectin, collagen, platelet-reactive protein, transforming growth factor (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), IGF-binding protein (IGFBP), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), neutral differentiation factor (NDF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), heparin-bound EGF (HBEGF), platelet-reactive protein, von Willebrand factor-C, heparin and heparin sulfate, and hyaluronic acid. According to some embodiments, one or more additional therapeutic agents include, but are not limited to, antitumor agents, antiviral agents, antibacterial agents, antimycobacterial agents, antifungal agents, antiproliferative agents, or antiapoptotic agents. Therapeutic agents included in the pharmaceutical compositions of the present invention are well known in the art. See, for example, *The Pharmacological Basis of Therapeutics*, 9th edition, edited by Goodman and Gilman, McGraw-Hill, 1996, the contents of which are incorporated herein by reference.

[0148] According to some embodiments, the additional one or more therapeutic agents are compounds, compositions, biological agents, etc. According to some embodiments, the additional one or more therapeutic agents enhance, stabilize, synergize with, or replace the ability of human sCD59 protein to protect cells from MAC deposition. According to some embodiments, the additional one or more therapeutic agents are provided simultaneously with a pharmaceutical composition containing human sCD59 protein. According to some embodiments, the additional one or more therapeutic agents are provided after the pharmaceutical composition containing human sCD59 protein. According to some embodiments, the additional one or more therapeutic agents are provided before the pharmaceutical composition containing human sCD59 protein. According to some embodiments, the additional one or more therapeutic agents are used to treat the same, concurrent, or related symptoms, conditions, or diseases.

[0149] According to some embodiments, the pharmaceutical compositions of the present invention comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, any and all solvents, diluents or other liquid solvents, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., provided they are suitable for the desired specific dosage form. Remington's Pharmaceutical Sciences, edited by Gennaro (Mack Publishing, Easton, Pa., 1995), provides information on various carriers for formulating pharmaceutical compositions and known techniques for their preparation. Examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as glucose and sucrose; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; and phosphate buffer solutions, as well as other non-toxic, compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and colorants, release agents, coating agents, preservatives, and antioxidants may also be present in the composition at the formulator's discretion.

[0150] According to some embodiments, the pharmaceutical composition further comprises a peptide (POD) for whole-body delivery, the pharmaceutical composition being operatively linked to a compound to obtain a conjugated compound such that the POD comprises a protein transduction domain (PTD). For example, POD compositions are shown in: PCT7US2008 / 010179, filed August 28, 2008, by Kumar-Singh et al.; or U.S. Patent Publication 2010 / 0209447, published August 19, 2010, by Kumar-Singh et al., each of which is incorporated herein by reference in its entirety.

[0151] According to some embodiments, the pharmaceutical composition comprises a dose of viral vector particles administered to the affected eye. In a further embodiment, the pharmaceutical composition comprises approximately 8.2 × 10⁻⁶ particles. 10 A dose of viral particles representing one vector genome (vg). In a further embodiment, the pharmaceutical composition comprises approximately 4.1 × 10^6 viral particles. 11 A dose of viral particles per vg.

[0152] According to some embodiments, the pharmaceutical composition further comprises at least one therapeutic agent selected from the group consisting of: anti-inflammatory agents, antitumor agents, antiviral agents, antibacterial agents, antimycobacterial agents, antifungal agents, antiproliferative agents, and antiapoptotic agents.

[0153] According to some embodiments, the present 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 of an amino acid encoding a GPI anchoring domain of a protein, such that the resulting vector encodes an engineered recombinant membrane-independent (i.e., soluble) CD59 (sCD59) protein, and the vector being a viral vector or a synthetic gene delivery vector; and contacting at least one ocular tissue of the subject with the composition, such that the cells of the tissue locally express and secrete CD59, thereby treating the subject's GA.

[0154] According to some embodiments, the human sCD59 protein expression source is administered via ocular injection. Injectable formulations can be prepared using suitable dispersants or wetting agents and suspending agents according to known techniques, such as sterile injectable aqueous or oily suspensions. According to some embodiments, the injectable formulation is a sterile injectable formulation. A sterile injectable formulation can be a sterile injectable solution, suspension, or emulsion in a non-toxic, non-enteric-acceptable diluent or solvent, such as a solution of 1,3-butanediol. Acceptable solvents and media that can be used are water, Ringer's solution (United States Pharmacopeia), and isotonic sodium chloride solution. Furthermore, sterile, non-volatile oils are typically used as solvents or suspension media. Any mild, non-volatile oil can be used, including but not limited to synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid are used in the preparation of injectable formulations. Injectable formulations can be sterilized, for example, by filtration through a bacterial retention filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0155] According to some embodiments, sterile injectable formulations include excipients. Such excipients include, but are not limited to, suspending agents (e.g., sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and gum arabic), dispersants, or wetting agents including naturally occurring phospholipids (e.g., lecithin), or condensation products of ethylene oxide and fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide and long-chain fatty alcohols (e.g., heptadecetylacetoxycetyl alcohol), or condensation products of ethylene oxide and esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitan monooleate), or condensation products of ethylene oxide and esters derived from fatty acids and hexitols (e.g., polyvinyl sorbitan monooleate).

[0156] Sterile injectable formulations can be sterile injectable solutions or suspensions in non-toxic, non-enteric-acceptable diluents or solvents, such as solutions of 1,3-butanediol. A solution is generally considered a homogeneous mixture of two or more substances; it is usually a liquid, although not essential. In a solution, the molecules of the solute (or dissolved substance) are uniformly distributed among the molecules of the solvent. A suspension is a dispersion (mixture) in which a finely dispersed class of substances is combined with another class of substances, wherein the former is so finely dispersed and mixed that it does not rapidly precipitate. In everyday life, the most common suspension is a suspension of solids in liquid water. Acceptable solvents and media that can be used are water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, non-volatile oils are often used as solvents or suspension media. For parenteral applications, particularly suitable solvents consist of solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants. Suitable lipophilic solvents or media include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or reagents that increase the solubility of the compound to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be present in powder form for mixing with a suitable solvent (e.g., sterile, pyrogen-free water) prior to use.

[0157] According to some embodiments, the present 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 active agents, the liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol, and mixtures thereof. In addition to inert diluents, ocularly delivered pharmaceutical compositions may also include adjuvants, such as wetting agents, as well as emulsifiers and suspending agents.

[0158] According to some embodiments, the pharmaceutical composition of the present invention may be in the form of a sterile injectable aqueous or oily suspension. Injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques.

[0159] According to some embodiments, the present invention includes ophthalmic devices, surgical devices, auditory devices, or products containing the disclosed compositions (e.g., gauze bandages or strips), and methods of manufacturing or using such devices or products. These devices may be coated, impregnated, combined, or otherwise treated with the pharmaceutical compositions described herein.

[0160] According to some embodiments, the composition is administered in a pharmaceutically acceptable solution, which may conventionally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optional other therapeutic agents.

[0161] According to some embodiments, the pharmaceutical composition is an aqueous suspension or emulsion mixed with excipients suitable for preparing aqueous suspensions and emulsions. Such excipients include, but are not limited to, suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and gum arabic; dispersants or wetting agents may be naturally occurring phospholipids (such as lecithin), or condensation products of alkyl esters with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecetylidene cetyl alcohol), or condensation products of ethylene oxide with esters derived from fatty acids and hexitols (such as polyoxyethylene sorbitan monooleate), or condensation products of ethylene oxide with esters derived from fatty acids and hexitol anhydrides (e.g., polyvinyl sorbitan monooleate).

[0162] Solutions or suspensions intended for parenteral, intradermal, subcutaneous, intrathecal, or topical application may include, but are not limited to, sterile diluents such as water for injection, saline solutions, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and tonic agents such as sodium chloride or glucose. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Specific carriers for intravenous administration are physiological saline or phosphate-buffered saline (PBS).

[0163] Injectable formulations can be sterilized, for example, by filtration through a bacterial retention filter or by incorporating a sterilizing agent into a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media just before use. Injectable formulations can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques, such as sterile injectable aqueous or oily suspensions. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic, non-enteric-acceptable diluents or solvents, such as solutions as 1,3-butanediol. Acceptable solvents and media that can be used are water, Ringer's solution (United States Pharmacopeia), and isotonic sodium chloride solution. Furthermore, sterile, non-volatile oils are typically used as solvents or suspension media. For this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid are used in the preparation of injectable formulations.

[0164] Formulations intended for parenteral administration include aqueous and non-aqueous sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners. Formulations may be available in single-dose or multi-dose containers (e.g., sealed ampoules and vials) and may be stored under lyophilized (freeze-dried) conditions, requiring the addition of a sterile liquid carrier (e.g., saline, water for injection) only shortly before use. Temporary injectable solutions and suspensions may be prepared from the aforementioned types of sterile powders, granules, and tablets.

[0165] In addition to active compounds, suspensions may also contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitol esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar, tragacanth gum and mixtures thereof.

[0166] The pharmaceutical compositions of the present invention may further comprise conventional excipients, namely pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral administration that do not react adversely with the active compound. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, vegetable oils, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silica, viscous paraffin, fragrance oils; monoglycerides and diglycerides of fatty acids, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0167] The pharmaceutical compositions of the present invention are sterilizable and, if desired, can be mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavoring agents, and / or aromatic substances, which do not react adversely with the active compound. For parenteral application, suitable solvents include solutions, such as oily or aqueous solutions, as well as suspensions, emulsions, or implants. Aqueous suspensions may contain substances that increase the viscosity of the suspension and include, for example, but not limited to, sodium carboxymethyl cellulose, sorbitol, and / or dextran. Optionally, the suspension may also contain stabilizers. These compositions may also contain adjuvants, including preservatives, wetting agents, emulsifiers, and dispersants. Antimicrobial activity can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenolic sorbic acid, etc. Isotonic agents, such as sugars, sodium chloride, etc., may also be desired. Prolonged absorption of injectable drug forms can be achieved by using agents with delayed absorption, such as aluminum monostearate and gelatin.

[0168] According to some embodiments, the pharmaceutical composition of the present invention comprises a therapeutically effective amount of human sCD59 and other therapeutic agents optionally contained in a pharmaceutically acceptable carrier. According to some embodiments, the components of the pharmaceutical composition can also be mixed in a manner that does not involve interactions that would significantly impair the desired pharmaceutical efficacy.

[0169] According to some embodiments, the pharmaceutical compositions of the present invention comprise pharmaceutically acceptable salts. Pharmaceutically acceptable salts are those suitable for use in contact with human and lower animal tissues within a reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, etc., and with a reasonable efficacy / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail by PH Stahl et al. in “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” (Wiley VCH, Zurich, Switzerland: 2002).

[0170] sequence describe sequence SEQ ID NO Membrane-independent CD59, nucleic acid ATGGGAATCCAAGGAGGGTCTGTCCTGTTCGGGCTGCTGCTCGTCCTGGCTGTCTTCTGCCATTCAGGTCATAGCCTGCAGTGCTACAACTGTCCTAACCCAACTGCTGACTGCAAAACAGCCGTCAATTGTTCATCTGATTTTGATGCGTGTC TCATTACCAAAGCTGGGTTACAAGTGTATAACAAGTGTTGGAAGTTTGAGCATTGCAATTTCAACGACGTCACAACCCGCTTGAGGGAAAATGAGCTAACGTACTACTGCTGCAAGAAGGACCTGTGTAACTTTAACGAACAGCTTGAAAATTAA SEQ ID NO: 1 Membrane-independent CD59, amino acids MGIQGGSVLFGLLLVLAVFCHSGHSLQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNKCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLEN SEQ ID NO: 2 Example The embodiments and implementations described herein are for illustrative purposes only and are intended to suggest to those skilled in the art various modifications or variations to be considered, which are included within the spirit and scope of this application and within the scope of the appended claims.

[0171] Example 1: Evaluation of intravitreal JNJ-81201887 (AAVCAGsCD59) compared to sham surgery in a phase 2b clinical trial. It is being tested for the treatment of geographic atrophy (GA) secondary to age-related macular degeneration (AMD). This article provides an ongoing, non-limiting example of a phase 2b, randomized, double-blind, multicenter, dose-range, sham-controlled clinical trial to evaluate intravitreal JNJ-81201887 (AAVCAGsCD59) versus sham surgery for the treatment of geographic atrophy (GA) secondary to age-related macular degeneration (AMD).

[0172] Description of the compound The investigational gene therapy JNJ-81201887 (AAVCAGsCD59) is a recombinant adeno-associated virus serotype 2 (rAAV2) with a transgene encoding soluble CD59 (sCD59).

[0173] therapeutic goals and endpoints Target end main • Changes in GA lesion growth in eyes treated with JNJ-81201887 compared to a sham control. • Studies of changes in the square root area of ​​GA lesions in the eye using fundus autofluorescence (FAF) measurements secondary • Effects of JNJ-81201887 on Low-Light Visual Acuity (LLVA) • Changes in LLVA (Early Treatment Diabetic Retinopathy Study (ETDRS) Alphabetical Score) • Effects of JNJ-81201887 on visual function and retinal function • Changes in reading speed (Radner visual acuity chart) • Changes in retinal sensitivity via intermediate visual microfield testing (macular integrity assessment (MAIA)) • Effects of JNJ-81201887 on visual acuity (best corrected visual acuity (BCVA)) • Changes in BCVA (ETDRS letter rating) • The impact of JNJ-81201887 on functional reading independence and vision-related quality of life (VRQoL) (patient-reported outcomes (PRO)) • Changes in PRO: ○ Functional Reading Independence (FRI) Index ○ National Eye Institute Visual Function Questionnaire-25 (NEI-VFQ-25) Composite Score Overall Design This is a phase 2b, randomized, double-blind, multicenter, dose-range, sham-controlled study in participants aged 60 years or older with GA secondary to AMD to evaluate the efficacy and safety of a single intravitreal injection of the gene therapy JNJ-81201887 (AAV2CAGsCD59) in the study eye after 18 months. The JNJ-81201887 gene therapy will be administered to a single eye in each participant.

[0174] Participants meeting all inclusion criteria will be randomly assigned in a 1:1:1 ratio (stratified by lesion continuity [multifocal vs. non-multifocal] and baseline GA area [2.5 to 10 mm² or >10 to 17.5 mm²]) to one of the two dose levels of JNJ-81201887 (low dose: 8.2 × 10⁻⁶). 10 One dose per vg; High dose: 4.1 × 10 11 (vg dose) or sham surgery group. vg = vector genome.

[0175] Participants will receive a 20-day course of oral prednisone starting on day 1, followed by a single periocular corticosteroid injection on day 4 to prevent intraocular inflammation (except for those in the sham surgery group who will receive a matched placebo and a sham periocular injection). 40 mg triamcinolone (TA) is recommended as the periocular corticosteroid injection.

[0176] Study participants will undergo a 28-day screening period during which all baseline procedures / tests will be performed on or before day 1 / randomization (and the steroid / placebo regimen will begin after randomization), followed by dosing on day 4. Participants will be followed up for 18 months. The study duration is approximately 19 months.

[0177] Study population Number of participants The goal of this study is to enroll 300 participants.

[0178] Selection criteria Each potential participant must meet all of the following research recruitment criteria: age 1. 60 years old or older.

[0179] Participant types and disease characteristics Study the eye 2. A non-central fovea (i.e., not involving the center point of the fovea) secondary to AMD, with an area measured at 2.5 mm. 2 Up to 17.5mm 2 (1 and 7 optic disc areas, respectively), determined by screening images from FAF and SD-OCT.

[0180] 3. If the GA is multifocal, then at least one lesion must be ≥1.25mm. 2 (0.5 times the area of ​​the viewing plate).

[0181] 4. GA can capture a complete image using FAF with a 30-degree field of view centered on the fovea.

[0182] 5. GA can be measured separately from any area of ​​peri-nipple atrophy, as assessed by CRC.

[0183] 6. Any mode of superautofluorescence (superAF) is present in the GA boundary region (lack of superAF will be excluded).

[0184] 7. Sufficiently clear ocular media, adequate fixation, and pupillary dilation are required to allow for sufficiently high-quality fundus imaging.

[0185] 8. Using the ETDRS visual acuity chart, the best corrected distance visual acuity (BCVA) is 35 letters or better.

[0186] a. If both eyes meet the criteria, the eye with poorer vision due to GA will be used as the study eye.

[0187] b. If both eyes meet the criteria and have the same BCVA, the right eye will be designated as the study eye.

[0188] opposite eye 9. The other eye must have a counting finger or better BCVA.

[0189] system 10. Based on physical examination (including assessment of vital signs, medical history, and clinical laboratory tests performed at screening), the participant is medically stable. If these results are outside the normal reference range, the investigator may decide at their discretion whether to include the participant in the study (i.e., if the investigator considers the abnormality or deviation from the normal value to be clinically significant or appropriate and reasonable for the study population), except in the following cases where the participant must be excluded: alanine aminotransferase, aspartate aminotransferase, or gamma-glutamyl transferase >2 times the ULN (upper limit of normal), hemoglobin <9 g / dL, white blood cell count <2,500 / mm³. 3 Platelet count <80,000 / mm 3 Or hematocrit >54% (male) or >49% (female).

[0190] Gender and contraception / barrier requirements 11. Male or female (based on their reproductive organs and functions as specified by the entire set of chromosomes).

[0191] 12. Women of childbearing potential must have a negative result for high-sensitivity serum (β-human chorionic gonadotropin) in the sample collected at the time of screening, and a negative result for urine pregnancy test on day 4 prior to the study intervention.

[0192] 13. Women must be (a) infertile or (b) fertile and are using a highly effective method of contraception (with a failure rate of <1% per year if used correctly and consistently) and agree to continue using a highly effective method of contraception throughout the study period.

[0193] 14. Women must agree not to donate eggs (ovules, oocytes) or freeze them for future assisted reproductive purposes for 18 months from the date of administration.

[0194] 15. Male participants must use condoms during any activity that could result in semen flowing into a fertile woman for 18 months from the date of administration. Male participants should also be advised of the benefits of using highly effective contraception for their fertile female partners, as condoms may break or leak.

[0195] 16. Male participants must agree not to impregnate their female partners or donate sperm for reproductive purposes for 18 months from the date of administration.

[0196] Informed Consent Form 17. Must sign (or have a legally acceptable representative sign) an ICF indicating that the participant understands the purpose and required procedures of the study, and is willing to participate in the study and be able to complete all necessary assessments during the study.

[0197] 18. If a participant agrees to provide an optional DNA sample for the study (where permitted by local regulations), a consent form must be provided (or signed by its legally acceptable representative). Refusal to consent to an optional DNA sample for research does not preclude a participant from participating in the study.

[0198] 19. Willing and able to comply with prescribed lifestyle restrictions.

[0199] Exclusion criteria Any potential participant who meets any of the following criteria will be excluded from participation in the study: Medical conditions Study the eye 1. History or presence of retinal diseases other than GA: diabetic retinopathy, central serous chorioretinopathy, hereditary retinal degeneration, toxic macular degeneration (i.e., hydroxychloroquine macular degeneration), arterial and venous occlusive diseases, presence or previous repair of macular holes or choroidal melanoma.

[0200] 2. Presence of macular fibrosis or retinal epithelial tears, clinically relevant myopic degeneration, or vitreous hemorrhage. Benign vitreous lesions (i.e., posterior vitreous detachment) or benign peripheral retinal lesions (i.e., cobblestone degeneration, lattice degeneration, etc.) are permissible.

[0201] 3. Investigate the history of previous transpulsive thermotherapy, photodynamic therapy, or external beam radiation therapy to the ocular region.

[0202] 4. Previous treatment with any thermal laser in the macular region, regardless of the indication.

[0203] 5. History of retinal detachment (with or without repair).

[0204] 6. History of vitrectomy, corneal transplantation, trabeculectomy, or drainage tube implantation.

[0205] 7. Uncontrolled glaucoma (defined as IOP>22 despite treatment with more than 2 anti-glaucoma drugs) or advanced glaucoma (cup-to-disc ratio>0.8).

[0206] 8. Known history of steroid-induced ocular hypertension / glaucoma.

[0207] 9. Within 3 months of the study intervention, the patient had undergone any intraocular or periocular surgery or intraocular laser surgery (i.e., selective laser trabeculoplasty (SLT), yttrium aluminum garnet (YAG)).

[0208] 10. Any concurrent eye disease that researchers believe requires surgery within 18 months or may affect the interpretation of test results during the study.

[0209] 11. Aphakia or posterior capsule absence, unless caused by yttrium aluminum garnet (YAG) laser posterior capsulotomy following a previous posterior chamber intraocular lens implantation.

[0210] Any eye 12. History of uveitis and / or vitreitis or inflammatory eye disease. Mild postoperative inflammation lasting no more than 2 months after a previous interventional ocular surgery is permissible.

[0211] 13. Active infectious conjunctivitis, keratitis, scleritis or endophthalmitis.

[0212] 14. Any signs of diabetic retinopathy or central serous chorioretinopathy.

[0213] 15. A history of exudative MNV in either eye (also known as active CNV or wet AMD).

[0214] system 16. Within 6 months prior to the intervention, the patient had experienced acute coronary syndrome, myocardial infarction or coronary revascularization, cerebrovascular accident, or transient ischemic attack.

[0215] 17. Uncontrolled hypertension is defined as systolic blood pressure ≥170 mmHg or diastolic blood pressure ≥100 mmHg.

[0216] 18. A predisposition or history of increased risk of infection (e.g., history of splenectomy or chronic immunosuppression).

[0217] 19. A known history of complement deficiency, or current / previous treatment with complement inhibitors.

[0218] 20. Having an active malignant tumor within the past 12 months, except for cervical carcinoma in situ that has been appropriately treated, non-melanoma skin cancer, and prostate cancer with a Gleason score ≤6 and a stable PSA (prostate-specific antigen) for >12 months.

[0219] 21. History of allergy to fluorescein that cannot be relieved by treatment.

[0220] 22. Pregnant, breastfeeding, or planning to become pregnant within 18 months of taking the medication.

[0221] 23. Medical conditions (e.g., uncontrolled diabetes, known congestive heart failure, clinically significant valvular heart disease, clinically significant heart rhythm or conduction abnormalities) or mental illnesses, or any condition that may impair the participant's health or may prevent, limit or obstruct the assessments prescribed in the protocol.

[0222] 24. Known hypersensitivity, allergy or intolerance to JNJ-81201887 or its excipients.

[0223] Previous / companion therapy Study the eye 25. Previous history of intravitreal drug delivery, implantation, complement antidotes, or device implantation. A single intraoperative or perioperative administration of corticosteroids for cystoid macular edema prophylaxis is permitted at least 6 months prior to enrollment during cataract surgery.

[0224] Any eye 26. Has previously received any complement inhibitor treatment (research or non-research) via any route (e.g., ocular or systemic).

[0225] 27. Previous treatment for any type of exudative AMD / MNV.

[0226] system 28. According to local prescribing information, there are contraindications to the use of corticosteroids.

[0227] 29. No unauthorized treatments have been administered prior to the planned dose study intervention.

[0228] 30. Has previously received any gene therapy treatment through any route (e.g., eye or whole body).

[0229] 31. Treatment for active systemic or local infections.

[0230] 32. Received or planned to receive a live attenuated vaccine within 4 weeks of study enrollment, or planned to receive a live attenuated vaccine at any time during the study. Non-live vaccines are permitted.

[0231] Previous / companion clinical research experience Study the eye 33. Previous participation in an ophthalmic interventional clinical trial for AMD (in which the study participant was randomly assigned to the study treatment group or the sham surgery / placebo group), except where: (a) the intervention in the previous ophthalmic interventional clinical trial was the administration of vitamins or minerals; or (b) there is documentation (written confirmation from the research center or sponsor of the previous trial) indicating that the study participant 1) was randomized and received sham surgery / placebo treatment in the previous ophthalmic interventional clinical trial, and 2) is no longer participating in that trial or its follow-up.

[0232] system 34. Have received an investigational intervention within 6 weeks or 5 half-lives (whichever is longer) prior to the planned dose of the investigational intervention, or are currently participating in an investigational study.

[0233] Other exclusion criteria 35. Employees of a researcher or research center who are directly involved in the proposed research or other research under the guidance of that researcher or research center, and family members of the employee or researcher.

[0234] Intervention group and duration In a study of two doses of JNJ-81201887 administered via a single intravitreal fluid injection in the eye, one of the following intensities was used. One dose : Group A (low dose): 8.2 × 10 10 VG dose Group B (high dose): 4.1 × 10 11 VG dose Sham surgery / placebo control : Group C: A simulated single sham surgery involving intravitreal injection in the eye (using a needleless syringe) (placebo). Prevention of intraocular inflammation : Participants in Group A (low dose) and Group B (high dose) will begin a 20-day course of oral prednisone 3 days before the study intervention administration date and receive a single periocular corticosteroid injection on the study intervention administration date (day 4) to prevent intraocular inflammation.

[0235] Participants in group C (single sham surgery) will receive a 20-day course of oral prednisone-matched placebo and a pseudo-corticosteroid periorbital injection on the day of the sham surgery to maintain blindness.

[0236] Therapeutic effect evaluation The following are the primary and secondary efficacy evaluations.

[0237] Retinal imaging • Blue Light FAF (Heidelberg) • Spectral domain optical coherence tomography (SD-OCT) vision • Best corrected visual acuity (BCVA, using the Early Treatment in Diabetic Retinopathy Research [ETDRS] visual acuity chart) • Best corrected low-light visual acuity (LLVA) • Low-light visual impairment (LLVD) Vision and retinal function • Reading speed (Radner Vision Chart) • Intermediate visual micro-field inspection (MAIA; if available) • Patient-Reported Outcomes (PRO): Functional Reading Independence (FRI) Index and National Eye Institute Visual Function Questionnaire-25 (NEI-VFQ-25) Safety assessment Safety assessments include ocular adverse events of interest (including intraocular inflammation and new exudative macular neovascularization [MNV], as described in the protocol text), systemic adverse events, physical examination (including vital signs), ocular examination, ocular imaging parameters (e.g., FAF, SD-OCT, fluorescein angiography, and color fundus photography), laboratory assessments (clinical chemistry and hematology measurements), and pregnancy testing (if applicable).

[0238] Statistical methods Sample quantity determination Based on a meta-analysis of sham-operated groups from completed GA clinical trials and natural history studies with comparable inclusion / exclusion criteria, the estimated mean and standard deviation of the change in GA area (square root converted) relative to baseline at 18 months were 0.48 mm and 0.27 mm, respectively. Assuming a 15% dropout rate at 18 months, it was estimated that each group of 100 participants would have an 87% confidence level in detecting at least one active treatment, which would show an inter-group difference of 0.12 mm at a significance level of 0.025 (one-sided) (corresponding to a 25% reduction in growth).

[0239] Analysis settings For the purposes of this analysis, the following population group is defined: crowd describe Full Analysis Set (FAS) All participants in this study were randomized. Following the intention-to-treat principle, participants will be analyzed based on their randomized study intervention (JNJ-81201887 - high-dose, low-dose, or sham surgery), which may differ from the study intervention they received. Security Analysis Set (SAF) All participants, randomly assigned to either an intravitreal injection or a sham surgery, received either the research intervention or the intervention. Participants were assessed based on the research intervention they received. Immunogenicity analysis set All randomized participants received the intravitreal injection study intervention and had at least one suitable sample for assessing the immune response to JNJ-81201887. Participants were assessed based on the study intervention received. Virus Emission and Biodistribution Analysis Set All randomly assigned participants received an intravitreal injection study intervention and had at least one suitable sample for viral genome detection. Participants were assessed based on the study intervention they received. Efficacy Analysis For the primary power analysis of changes in sqrt-GAA in the eye, a repeated measures mixture model (MMRM) will be used, including treatment, time (a categorical variable via visits), treatment-time interaction, a stratification factor (GA lesion continuity) as a fixed effect, and baseline sqrt-GAA as a covariate. Intra-subject variability will be modeled using unstructured covariance. The inter-treatment differences in the least-squares means at month 18 and their two-sided 95% confidence intervals will be presented. The primary endpoint will be tested using the Dunnett step-down procedure, controlling for the overall type 1 error rate (with a two-sided alpha level of 0.0499 at the final analysis).

[0240] Security Analysis Safety will be summarized by physical examination (including vital signs), ocular examination, clinical chemistry, hematological measurements, and changes in adverse events and SAEs relative to baseline during ocular and systemic treatment, including ocular adverse events of interest (intraocular inflammation and new-onset exudative MNV, as well as other adverse events outlined in the main body of this report). In addition, certain imaging parameters will be evaluated periodically to monitor post-injection changes in the retina, including FAF, SD-OCT, fluorescein angiography, and color fundus photography, and changes and abnormalities in ocular examination results will be summarized descriptively. Observations and changes relative to baseline in clinical laboratory tests and vital signs will be summarized using descriptive statistics up to month 18. The number and percentage of participants with an intraocular pressure (IOP) ≥25 mm Hg at at least one post-baseline visit and participants with an IOP increase of ≥10 mm Hg relative to baseline will be presented. The number and percentage of participants with anterior chamber cells and / or vitreous cells and vitreous opacities (at least one of these) scores ≥2+ in the studied eye at baseline and at least one post-baseline visit will be presented. For intraocular inflammation scores (0, 0.5, 1+, 2+, 3+, 4+), a deviation table from baseline to the worst post-baseline value will be presented. The number and percentage of participants with abnormal slit-lamp biomicroscopy and indirect / mydriatic fundus examination results that occurred during treatment will be presented over time.

[0241] Immunogenicity For all participants who received the intravitreal injection study intervention (JNJ-81201887), the incidence of antibodies against the sCD59 transgenic protein and the AAV2 viral capsid protein will be summarized.

[0242] A list of participants who tested positive for antibodies against the sCD59 transgenic protein and the anti-AAV2 viral capsid protein will be provided. For participants who tested positive for antibodies against either the sCD59 transgenic protein or the anti-AAV2 viral capsid protein, the antibody titers against either protein will be summarized.

[0243] For all participants who received the intravitreal injection study intervention (JNJ-81201887), the incidence of cellular immune responses to the AAV2 viral capsid and sCD59 transgenic protein will be summarized. A list of participants who tested positive for cellular immune responses to the AAV2 viral capsid and sCD59 transgenic protein will be provided.

Claims

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

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

3. The method according to claim 1 or 2, wherein the pharmaceutical composition comprises about 8.2 × 10⁻⁶ ppm. 10 The viral particle dose of each vector genome (vg).

4. The method according to any one of the preceding claims, wherein the pharmaceutical composition comprises about 4.1 × 10 11 The viral particle dose of each vector genome (vg).

5. The method according to any one of the preceding claims, wherein the measurement of the GA covering the affected eye is 2.5 mm. 2 Up to 17.5mm 2 The area.

6. The method according to any one of the preceding claims, wherein the GA is not centrally recessed.

7. The method according to any one of the preceding claims, wherein the GA is multifocal and has at least one lesion ≥1.25 mm 2 Pathological lesions.

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

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

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

11. The method according to any one of the preceding claims, wherein the pharmaceutical composition is administered to the GA patient's eye by a single intravitreal injection.

12. The method according to any one of the preceding claims, wherein the treatment is evaluated about 18 months after administration of the pharmaceutical composition.

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

14. The method according to 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 reduced by about 10 mg every 5 days.

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

17. The method of claim 16, wherein the corticosteroid is administered via a single periorbital 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 according to any one of the preceding claims, wherein the sCD59 protein lacks a glycosylphosphatidylinositol (GPI) anchoring domain.

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

1.

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

2.

23. The method according to any one of the preceding claims, wherein the nucleic acid encoding the sCD59 protein is operatively linked to a promoter sequence to express and secrete the sCD59 protein in cells of the GA-affected eye.

24. The method according to 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 carrier is AAV2.

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

28. A method for improving the growth of geographic atrophy (GA) lesions in a subject, the method comprising administering a pharmaceutical composition to the subject’s GA-affected eye via ocular injection, wherein the pharmaceutical composition comprises a nucleic acid encoding a soluble CD59 (sCD59) protein.

29. The method of claim 28, wherein the improvement is the change in the square root transformed area of ​​GA lesions (sqrt-GAA) relative to baseline at months 3, 6, 9, 12, 15, and 18.

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

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

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