Compstatin analogs for carrier-based therapy

Abstract

Compounds comprising peptides capable of binding to C3 protein and inhibiting complement activation are disclosed. The compounds comprise compstatin analogs that can be vectorized for use in vector-mediated therapies, such as viral vector-mediated gene therapies, as independent treatments for diseases involving aberrant complement activation, or in combination with other viral vector-mediated gene therapies, to reduce vector-induced complement activation, or in combination with other viral vector-mediated gene therapies. And provide additive or synergistic therapeutic benefits when combined with other drug patterns in the same or more carriers. Also disclosed are nucleic acids encoding the compstatin analogs, as well as vectors, pharmaceutical compositions and methods of using the compstatin analogs.

Description

TECHNICAL FIELD

[0001] The present invention relates to the activation of the in vivo complement cascade. Specifically, the present invention provides compstatin analogs that can be vectorized for use in vector-mediated therapies (such as virus vector-mediated gene therapy), as monotherapy for diseases involving abnormal complement activation, or in combination with other virus vector-mediated gene therapies, to reduce vector-induced complement activation and provide additive or synergistic therapeutic benefits when combined with other drug modalities in the same or multiple vectors. These novel molecules containing natural amino acids of compstatin can be used for therapeutic C3 modulation in systemic or local applications. BACKGROUND OF THE INVENTION

[0002] Throughout this specification, various publications are cited, including patents, published applications, technical articles, and academic articles. Each of these cited publications is hereby incorporated by reference in its entirety.

[0003] Virus vector gene therapy uses modified viruses as drug delivery mediators to introduce specific DNA sequences into cells. For a long time, this technology has attracted interest due to its potential advantages over traditional modalities. Many types of therapeutic agents (such as therapeutic peptides, enzymes, antibodies, and regulatory RNAs) can be encoded in the DNA sequence, which can be rapidly designed and synthesized once the target is identified. The delivery of these vector therapies requires only one administration or at a much lower frequency than traditional treatment modalities, thereby also improving patient compliance.

[0004] Almost all currently available gene therapies use one of three vector types: adeno-associated virus (AAV) vectors, adenovirus vectors, or lentivirus (retrovirus) vectors. AAV and adenovirus vectors are commonly used in gene therapies directly administered to patients by infusion or local application (in vivo), where AAV is the most popular vector in areas other than oncology and vaccines. Although the applicability of virus vector-based gene therapies is expanding to new disease areas and clinical conditions, there is an unmet clinical need for the development of effective countermeasures that can mitigate vector-induced immune-related adverse events.

[0005] For example, maladaptive host immune responses to AAV capsids or transgenes involve innate and adaptive immune pathways and can limit the transduction efficacy and therapeutic efficacy of AAV-based therapies. Complement activation is considered a key pathogenic driver of acute immunotoxicity associated with AAV gene therapy (Hamilton BA et al., 2021, Front Immunol. 2021 May 17;12:675897. doi: 10.3389 / fimmu.2021.675897. eCollection 2021. PMID: 34084173; Muhuri M et al., J Clin Invest, 2021, Jan 4;131(1):e143780. doi: 10.1172 / JCI143780. PMID: 33393506; Smith CJ et al., Front Immunol. 2022, Sep 16;13:999021. doi: 10.3389 / fimmu.2022.999021. eCollection 2022. PMID: 36189251). Pre-existing neutralizing or non-neutralizing IgM and IgG antibodies against AAV capsids or transgenes can effectively trigger complement activation via the classical pathway, leading to C3 activation, amplification of the complement response via the alternative pathway, and downstream activation of the terminal pathway (i.e., direct MAC-mediated cytotoxicity). C3 activation occupies a central position in this cascade of events that together limit the transduction efficiency of AAV particles and impair the clinical efficacy of AAV therapies. C3 activation can lead to macrophage activation, increased AAV uptake via CR3-dependent phagocytosis, and direct AAV toxicity via activation of downstream lytic pathways. In addition, C3-derived fragments can modulate the antigen presentation pathway and B cell responses, thereby affecting the production of anti-AAV antibodies and promoting AAV-directed cytotoxic T cell responses. Given the multifaceted role of C3 in these processes that affect both AAV survival and efficient tissue homing, C3 inhibition appears to be a central therapeutic modality for enhancing the clinical potential of AAV-based systemic therapies.

[0006] The human complement system contributes to a wide range of pathologies, from autoimmune, age-related, and inflammatory disorders to graft- and biomaterial-induced complications, making it a prime target for therapeutic intervention. Despite growing interest, the development of complement-targeted drugs has been slow, and two related anti-C5 antibodies (eculizumab, ravulizumab) approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and other indications have long been the only clinical options. Until 2021, with the approval of pegcetacoplan by the FDA and EMA Apellis), a second class of complement inhibitors with different mechanisms became available. Compared to existing therapies, pegcetacoplan acts upstream in the complement cascade by attenuating the activation of the central component C3 to provide broader control of complement effectors. Given the diverse involvement of complement in pathologies, there is high anticipation for expanding the therapeutic intervention points within the cascade. Recently, two additional complement inhibitors, namely an anti-Cls mAb (sutimlimab, Enjaymo) and a small molecule C5aR1 antagonist (avacopan, Tavneos), were approved for complement-mediated diseases such as cold agglutinin disease and ANCA-associated vasculitis, respectively.

[0007] Complement primarily functions as a rapid host defense system to eliminate microbial invaders and apoptotic cells. After initiation through various means including immune complexes (classical pathway) or microbial signatures (lectin pathway), the cascade converges at the activation of plasma protein C3 by convertases. C3 cleavage releases the anaphylatoxin C3a and generates an opsonin fragment (C3b), which covalently attaches to the surface of target cells. The cooperative binding of protease factor B (FB) and factor D (FD) to C3b generates the major C3 convertase (i.e., C3bBb) to activate more C3. In the absence of regulators, this process forms an amplification loop (alternative pathway) to rapidly opsonize surfaces with C3b. Although C3b and its degradation fragments are directly involved in phagocytosis and adaptive immune signaling, C3b also provides a platform for the formation of the C5 convertase. Cleavage of C5 generates the inflammatory mediator C5a and produces the membrane attack complex (MAC), which lyses or damages susceptible cells. Although these potent effector functions provide an important layer of antimicrobial defense, any excessive or misguided complement activation can trigger clinical complications by inducing tissue damage, inflammation, and adverse immune responses. Depending on the disorder, pathway- or effector-specific inhibition may be sufficient, while other conditions require a method to more broadly inhibit complement activity.

[0008] Compstatin family C3 inhibitors are particularly suitable for broad complement inhibition as it potently impairs convertase-mediated C3 activation through all pathways and blocks the generation of most effectors. Compstatin was originally derived from phage display as a disulfide-bridged 13-amino acid peptide with micromolar binding affinity for C3 and was optimized to improve affinity, efficacy, and pharmacokinetic properties. Replacement of residues in the cyclic core generates compstatin analogs with significantly enhanced target affinity. Compstatin Cp05 (SEQ ID NO: 2) is pegcetacoplan ( Apellis) established the basis in which two Cp05 units were bridged with a 40 kDa PEG moiety to reduce renal clearance. Finally, N-methylation of the backbone and addition of D-Tyr to the N-terminus yielded the analogue Cp40 (SEQ ID NO: 3), which is characterized by picomolar affinity and improved pharmacokinetic properties in the absence of PEGylation.

[0009] Recently, new analogues of Cp40 have been developed, which have enhanced solubility and improved pharmacokinetic properties. PEGylation using small PEG moieties or addition of Lys residues has been shown to increase the solubility of Cp40 at physiological pH (~7.4) without affecting the favorable C3 inhibitory activity of Cp40. In addition, compared to Cp40, the new Cp40-based Compstatin derivatives showed similar or extended half-lives after subcutaneous (sc) administration to NHPs, resulting in a longer saturation time of plasma C3 (the length of time when the molar concentration of the Cp40 analogue is equal to or higher than the molar concentration of plasma C3). These improved properties facilitate the subcutaneous administration of Cp40-based derivatives, thus leveraging patient compliance during chronic C3-targeted interventions and expanding the possible routes of Cp40 delivery, thereby broadening its potential uses in various indications. Recent studies in severely COVID-19 patients have shown that administration of Cp40 can produce complete and sustained systemic C3 inhibition, saturating the plasma concentration of C3 during treatment. Inhibition of C3 by Cp40 leads to broad anti-inflammatory effects and significantly attenuates the thrombotic response (i.e., neutrophil extracellular trap (NET) release) (Skendros P, Germanidis G et al., Sci Adv, 2022, Aug 19; 8(33): eabo2341. doi: 10.1126 / sciadv.abo2341. Epub 2022 Aug 17. PMID: 35977025). In addition, local (intragingival) administration of Cp40 to adult patients with periodontal inflammation resulted in a significant reduction in key clinical indicators of gingival inflammation and markers of inflammatory tissue damage, with a sustained therapeutic effect extending up to 90 days after cessation of treatment (Hasturk H et al., J Clin Invest, 2021, Dec 1; 131(23): el52973. doi: 10.1172 / JCI152973. PMID: 34618684).

[0010] Although compstatin itself is a peptide composed of natural amino acids, the above-mentioned compstatin analogs contain unnatural components, the inclusion of which increases their potency by hundreds of times compared to compstatin and confers a number of pharmacokinetic benefits. These advantages, combined with their small size, make Cp40 and its derivatives particularly attractive for drug development. However, the presence of unnatural components in these analogs has hindered their expression and preparation from DNA sequences in viral vectors or other expression vectors. Thus, the development of new analogs that are easy to vectorize but still have activity and pharmacokinetic characteristics comparable to those of potent Cp40 and derivatives would be an advance in the art. Summary of the Invention

[0011] The present invention provides analogs of the complement inhibitory peptide compstatin that are easily vectorized as a fusion moiety for a single therapeutic modality or in combination with other therapeutic modalities (such as anti-VEGF agents) and are prepared by expression, while having potent complement inhibitory activity and desirable pharmacokinetic properties.

[0012] One aspect of the present invention relates to a compstatin analog comprising a peptide having the amino acid sequence Xaa1-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Xaa2-His-Arg-Cys]-Xaa3-Xaa4 (SEQ ID NO: 4), wherein Xaa1 is absent or comprises the dipeptide Tyr-Ile, Xaa2 is Ala or Glu, Xaa3 is absent or is Ile, Xaa4 is absent or represents one, two, or three Lys residues, and wherein the Cys residues form a disulfide bond to form a cyclic peptide comprising the sequence within the square brackets. Specifically, the compstatin analog may have the amino acid sequence Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Xaa2-His-Arg-Cys]-Ile-Xaa4 (SEQ ID NO: 5), wherein Xaa2 is Ala or Glu, and Xaa4 represents two or three Lys residues. Embodiments include any one of SEQ ID NO: 6 and / or SEQ ID NO: 7 and SEQ ID NOs: 8-16.

[0013] One aspect of the present invention relates to a polynucleotide comprising a sequence encoding the above-mentioned compstatin analog or peptide. In one embodiment, the polynucleotide encodes SEQ ID NO: 6 or SEQ ID NO: 7. In one embodiment, the polynucleotide is selected from SEQ ID NO: 17 and SEQ ID NO: 18.

[0014] In certain embodiments, the campstatin analog polynucleotide is configured in an expression cassette or vector. The vector can be an expression vector. It can be suitable for expression in a prokaryotic or eukaryotic expression system. In certain embodiments, the vector is used for gene therapy and is selected from retroviruses, adenoviruses, adeno-associated viruses (AAV), and herpes simplex virus-1. In certain embodiments, the vector is an adeno-associated virus (AAV) vector. The AAV vector can be an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector or any variant thereof.

[0015] Another aspect of the invention relates to a vector or vectors, wherein at least one vector comprises a polynucleotide encoding a campstatin analog comprising any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8-16. The campstatin analog polynucleotide can have the sequence of SEQ ID NO: 17 or SEQ ID NO: 18. The vector can further comprise at least one insertion site for at least one transgene for delivering gene therapy. The vector can be selected from retroviruses, adenoviruses, adeno-associated viruses (AAV), and herpes simplex virus-1. In certain embodiments, the vector is an adeno-associated virus (AAV) vector. The AAV vector can be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vectors or any variant or combination thereof.

[0016] In certain embodiments, the above-mentioned vector comprises at least one other transgene for gene therapy. The transgene can encode, for example, a therapeutic protein, enzyme, hormone, clotting factor, cytokine, or growth factor. In various embodiments, the gene therapy is used to treat blood disorders, ocular disorders, autoimmune diseases, muscle disorders, neurological disorders, or cancer. In certain embodiments, the campstatin analog polynucleotide and / or the transgene is suitable for tissue- or organ-specific expression.

[0017] In certain embodiments, the campstatin analog polynucleotide and the transgene are arranged on the vector to produce a fusion protein comprising the campstatin analog and the transgene product. The fusion protein can comprise the campstatin analog directly linked to the transgene product, or it can comprise the campstatin analog linked to the transgene product through a linker or spacer.

[0018] In certain embodiments, the transgene encodes a VEGF inhibitor. The VEGF inhibitor may comprise the extracellular domain of a VEGF receptor. In other embodiments, the VEGF inhibitor comprises an antibody fragment selected from Fab, F(ab’)2, Fv, scFv, or a single domain antibody. The antibody fragment may be a Fab comprising a VL domain, a CL domain, a VH domain, and a CH1 domain. In certain embodiments, the vector encodes a fusion protein comprising the campstatin analog directly or indirectly fused to one, two, three, or four of the following: (i) the VL of the Fab; (ii) the CL of the Fab; (iii) the VH of the Fab; or (iv) the CH1 of the Fab. The vector may encode a fusion protein comprising the campstatin analog directly or indirectly fused to the N-terminus of the VL of the Fab, the C-terminus of the CL of the Fab, the N-terminus of the VH of the Fab, or the C-terminus of the CH1 of the Fab.

[0019] Another aspect of the invention relates to a pharmaceutical composition comprising at least one of the vectors described above and a pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated for administration by a route selected from subcutaneous, intradermal, intravenous, intraocular (including intravitreal and subretinal), intracerebral, intraperitoneal, intramuscular injection, periodontal administration (including gingival administration or infiltration injection within the interdental papilla), intranasal, epidural, oral, sublingual, intrathecal, intravaginal, transdermal, rectal, inhalation, or topical.

[0020] The pharmaceutical composition may be formulated for systemic administration, or it may be formulated for local administration. The local administration may be directed to the brain and / or central nervous system, the eye, the lung and / or respiratory system, the heart and / or vascular system, the lymphatic system, the kidney, the spleen, the pancreas, the liver, the gastrointestinal system, the periodontal tissue, the skin, the bone, the joint, or the synovial fluid, or any combination thereof. In addition to the above vectors, the pharmaceutical composition may further contain a campstatin analog peptide as described herein, which may have a sequence selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

[0021] Another embodiment relates to a pharmaceutical composition comprising a camptastatin analogue having a sequence selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 in a pharmaceutically acceptable carrier. Such a composition can be used for certain therapeutic applications as described herein. Such a pharmaceutical composition can be formulated for administration by a route selected from subcutaneous, intradermal, intravenous, intraocular (including intravitreal, subretinal), intracerebral, intraperitoneal, intramuscular injection, periodontal administration (including gingival administration or infiltration injection into the interdental papilla), intranasal, epidural, oral, sublingual, intrathecal, intravaginal, transdermal, rectal, inhalation or topical. It can be configured for systemic or local administration, and the local administration can be directed to the brain and / or central nervous system, eye, lung and / or respiratory system, heart and / or vascular system, lymphatic system, kidney, spleen, pancreas, liver, gastrointestinal system, periodontal tissue, skin, bone, joint or synovial fluid or any combination thereof. The above pharmaceutical composition containing a camptastatin analogue can further comprise at least one carrier as described herein.

[0022] Another aspect of the invention relates to a kit comprising a plurality of pharmaceutical compositions, wherein at least one of the pharmaceutical compositions is a peptide-containing composition, the peptide-containing composition comprising a camptastatin analogue having a sequence selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 in a pharmaceutically acceptable carrier, and at least one of the pharmaceutical compositions is a carrier-containing composition, the carrier-containing composition comprising a carrier for gene therapy as described herein in a pharmaceutically acceptable carrier. In one embodiment, at least one of the peptide-containing composition and the carrier-containing composition is formulated for systemic administration. In another embodiment, at least one of the peptide-containing composition and the carrier-containing composition is formulated for local administration. In another embodiment, the peptide-containing composition is formulated for a selected route of administration and the carrier-containing composition is formulated for a route of administration different from the route of administration selected for the peptide-containing composition. The kit can further comprise instructions for administering the peptide-containing composition according to a schedule different from the schedule for administering the carrier-containing composition.

[0023] According to another aspect of the invention, the kit comprises a plurality of pharmaceutical compositions, each of which is a carrier-containing composition, the carrier-containing composition comprising a carrier as described herein in a pharmaceutically acceptable carrier. Such kits can include pharmaceutical compositions formulated for systemic administration, local administration or both. Such kits can include pharmaceutical compositions formulated for different routes of administration. They can also include instructions for administering one composition according to a schedule different from the schedule for administering another composition.

[0024] Another aspect of the present invention relates to a method of treating a subject suffering from or at risk of a complement-mediated disorder, the method comprising administering to the subject a composition comprising at least one vector, the vector comprising a compstatin analog polynucleotide encoding a compstatin analog of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. The subject can be a non-human primate or a human. In certain embodiments, after administering the composition, the level of complement activity in the subject or in a biological sample from the subject is reduced relative to the level in the subject before administering the composition or in an equivalent subject that has not received the composition. The level of complement activity can be reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, 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% or at least 90% relative to the level in the subject before administration or in an equivalent subject that has not received the composition.

[0025] In certain embodiments, the composition is administered systemically to the subject. In other embodiments, the composition is administered locally to a tissue or organ of the subject.

[0026] In certain embodiments, the vector is an adeno-associated virus (AAV) vector. The AAV vector can be an AAV1, AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 vector.

[0027] In certain embodiments, the complement-mediated disorder is a chronic disorder. In certain embodiments, the complement-mediated disorder involves complement-mediated damage to red blood cells, optionally wherein the disorder is paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome. In certain embodiments, the complement-mediated disorder is an autoimmune disease, optionally wherein the disorder is multiple sclerosis. In certain embodiments, the complement-mediated disorder involves the kidney. These disorders can include membranoproliferative glomerulonephritis, lupus nephritis, IgA nephropathy (IgAN), primary membranous nephropathy (primary MN), C3 glomerulopathy (C3G), or acute kidney injury. In certain embodiments, the complement-mediated disorder involves the central or peripheral nervous system or the neuromuscular junction. Examples include neuromyelitis optica, Guillain-Barré syndrome, amyotrophic lateral sclerosis, multifocal motor neuropathy, or myasthenia gravis. In certain embodiments, the complement-mediated disorder involves the respiratory system. The respiratory disorder can be characterized by pulmonary fibrosis. In certain embodiments, the complement-mediated disorder involves the vascular system. The vascular system disorder can be characterized by vasculitis.

[0028] In certain embodiments of the method, the composition is administered to the eye of a subject having an eye disorder. The composition can be administered intravitreally. The eye disorder can be age-related macular degeneration (AMD). In certain embodiments, the eye has one or more of the following: (i) geographic atrophy, (ii) wet AMD, (iii) geographic atrophy and wet AMD, or (iv) intermediate AMD.

[0029] The method can include the following steps: (1) providing the subject; (2) administering the composition to the subject to produce the campastatin analog in the subject; and (3) measuring one or more parameters of the complement-mediated disorder. The measurement can be performed before, during, and / or after administering the composition. It can also or alternatively be performed on an equivalent subject not administered the composition.

[0030] In certain embodiments, the complement-mediated disorders are selected from atypical hemolytic uremic syndrome (aHUS), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), C3 glomerulopathy, complement-mediated nephropathy and glomerular inflammatory diseases, age-related macular degeneration (AMD), ocular disorders characterized by macular degeneration, choroidal neovascularization (CNV), retinal neovascularization (RNV), proliferative vitreoretinopathy, glaucoma, uveitis, ocular inflammation or any combination of these diseases, paroxysmal nocturnal hemoglobinuria (PNH), cold agglutinin disease (CAD), warm antibody autoimmune hemolytic anemia (wAIHA), sickle cell disease, transplant-associated thrombotic microangiopathy, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), autoimmune and autoinflammatory kidney diseases, autoimmune myocarditis, multiple sclerosis, traumatic brain and spinal cord injury, cerebral, intestinal and renal ischemia-reperfusion (IR) injury, spontaneous and recurrent miscarriage, antiphospholipid syndrome (APS), Parkinson's disease, Alzheimer's disease, neurodegenerative inflammatory disorders based on abnormal synaptic remodeling, microglial activity and cognitive decline, asthma, antinuclear cytoplasmic antigen-related pauci-immune vasculitis (Wegener's syndrome), non-lupus autoimmune skin diseases such as pemphigus, bullous pemphigoid and epidermolysis bullosa, post-traumatic shock, cancer, periodontitis, gingivitis and atherosclerosis.

[0031] In certain embodiments, the method comprises administering to the subject more than one dose of the composition. The method may comprise administering multiple doses of the composition to the subject at predetermined time intervals. Such time intervals may be within hours, days, weeks or months of each other.

[0032] Another aspect of the invention relates to a method of treating a subject having or at risk of having a disease or disorder having a complement-mediated component and one or more other components. The method comprises administering to the subject a composition comprising at least one vector, the vector comprising: (a) a compstatin analog polynucleotide encoding a compstatin analog of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, and (b) at least one transgene encoding a gene product for treating the one or more other components. The subject may be a non-human primate or a human.

[0033] In certain embodiments, after administration of the composition, complement activation and / or the level of the other component(s) in the subject or a biological sample from the subject is reduced or improved relative to the level before administration of the composition or in an equivalent subject that has not received the composition. The level of complement activation and / or other component(s) can be reduced or improved by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, 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%, or at least 90% relative to the level before administration or in an equivalent subject that has not received the composition.

[0034] In one embodiment, the other component is associated with a neovascular phenotype. In one embodiment, the composition is administered to the eye of a subject having an eye disorder. In one embodiment, the eye disorder is age-related macular degeneration (AMD). In one embodiment for treating an eye disorder, the transgene encodes a VEGF inhibitor.

[0035] In certain embodiments, the method involves the use of an adeno-associated virus (AAV) vector. The AAV vector can be an AAV1, AAV2, AAV3 (such as AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector. In certain embodiments, the transgene encodes a therapeutic protein, enzyme, hormone, clotting factor, cytokine, or growth factor.

[0036] In certain embodiments, the campastatin analog polynucleotide and the transgene are arranged on a single vector to produce a fusion protein comprising the campastatin analog and the transgene product. The campastatin analog can be directly linked or linked via a linker or spacer to the transgene product. The fusion protein can comprise a VEGF inhibitor. In other embodiments, the campastatin analog polypeptide and at least one other transgene are configured on two or more separate vectors.

[0037] In certain embodiments, the vector comprising the campastatin analog and one or more vectors comprising at least one other transgene are administered by different routes. In certain embodiments, the vector comprising the campastatin analog is administered systemically and one or more vectors comprising at least one other transgene are administered locally. In certain embodiments, the vector comprising the campastatin analog is administered before, during, or after administration of one or more vectors comprising at least one other transgene.

[0038] In certain embodiments, the method comprises the following steps: (1) providing the subject; (2) administering the composition to the subject, thereby generating the campastatin analog and the transgene in the subject; and (3) measuring one or more parameters of the complement-mediated disorder or other components. The measurement can be performed before, during, and / or after administering the composition and / or on equivalent subjects not administered the composition. In certain embodiments of this method, the campastatin analog polynucleotide or the vector comprising the campastatin analog polynucleotide is replaced with the campastatin analog peptide itself.

[0039] Another aspect of the invention relates to a method for enhancing the efficacy of gene therapy in a subject who will receive, is receiving, or has received gene therapy. The method comprises administering to the subject a vector comprising a campastatin analog polynucleotide, which generates a campastatin analog having SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 in the subject, thereby enhancing the efficacy of the gene therapy. In certain embodiments, in the subject, the efficacy of the gene therapy is improved over a selected time period. The selected time period can be, for example, at least about 1 week, 2 weeks, 4 weeks, 2 months, 3 months, 6 months, or 1 year. In certain embodiments, the efficacy of the gene therapy is evaluated by observing or measuring a reduction in the immune response against the gene therapy. In certain embodiments, the efficacy is evaluated by observing or measuring an improvement in the transduction of the viral vector carrying the transgene. In certain embodiments, the efficacy is evaluated by observing or measuring a reduction in the complement-mediated clearance of the viral vector carrying the transgene. In certain embodiments, the efficacy of the gene therapy is measured by: (i) comparing a subject who has received the vector comprising the campastatin analog polynucleotide with a control subject who has not received the vector comprising the campastatin analog polynucleotide, and / or (ii) comparing a subject during or after receiving the vector comprising the campastatin analog polynucleotide with the same subject before receiving the vector comprising the campastatin analog polynucleotide.

[0040] In certain embodiments of this method, the vector comprising the campastatin analog polynucleotide comprises at least one insertion site for at least one transgene for delivering gene therapy. The vector can be selected from retroviruses, adenoviruses, adeno-associated viruses (AAV), and herpes simplex virus-1. In one embodiment, the vector comprising the campastatin analog polynucleotide is an adeno-associated virus (AAV) vector. The AAV vector can be an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector or any variant or combination thereof.

[0041] In certain embodiments, the vector comprising the campastatin analog polynucleotide comprises at least one other transgene for gene therapy. In certain embodiments, the vector comprising the campastatin analog polynucleotide is different from another vector comprising the other transgene for gene therapy, and the vectors are administered together. In other embodiments, the vector comprising the campastatin analog polynucleotide is different from another vector comprising the other transgene for gene therapy, and the vectors are administered separately. Another embodiment of this aspect of the invention includes replacing the campastatin analog polynucleotide or the vector comprising the campastatin analog polynucleotide with the campastatin analog peptide itself.

[0042] Other features and advantages of the invention will be understood by reference to the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 . A graph is shown depicting the change over time in the individual grade 4 lesion ratio (expressed as % of grade 4 lesions at week 2) after laser-induced choroidal neovascularization (CNV) in a non-human primate (macaque) model. The arrow indicates intravitreal treatment (day 15). Three eyes (K-2434, K-2437, and K-2485) were treated with the campastatin analog CP50, and three eyes (K-2089, K-2482, and K-2468) were treated with vehicle. DETAILED DESCRIPTION

[0044] DEFINITIONS:

[0045] Various terms related to the methods and other aspects of the invention are used throughout the specification and claims. Unless otherwise indicated, these terms shall have their ordinary meaning in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0046] The following abbreviations may be used in this text: Ac, acetyl; BSA, bovine serum albumin; DCM, dichloromethane; DMF, dimethylformamide; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionization; Fmoc, 9-fluorenylmethoxycarbonyl; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; NHP, non-human primate; PBS, phosphate-buffered saline; RP-HPLC, reverse-phase high-performance liquid chromatography; Sar, N-methylglycine; s.c., subcutaneous; SPR, surface plasmon resonance; TFA, trifluoroacetic acid; UPLC-ESLMS, ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry; VBS, Veronal buffered saline; WFI, water for injection.

[0047] Unless the context clearly dictates otherwise, the singular forms of words include the plural forms, and vice versa. References in the absence of an explicit number generally include the plural forms of the corresponding terms. For example, a reference to "compound" or "method" includes a plurality of such "compounds" or "methods". Similarly, the word "comprising" should be interpreted inclusively rather than exclusively. Likewise, the terms "include" and "or" should both be interpreted as inclusive, unless the context clearly prohibits such an interpretation.

[0048] The term "comprising" or "including" is intended to include the embodiments covered by the terms "consisting essentially of" and "consisting of". Similarly, the term "consisting essentially of" is intended to include the embodiments covered by the term "consisting of". In addition, the term "consisting essentially of" limits the scope of an embodiment to the specified components or steps and those components or steps that do not materially affect the basic and novel features of the embodiment.

[0049] As used herein, the term "about", when referring to a measurable value such as an amount, duration, etc., is intended to cover variations of ±20% or ±10%, in some embodiments ±5%, and in some embodiments ±1% from the specified value, because such variations are suitable for the manufacture and use of the disclosed compounds and compositions.

[0050] As used herein, the term "campastatin" refers to a peptide comprising SEQ ID NO:1, i.e., I[CVVQDWGHHRC]T (cyclic C2-C12 formed by a disulfide bond as indicated by square brackets). The term "campastatin analog" refers to a modified campastatin that comprises substitutions of amino acids that are amenable to expression from DNA, as described in more detail herein. These analogs are sometimes referred to herein as being "proproteinogenic". When referring to the position of a particular amino acid or analog in campastatin or a campastatin analog, these positions are sometimes referred to as "positions" within the peptide, numbered from 1 (Ile in campastatin) to 13 (Thr in campastatin). For example, the Gly residue occupies "position 8".

[0051] The terms "pharmacological activity" and "biological activity" refer to the ability of the campastatin analogs of the invention to bind to C3 or a fragment thereof and inhibit complement activation. This biological activity can be measured by one or more of several assays well known in the art.

[0052] As used herein, the term "gene therapy" encompasses therapies for treating a disease or disorder by introducing exogenous DNA into the cells of an individual suffering from the disease or disorder. Gene therapy includes, for example, (1) replacing a dysfunctional or non-functional gene with a healthy copy of the gene, (2) inactivating a disease-causing gene that is malfunctioning and causing the disease; and / or (3) introducing a heterologous nucleic acid into cells, tissues or organs in the body to produce a therapeutic agent to aid in treating the disease.

[0053] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to all forms of nucleic acids, oligonucleotides, including DNA and RNA. Nucleic acids include genomic DNA, cDNA, antisense DNA / RNA, plasmid DNA, linear DNA (polynucleotides and oligonucleotides), chromosomal DNA, spliced or unspliced mRNA, rRNA, tRNA, inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-spliced RNA or antisense RNA), locked nucleic acid analogs (LNA), single-stranded and double-stranded oligodeoxynucleotides (ODN), immunostimulatory sequences (ISS), riboswitches and ribozymes. Nucleic acids include naturally occurring, synthetic and intentionally modified or altered polynucleotides.

[0054] The term "transgene" refers to a nucleic acid that is intended or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a nucleic acid encoding a campastatin analog of the invention, a "campastatin analog transgene", as well as any other heterologous nucleic acid encoding a protein, peptide or nucleic acid (e.g., miRNA, etc.). The terms transgene and heterologous nucleic acid / polynucleotide sequence are used interchangeably herein.

[0055] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of directing the expression of a gene operably linked thereto. Such vectors are referred to herein as "expression vectors". Those of ordinary skill in the art understand that a "viral vector" as described herein contains viral components in addition to the transgene.

[0056] "Expression control element" refers to a nucleic acid sequence that affects the expression of an operably linked nucleic acid. Vector sequences and non-viral vectors can contain one or more "expression control elements". Generally, such elements are included to facilitate proper transcription of a heterologous polynucleotide and, optionally, translation (e.g., promoters, enhancers, intron splicing signals, maintaining the correct reading frame of a gene to allow in-frame translation of the mRNA, and termination codons, etc.).

[0057] As used herein, "operably linked" refers to the relationship between two or more nucleic acid sequences in which certain nucleic acid sequences (e.g., control elements) affect the characteristics of another nucleotide sequence (e.g., affect the expression of a transgene). Operably linked sequences include both expression control elements contained within or adjacent to a transgene and expression control elements that act in trans or at a distance to control the expression of a transgene. Expression control elements as used herein include appropriate transcription initiation, termination, promoter, and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and sequences that enhance secretion of the encoded product when needed. For example, when used herein, a nucleic acid sequence (e.g., a campstatin coding sequence or other transgene) and a regulatory sequence are considered to be operably linked when covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequence.

[0058] As used herein, "pharmaceutically acceptable salt" or "pharmaceutically acceptable ester" refers to derivatives of the disclosed compounds, wherein the parent compound is modified in the form of an ester or an acid or base salt, which is compatible with any other components of the pharmaceutical composition and is harmless to the subject to whom the composition is to be administered. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, base or organic salts of acidic residues such as carboxylic acids, and the like. Thus, the term "acid addition salt" refers to the corresponding salt derivative of the parent compound prepared by the addition of an acid. Pharmaceutically acceptable salts include, for example, conventional salts or quaternary ammonium salts of the parent compound formed from inorganic or organic acids. For example, such conventional salts include, but are not limited to, salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc., and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, hydroxyethanesulfonic acid, etc. Certain acidic or basic compounds of the present invention may exist as zwitterions. All forms of the compounds, including free acids, free bases, and zwitterions, are contemplated within the scope of the present invention.

[0059] As used herein, the terms "topical administration" or "topical delivery", when referring to the delivery of a campstatin analog peptide or its encoding polynucleotide described herein, refer to a delivery that can rely on the transport of the peptide or polynucleotide through the vascular system to its intended target tissue or site.

[0060] When used herein, "intraocular administration" or "ophthalmic administration" of a pharmaceutical composition includes any route of administration characterized by introduction into the eye, including subretinal administration and intravitreal administration. The term "intravitreal administration" of a pharmaceutical composition includes any route of administration characterized by introduction into the vitreous cavity of the eye. The "vitreous" is a gel-like substance within the vitreous cavity that fills the space between the lens and the retina and helps the eye maintain its shape.

[0061] When used herein, "intramuscular administration" of a pharmaceutical composition includes any route of administration characterized by introduction into muscle.

[0062] As used herein, "periodontal administration" of a pharmaceutical composition refers to administration (e.g., by injection, topical application, or biodegradable implant) into the tissues around and / or adjacent to one or more teeth, and includes "gingival administration" and "interdental papilla infiltration". As used herein, "gingival administration" of a pharmaceutical composition includes any route of administration characterized by introduction into the gingiva or within the gingiva. "Interdental papilla infiltration" or "interdental papilla infiltration injection" is a form of gingival administration that refers to the administration of a pharmaceutical composition into the interdental papilla, which is the gingival tissue at the coronal aspect of the free gingival margin present on the buccal and lingual surfaces of the teeth.

[0063] As used herein, "oral administration" or "enteral administration" of a pharmaceutical composition includes any route of administration characterized by introduction into the gastrointestinal tract. "Oral administration" includes oral feeding as well as orogastric or intragastric tube feeding. "Oral administration" or "enteral administration" may also include sublingual, buccal, intranasal, pulmonary, or rectal administration, as well as other routes known in the art.

[0064] The term "treatment" refers to any indication of success in the treatment or amelioration of a disease or disorder. Treatment can include, for example, reducing or alleviating the severity of one or more symptoms of a disease or disorder, or can include reducing the frequency with which an individual (e.g., a human patient) experiences symptoms of a disease, defect, disorder, or adverse condition.

[0065] The term "prevention" refers to the prevention of a disease or disorder in an individual (e.g., a human patient). For example, if an individual at risk of developing an inflammatory disease is treated with a compound and / or method of the present invention and subsequently does not develop the disease or disorder, then the disease has been prevented in that individual.

[0066] The term "treatment or prevention" is sometimes used herein to refer to methods that result in some degree of treatment or amelioration of a disease or disorder and contemplates a range of outcomes directed thereto, including but not limited to complete prevention of the disorder.

[0067] The term "parameter" as used herein refers to any observable or measurable body function measured using suitable measurement techniques available in the art. One of ordinary skill in the art will understand that measurement of one or more "parameters" of a body function can be used to detect a particular dysfunction as compared to average normal parameters, and can also be used to determine whether the body function has improved after or during treatment. Such parameters can be general, such as body temperature, blood pressure, pulse (heart rate), and respiratory rate (breathing frequency), or can be specific to a particular organ, tissue, or disease or disorder, such as the results of functional tests of blood or other organs / tissues.

[0068] "Effective amount" or "sufficient amount" means an amount that, when administered in a single or multiple doses, alone or in combination with one or more other compositions (therapeutic or immunosuppressive agents such as drugs), treatments, regimens or therapeutic agents, provides a detectable response for any duration (long-term or short-term) and provides an expected or desired result or benefit to a subject to any measurable or detectable degree or for any duration (e.g., minutes, hours, days, months, years or a lifetime).

[0069] The terms "therapeutically effective amount" or "therapeutically effective dose" mean an amount of a pharmaceutical composition sufficient to provide a beneficial effect to an individual to whom the pharmaceutical composition is administered. A therapeutically effective amount can be determined empirically and in a conventional manner according to the stated purpose. For example, in vitro assays can optionally be employed to assist in identifying an optimal dosage range. A person skilled in the art can determine the selection of a specific effective dose based on consideration of several factors (e.g., through clinical trials), including the disease to be treated or prevented, the symptoms involved, the weight of the patient, the immune status of the patient and other factors known to the skilled artisan. The exact dosage to be used in the formulation also depends on the route of administration and the severity of the disease and should be decided according to the judgment of the physician and the circumstances of each patient. The effective dose can be extrapolated from a dose-response curve derived from in vitro or animal model test systems.

[0070] Description:

[0071] Complement activation is essential for a robust immune system. However, in certain circumstances, excessive or uncontrolled complement activation is a central component of a large number of pathological conditions. In addition, virus vector-based gene therapy is severely hampered by unwanted immune side effects involving complement activation. These immune responses can include antibody, B cell and / or T cell responses and can be specific for viral antigens of the virus vector, such as viral capsid or envelope proteins or peptides thereof (see, for example, Colella et al., 2018, Molecular Therapy: Methods & Clinical Development, 8: 87-104).

[0072] The present invention is in part derived from the inventors' development of proteogenic compstatin analogs with potent complement inhibitory activity, the encoding nucleic acids of which can be introduced into nucleic acid constructs such as viral vectors and expressed in cells. These constructs can be used as standalone agents to provide long-term gene therapy for treating complement activation-mediated diseases, or inserted into viral vectors in combination with sequences encoding other therapeutic modalities (such as anti-VEGF agents). The combination of such proteogenic compstatin molecules with other therapeutic modalities can provide additive or synergistic therapeutic benefits in diseases driven by complement activation dysregulation and abnormal neovascularization responses, such as by elevating VEGF levels or enhancing VEGF receptor signaling. Such combinations can enhance viral vector transduction, prevent C3 opsonization of the capsid, and / or mitigate or prevent immune responses (such as antibody, B cell, and / or T cell responses).

[0073] Peptides, transgenes, and vectors:

[0074] Certain embodiments of the present invention relate to proteogenic derivatives of the compstatin analog Cp40 (SEQ ID NO: 3; described in WO2013 / 036778). The peptides of the present disclosure are represented by the sequences shown below.

[0075] Xaa1-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Xaa2-His-Arg-Cys]-Xaa3-Xaa4 (SEQ ID NO: 4)

[0076] In the above sequence, Xaa1 is absent or comprises the dipeptide Tyr-Ile, Xaa2 is Ala or Glu, Xaa3 is absent or is Ile, and Xaa4 represents one, two, or three Lys residues. The Cys residues form a disulfide bond to form a cyclic peptide consisting of the sequence within the square brackets.

[0077] One embodiment is represented by the following sequence:

[0078] Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Xaa2-His-Arg-Cys]-Ile-Xaa4 (SEQ ID NO: 5)

[0079] Exemplary sequences of the present disclosure include Cp50 and Cp51 as shown below:

[0080] Cp50: Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys]-Ile-Lys-Lys-Lys (SEQ ID NO: 6)

[0081] Cp51: Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Glu-His-Arg-Cys]-Ile-Lys-Lys-Lys (SEQ ID NO: 7)

[0082] Other exemplary sequences include:

[0083] Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile-Lys-Lys (SEQ ID NO: 8)

[0084] Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile-Lys (SEQ ID NO: 9)

[0085] Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile (SEQ ID NO: 10)

[0086] Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys] (SEQ ID NO: 11)

[0087] [Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile-Lys-Lys-Lys (SEQ ID NO: 12)

[0088] [Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile-Lys-Lys (SEQ ID NO: 13)

[0089] [Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile-Lys (SEQ ID NO: 14)

[0090] [Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile (SEQ ID NO: 15)

[0091] [Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys](SEQ ID NO:16)

[0092] As previously described, Cp40-KK and Cp40-KKK not only exhibit improved solubility compared to unmodified Cp40, but they also exhibit increased plasma and vitreous retention and enhanced C3 binding compared to Cp40 and other Cp40-based analogs (see WO 2019 / 195712). In fact, Cp40-KKK has been shown to have an in vivo residence time of equal to or greater than three months after intravitreal administration. Notably, both Cp40-KK and Cp40-KKK analogs exhibit significantly improved pharmacokinetic properties compared to Cp40.

[0093] The proproteinogenic peptides Cp50 and Cp51 of the examples have been found to have in vitro and in vivo properties similar to Cp40-KK and Cp40-KKK. This is surprising considering the absence of unnatural residues within the backbone and at the Cp40 termini. Table 1 below shows some of these properties compared to Cp40 and other campstatin analogs.

[0094]

[0095] a OD (280 nm) measured at saturation; DPBS: Dulbecco's phosphate buffered saline,

[0096] b Retention time measured on a C18 column during HPLC.

[0097] The nucleic acid encoding Cp50 (YICIWQDWGAHRCIKKK (SEQ ID NO: 6)) is set forth below, showing all possible codons for each residue:

[0098]

[0099] The nucleic acid encoding Cp51 (YICIWQDWGEHRCIKKK (SEQ ID NO: 7)) is set forth below, showing all possible codons for each residue:

[0100]

[0101] The campstatin analogs of the present invention are produced by expressing a nucleic acid encoding the peptide inserted into a vector. The vector can be non-viral or viral. In certain embodiments, the vector is a viral vector as discussed below. The vector contains one or more polypeptides encoding the campstatin analogs disclosed herein.

[0102] In certain embodiments, the campstatin analogs described herein are pre-generated (i.e., not in the patient's cells or tissues) and incorporated into pharmaceutical compositions for use in the various combination therapies described herein. These campstatin analogs can be prepared by various synthetic methods of peptide synthesis by condensation of one or more amino acid residues according to conventional peptide synthesis methods.

[0103] Alternatively, since the campstatin analogs disclosed herein are composed of naturally occurring amino acids, they can be produced by expressing the encoding polynucleotide in a suitable prokaryotic or eukaryotic system. For example, a DNA construct can be inserted into a plasmid vector suitable for expression in bacterial cells (e.g., Escherichia coli (E. coli)) or yeast cells (e.g., Saccharomyces cerevisiae), or into a baculovirus vector for expression in insect cells or a viral vector for expression in mammalian cells. Such vectors contain the regulatory elements required for expression of the DNA in the host cell, which are positioned in a manner that allows expression of the DNA within the host cell. Such regulatory elements required for expression are well known in the art and include promoter sequences, transcription initiation sequences, and optionally enhancer sequences. Peptides produced by gene expression in recombinant prokaryotic or eukaryotic systems can be purified by methods known in the art.

[0104] The above-described campstatin analog polynucleotides and polypeptides are advantageous in several respects. For example, as described above, these campstatin analogs exhibit complement inhibitory activity and can even rival the most potent campstatin analogs. Thus, complement inhibition can be achieved at lower doses. In addition, these campstatin analogs are expected to have the same or similar pharmacokinetic characteristics as their analogous counterparts Cp40-KK and Cp40-KKK. Furthermore, due to the small size of the encoding polynucleotide, they can be added to any vector without occupying a substantial amount of vector space that may be required for one or more transgenes of interest. This is particularly important for vectors with limited insertion space (e.g., AAV vectors). Thus, vector systems having the campstatin analog polynucleotide as a standard component can be constructed and advantageously used to enhance the efficacy of these vectors in delivering any gene therapy.

[0105] Thus, in one embodiment, the vector comprises at least one insertion site for a campastatin analog peptide and another polypeptide such as for gene therapy. As described in the definitions, such polypeptides are sometimes referred to herein as "transgenes". In certain embodiments, the campastatin polypeptide and other polypeptides are included in a single vector, while in other embodiments they are included on multiple vectors. If included on multiple vectors, the vectors can be combined into a single pharmaceutical composition and administered together. Alternatively, the multiple vectors can be administered separately.

[0106] In certain embodiments, the campastatin polynucleotide is arranged with one or more other transgenes to produce a fusion protein, wherein the campastatin analog is linked to the other gene product. Such an arrangement can be advantageous in certain cases, such as when the linkage confers a benefit to the campastatin analog, such as targeting a specific tissue, cell, or intracellular location, or improving one or more other pharmacokinetic parameters.

[0107] In some embodiments, the campastatin analog is fused or conjugated to the N-terminus of the heterologous polypeptide, while in other embodiments it is fused or conjugated to the C-terminus of the heterologous polypeptide. In some embodiments, the fusion protein or protein conjugate comprises a linker (e.g., a flexible linker) between the campastatin analog and the heterologous polypeptide. In some embodiments, the fusion protein or protein conjugate lacks a linker and the campastatin analog is directly fused or conjugated to the heterologous polypeptide. The linker can have any sequence and length that allows each polypeptide to retain biological activity (e.g., not sterically hindered).

[0108] In certain cases, the fusion protein comprises a campastatin analog of the invention and a VEGF control gene product. In addition to vascular endothelial growth factor A (e.g., VEGF-A), such fusion proteins are also capable of specifically binding to C3 and / or C3b. In certain cases, the fusion protein reduces or inhibits angiogenesis compared to a reference polypeptide. In certain cases, the reference polypeptide includes an antibody (or fragment thereof) known as ranibizumab (sold by Genentech under the name).

[0109] Vectors for expressing the campothecin polynucleotides of the present invention contain regulatory elements required for expressing the DNA coding sequences in a host cell, positioned in a manner that permits the expression of the DNA within the host cell. Expression control elements or sequences include appropriate transcriptional initiation, termination, promoter, and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (poly A) signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and optionally sequences that enhance the secretion of the encoded product. The term "expression cassette" is sometimes used herein to describe the campothecin polynucleotides or other transgenes, as well as the expression control elements used to facilitate and control their expression in a host cell.

[0110] In certain embodiments, the campothecin polynucleotides and / or other transgenes of the present invention are carried on viral vectors. Examples of viral vectors include, but are not limited to, retroviral vectors (e.g., Moloney murine leukemia virus (MMLV), Harvey murine sarcoma virus, murine mammary tumor virus, Rous sarcoma virus), adenoviral vectors, adeno-associated virus (AAV) vectors, SV40-type viral vectors, polyomavirus vectors, Epstein-Barr virus vectors, papillomavirus vectors, herpesvirus vectors, vaccinia virus vectors, and poliovirus vectors.

[0111] In certain embodiments, the selected vector is a viral vector primarily used for gene therapy. The campothecin polynucleotides and / or other transgenes can be incorporated into any type of viral vector used in gene therapy, such as recombinant retroviruses, adenoviruses, adeno-associated viruses (AAV), and herpes simplex virus-1.

[0112] Retroviruses are enveloped viruses belonging to the family Retroviridae. Once inside a host cell, the virus replicates by transcribing its RNA into DNA using viral reverse transcriptase. The retroviral DNA replicates as part of the host genome and is called a provirus. Selected nucleic acids can be inserted into the vector and packaged into retroviral particles using techniques known in the art. Protocols for producing replication-defective retroviruses are known in the art.

[0113] In some embodiments, the retrovirus is a lentivirus. Lentiviruses include human immunodeficiency virus (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia (EIA), and visna virus. Vectors derived from lentiviruses can achieve significant levels of nucleic acid transfer in vivo and have been used for in vivo delivery to the eye (Campochiaro et al., 2017, Hum Gene Ther. 28:99-111).

[0114] Viral vectors based on herpes simplex virus (HSV) are also suitable for the uses provided herein. Many replication-deficient HSV vectors contain deletions that remove one or more immediate-early genes to prevent replication. The advantage of herpes vectors is that they can enter a latent state, resulting in long-term DNA expression, and their large viral DNA genome, which can accommodate up to 25 kb of foreign DNA.

[0115] In some embodiments, the vector is an adenovirus vector. Adenoviruses are a large group of viruses that contain double-stranded DNA. They replicate in the host cell nucleus, using the host cell machinery to synthesize viral RNA, DNA, and proteins. Adenoviruses are known in the art to infect both replicating and non-replicating cells, accommodate large transgenes, and encode proteins without integrating into the host cell genome. By deleting selected genes required for viral replication, the virus can be made replication-deficient. The dispensable non-essential E3 region is also often deleted to make additional space for larger DNA inserts. The adenovirus on which the viral vector may be based can be from any source, any subgroup, any subtype, a mixture of subtypes, or any serotype. For example, the adenovirus can be subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22 - 30, 32, 33, 36 - 39, and 42 - 48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), an unclassified subgroup (e.g., serotypes 49 and 51), or any other adenovirus serotype. Adenovirus serotypes 1 to 51 can be obtained from the American Type Culture Collection (Manassas, VA). Non-group C adenoviruses, and even non-human adenoviruses, can be used to prepare replication-deficient adenovirus vectors.

[0116] In certain embodiments, recombinant AAV (rAAV) is the selected vector. AAV particles contain a linear single-stranded AAV nucleic acid genome bound to an AAV capsid protein shell. AAV cannot replicate without a helper virus, which can be an adenovirus, vaccinia, or herpesvirus. In the absence of a helper virus, AAV inserts its genome into the host cell chromosome, presenting a latent state. Subsequent infection with the helper virus rescues the latent integrated copy, which then replicates to produce infectious virus progeny.

[0117] A recombinant adeno-associated virus (rAAV) vector consists of a recombinant viral genome and a capsid protein. The rAAV genome is assembled from polynucleotides encoding the transgene, regulatory elements, and viral elements required for packaging the rAAV genome. Methods for constructing the rAAV genome are known in the art. The AAV expression vector can consist of AAV inverted terminal repeats (ITRs), which are flanked by restriction sites for inserting the transgene either directly using the available restriction sites or by excising the transgene with a restriction enzyme, then polishing the ends and optionally ligating it into the AAV expression vector using a linker. The transgene is integrated into the AAV expression vector together with one or more of the above-described expression control elements (including, for example, enhancers, promoters, and / or post-transcriptional regulatory sequences (PREs)) flanking the AAV ITRs.

[0118] Methods for preparing rAAV vectors with specific capsid proteins are known in the art. Briefly, viral particles are prepared by providing in trans the components required to package the rAAV genome in the capsid, or the required components can be provided by engineered host cells. Some or all of the required elements can be under the control of inducible or constitutive promoters. The recombinant AAV genome, rep sequences, cap sequences, and helper functions for producing rAAV can be delivered into packaging host cells using any suitable genetic element (vector). Generally, recombinant AAV is produced by transfecting host cells with a recombinant AAV genome (containing the transgene) to be packaged in the AAV particle, an AAV helper function vector, and a helper function vector. The AAV helper function vector encodes AAV helper function sequences (i.e., rep and cap), which act in trans for productive AAV replication and encapsidation. The helper function vector usually encodes nucleotide sequences of non-AAV-derived viral and / or cellular functions required for AAV replication, including those elements involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly.

[0119] The AAV vectors described herein typically contain an rAAV genome encoding one or more transgenes, which are operably linked to one or more regulatory elements in a manner that permits transcription, translation, and / or expression of the transgene in a target cell or tissue, and are flanked by 5' and 3' ITRs. The length of the ITR sequence is typically about 145 bp. The AAV ITR sequence can be modified, for example, by inserting, deleting, or substituting one or more nucleotides using standard molecular biology techniques, provided that the modification of the ITR sequence does not interfere with the function of the AAV vector. The AAV ITR can be derived from any of several AAV serotypes. The AAV ITR sequences at the 3' and 5' ends can be the same or can be derived from different AAV serotypes.

[0120] Any suitable AAV serotype or combination of AAV serotypes can be used in the methods and compositions described herein. Several AAV serotypes have been characterized, including AAV1, AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11 and variants thereof. In some embodiments, the AAV vector is an AAV2 / 6, AAV2 / 8, or AAV2 / 9 vector (e.g., an AAV6, AAV8, or AAV9 serotype having AAV2 ITRs). Other AAV vectors are described, for example, in Sharma et al., Brain Res Bull. 2010 Feb 15;81(2-3):273. Generally, any AAV serotype can be used to deliver the transgene described herein. However, the serotypes have different tropisms, e.g., they preferentially infect different tissues. In certain embodiments, an AAV serotype that targets the cell type or organ of interest can be employed. For example, serotypes that target the central nervous system (among other targets) include, but are not limited to, AAV1, AAV2, AAV4, AAVS, AAV6, AAV7, AAV8, AAV9, and AAV10. As another example, favorable retinal gene transfer has been observed using serotypes AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, and AAV9. AAV7 and AAV8 have been shown to achieve excellent long-term transduction of the retina and anterior chamber structures (Lebherz C, Maguire A, Tang W, Bennett J, Wilson JM. J Gene Med. 2008 Apr;10(4):375-82. doi:10.1002 / jgm.1126. PMID:18278824).

[0121] A promoter operably linked to a transgene can be inducible or constitutive. Inducible promoters allow for the regulation of gene expression and can be regulated by an exogenous environment or compound. Constitutive promoters are unregulated promoters that allow for the continuous transcription of their associated genes. Examples of inducible promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, and the T7 polymerase promoter system, the ecdysone-inducible promoter system, and the tetracycline repressor-inducible system. Examples of constitutive promoters include the chicken β-actin promoter, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, and the β-actin promoter. In certain embodiments, if it is preferred to express a selected transgene to mimic native expression, the native promoter of the transgene or a fragment thereof can be used.

[0122] In some embodiments, the regulatory sequence confers the ability to express genes in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are known in the art. For example, promoters that are active in the liver include the transthyretin (TTR) gene promoter, the human α1-antitrypsin (hAAT) promoter, the lipoprotein A-I promoter, the hepatitis B virus core promoter, the alpha-fetoprotein (AFP), the human factor IX promoter; the thyroxine-binding globulin (TBG) promoter, the TTR minimal enhancer / promoter, the α-antitrypsin promoter, and the LSP1 promoter. Examples of enhancers that are active in the liver are the apolipoprotein E (ApoE) hepatic control regions 1 (HCR-1) and 2 (HCR-2). As another example, suitable neuron-specific promoters include, but are not limited to, neuron-specific enolase (NSE) (GenBank accession number: X51956) and the human neurofilament light chain promoter (NEFL) (GenBank accession number: L04147). Glial-specific promoters include, but are not limited to, the glial fibrillary acidic protein (GFAP) promoter (GenBank accession number: M65210), the S100 promoter (GenBank accession number: M65210), and the glutamine synthetase promoter (GenBank accession number: X59834).

[0123] The transgene of the gene therapy viral vector described herein can encode a functional version of any protein that causes a disease or disorder in a subject due to certain defects in the endogenous version in the subject, including defects in the expression of the endogenous version.

[0124] Therapeutic proteins also include myophosphorylase, glucocerebrosidase, fibroblast growth factor receptor 3, huntingtin, HFE protein, CFTR, ataxin, VMD2, hemoglobin, phenylalanine hydroxylase, fibrillin, myotonic dystrophy protein kinase, lignoceryl coenzyme A ligase, dystrophin, methyl CpG-binding protein 2, β-hemoglobin, myotubularin, cathepsin A, factor IX, lipoprotein lipase, β-galactosidase, ornithine transcarbamylase, iduronate-2-sulfatase, acid α-glucosidase, UDP-glucuronosyltransferase 1-1, GlcNAc-1-phosphotransferase, GlcNAc-1-phosphotransferase class, mucolipin-1, microsomal triglyceride transfer protein, sphingomyelinase, acid ceramidase, lysosomal acid lipase, α-L-iduronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, N-acetylglucosamine-1-phosphate N-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, α-mannosidase, α-galactosidase A, cystic fibrosis transmembrane conductance regulator, and respiratory proteins.

[0125] As a further example, therapeutic proteins also include functional versions of proteins associated with the following disorders: disorders of lipid and sphingolipid degradation (e.g., beta-galactosidase-1, beta-hexosaminidase A and B, GM2 activator protein, beta-galactosidase A, glucocerebrosidase, glucocerebrosidase, glucocerebrosidase, arylsulfatase A, galactosylceramidase, sphingomyelinase, sphingomyelinase, NPC1, HEI protein (cholesterol transport defect), acid ceramidase, and lysosomal acid lipase); disorders of mucopolysaccharide degradation (e.g., L-iduronidase, L-iduronidase, L-iduronidase, iduronate sulfatase, heparan N-sulfatase, N-acetylglucosaminidase, acetyl-CoA-glucosaminidase, acetyltransferase, N-acetylglucosamine 6-sulfatase, galactosamine 6-sulfatase, arylsulfatase B, glucuronidase); disorders of glycoprotein degradation (e.g., mannosidase, mannosidase, alpha-fucosidase, aspartylglucosaminidase, neuraminidase, lysosomal protective protein, lysosomal beta-N-acetylgalactosaminidase, lysosomal beta-N-acetylgalactosaminidase); lysosomal storage diseases (e.g., palmitoyl-protein thioesterase (at least 4 subtypes), lysosomal membrane protein, glucose-6-phosphatase, glucose-6-phosphate translocase, acid maltase, debranching enzyme amylo-1,6-glucosidase, N-acetylglucosamine-1-phosphate transferase, N-acetylglucosamine-1-phosphate transferase, ganglioside sialidase (neuraminidase), lysosomal cystine transporter, lysosomal cystine transporter, lysosomal cystine transporter, sialic acid transporter saposin A, B, C, D) and leukodystrophies (e.g., microsomal triglyceride transfer protein / apolipoprotein B, peroxisomal membrane translocation protein, peroxisome biogenesis protein, aspartoacylase, sterol-27-hydroxylase, proteolipid protein, ABC1 transporter, peroxisomal membrane protein 3 or peroxisome biogenesis factor 1, phytanoyl-CoA hydroxylase).

[0126] The viral vectors described herein can be used for gene editing. In such embodiments, the transgene of the viral vector is a gene editing transgene. Such a transgene encodes a factor or component involved in the gene editing process. Generally, this process results in a long-term or permanent modification of genomic DNA, such as targeted DNA insertion, replacement, mutagenesis, or removal. Gene editing can include delivering a nucleic acid encoding a DNA sequence of interest and using an endonuclease to insert the sequence of interest at a targeted site in genomic DNA. Thus, a gene editing transgene can include such nucleic acids encoding the DNA sequence of interest for insertion. In some embodiments, the DNA sequence for insertion is a DNA sequence encoding any one of the therapeutic proteins described herein. Additionally or alternatively, the gene editing transgene can include a nucleic acid encoding one or more components that can perform the gene editing process alone or in combination with other components, as known in the art.

[0127] The viral vectors described herein can be used for gene expression regulation. In such embodiments, the transgene of the viral vector is a transgene that regulates gene expression. Such a transgene encodes a gene expression regulator that can enhance, inhibit (e.g., silence), or modulate the expression of one or more endogenous genes. The endogenous genes can encode any one of the proteins described herein, provided that the protein is endogenous to the subject. Thus, the subject may have any one of the diseases or disorders described herein, in which gene expression regulation would be beneficial.

[0128] Gene expression regulators include DNA-binding proteins (such as artificial transcription factors and the transcriptional silencing protein NRF) and therapeutic RNAs. Therapeutic RNAs include mRNA translation inhibitors (antisense), RNA interference agents (RNAi), catalytically active RNA molecules (ribozymes), and RNAs that bind proteins and other molecular ligands (aptamers), among others.

[0129] Exemplary transgenes encode interfering RNAs, antisense RNAs, ribozymes, and aptamers that reduce the level of angiogenic factors in cells. For example, the RNAi can be a miRNA, shRNA, or siRNA that reduces the level of vascular endothelial growth factor (VEGF) in cells. For example, the RNAi can be a shRNA or siRNA that reduces the level of VEGF or VEGF receptor (VEGFR) in cells. RNAi reagents targeting VEGF include, for example, the RNAi described in U.S. Patent Publication No. 2011 / 0224282. For example, siRNAs specific for VEGF-A, VEGFR1, or VEGFR2 would be suitable. Suitable nucleic acid gene products also include ribozymes specific for VEGF-A, VEGFR1, or VEGFR2, antisense specific for VEGF-A, VEGFR1, or VEGFR2, siRNAs specific for VEGF-A, VEGFR1, or VEGFR2, and the like. miRNAs that reduce VEGF levels by regulating VEGF gene expression (e.g., by post-transcriptional repression or mRNA degradation) are also suitable as gene products. Examples of suitable miRNAs include, for example, miR-15b, miR-16, miR-20a, and miR-20b. See, for example, Hua et al., (2006) PLoS ONE 1:e116. Also suitable are anti-VEGF aptamers (e.g., EYEOOI). Regarding anti-VEGF aptamers, see, for example, Ng et al., (2006) Nature Reviews Drug Discovery 5:123; and U.S. Patents Nos. 6,426,335, 6,168,778, 6,147,204, 6,051,698, and 6,011,020. For example, the aptamer can be directed against VEGF 16s, which is the isoform primarily responsible for pathological ocular neovascularization and vascular permeability.

[0130] In some embodiments, the transgene encodes a polypeptide (e.g., an antibody or a fusion protein) that inhibits or reduces the activity of a pathogenic or disease-promoting polypeptide. For example, in some embodiments, the transgene encodes an anti-angiogenic polypeptide, including, for example, a vascular endothelial growth factor (VEGF) antagonist. Suitable VEGF antagonists include, but are not limited to, inhibitors of VEGFR1 tyrosine kinase activity, inhibitors of VEGFR2 tyrosine kinase activity, antibodies against VEGF, antibodies against VEGFR1, antibodies against VEGFR2, soluble VEGFR, etc. Antibodies specific for VEGF include, for example, bevacizumab (AVASTIN TM ) and ranibizumab (also known as rhuFAb V2). Also suitable for use are anti-angiogenic polypeptides such as endostatin, PEDF, and angiostatin.

[0131] Angiogenesis-inhibiting polypeptides include, for example, recombinant polypeptides comprising VEGF receptors. For example, a suitable angiogenesis-inhibiting polypeptide can be a soluble form of VEGFR-1, known as sFlt-1 (Kendall et al., (1996) Biochem. Biophys. Res Commun. 226:324). Suitable angiogenesis-inhibiting polypeptides also include the immunoglobulin-like (Ig) domain 2 of the first VEGF receptor (e.g., Flt1), alone or in combination with the Ig domain 3 of the second VEGF receptor (e.g., Flk1 or Flt4); the angiogenesis-inhibiting polypeptide can also include stabilizing and / or multimerizing components. Such recombinant angiogenesis-inhibiting polypeptides are described, for example, in U.S. Patent No. 7,521,049. Anti-VEGF antibodies suitable as heterologous gene products include single-chain Fv (scFv) antibodies. See, for example, U.S. Patent Nos. 7,758,859 and 7,740,844 (for anti-VEGF antibodies). Additional transgenes are described, for example, in Bordet et al., Drug Discov Today. 2019 Jun 5. pii:S1359-6446(18)30472-0 doi:10.1016 / j.drudis.2019.05.038. Such transgenes can be used to treat ocular disorders, such as age-related macular degeneration.

[0132] In certain embodiments, the campstatin analogs described herein are pre-prepared (i.e., not in the patient's cells or tissues) and incorporated into pharmaceutical compositions for use in the various combination therapies described herein. These campstatin analogs can be prepared by various synthetic methods of peptide synthesis, by condensation of one or more amino acid residues, according to conventional peptide synthesis methods.

[0133] Alternatively, given that the campstatin analogs disclosed herein are composed of naturally occurring amino acids, they can be produced by expressing the encoding polynucleotide in a suitable prokaryotic or eukaryotic system. For example, a DNA construct can be inserted into a plasmid vector suitable for expression in bacterial cells (e.g., Escherichia coli) or yeast cells (e.g., Saccharomyces cerevisiae), or into a baculovirus vector for expression in insect cells or a viral vector for expression in mammalian cells. Such vectors contain the regulatory elements required for expressing the DNA in the host cell, positioned in a manner that allows the DNA to be expressed within the host cell. Such regulatory elements required for expression are well known in the art and include promoter sequences, transcription initiation sequences, and optionally enhancer sequences. Peptides produced by gene expression in recombinant prokaryotic or eukaryotic systems can be purified by methods known in the art.

[0134] Pharmaceutical Compositions and Their Administration:

[0135] For the purpose of practicing one or more of the uses mentioned herein, another aspect of the invention relates to pharmaceutical compositions comprising a vector carrying the polynucleotide of a campstatin analog described and exemplified herein. Such pharmaceutical compositions may include an active ingredient (e.g., a viral vector containing a transgene) in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these ingredients. These pharmaceutical compositions may be delivered to a subject to permit the production of the polypeptide and the encoded protein. In certain embodiments, the pharmaceutical composition contains sufficient genetic material to enable the recipient to produce a therapeutically effective amount of the protein in the subject.

[0136] The dosage can vary and depends on the type, onset, progression, severity, frequency, duration or likelihood of the disease being treated, the desired clinical endpoint, previous or concurrent treatments, the overall health, age, gender, race or immune competence of the subject, and other factors that will be understood by the skilled artisan. The amount, quantity, frequency or duration of administration can be increased or decreased proportionally, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy and the status of the subject. The skilled artisan will understand the factors that can affect the dosage and timing required to provide an amount sufficient to provide a therapeutic or prophylactic benefit.

[0137] The dosage to achieve a therapeutic effect, e.g., the dosage of a viral vector such as rAAV in vector genomes per kilogram body weight (vg / kg), will vary depending on several factors, including but not limited to the route of administration, the choice of AAV vector serotype, the viral transduction efficiency in the selected tissue of interest, the level of heterologous polynucleotide expression required to achieve a therapeutic effect, the specific disease being treated, any host immune response to the viral vector, the host immune response to the heterologous polynucleotide or the expressed product (protein), and / or the stability of the expressed protein. Those skilled in the art can determine the dosage range of the vector genome or non-viral vector based on the above factors and other factors to treat patients suffering from a specific disease or disorder.

[0138] In embodiments utilizing an rAAV vector, the viral vector dosage will range from at least 1x10 8 vector genomes per kilogram (vg / kg) of subject body weight or more, e.g., 1x10 9 、1x10 10 、1x10 11 、1x10 12 、1x10 13 or 1x10 14One or more vector genomes per kilogram (vg / kg) of subject body weight to achieve a therapeutic effect. Specifically, the dose range of recombinant AAV is from about 1x10 11 to about 5x10 13 vg / kg, and within this range up to about 2x10 11 , or about 3x10 11 , or about 4x10 11 , or about 5x10 11 , or about 6x10 11 , or about 7x10 11 , or about 8x10 11 , or about 9x10 11 , or about 1x10 12 , or about 2x10 12 , or about 3x10 12 , or about 4x10 12 , or about 5x10 12 , or about 6x10 12 , or about 7x10 12 , or about 8x10 12 , or about 9x10 12 , or about 1x10 13 , or about 2x10 13 , or about 3x10 13 , or about 4x10 13 recombinant AAV vg / kg.

[0139] As used herein, a "dose unit" refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount, optionally combined with a pharmaceutical carrier, the predetermined amount being calculated to produce the desired effect (e.g., a prophylactic or therapeutic effect) when administered in one or more doses. In various embodiments, the dose unit can be contained in ampoules and vials, which can include liquid compositions or compositions in a lyophilized or freeze-dried state; for example, a sterile liquid carrier can be added before in vivo administration or delivery. A single dose unit can be contained in a multi-dose kit or container. Viral particles, non-viral vectors, and their pharmaceutical compositions can be packaged in single or multiple unit dosage forms for ease of administration and dose uniformity.

[0140] In certain embodiments, the dose is calculated based on the amount of therapeutic gene product produced in the target cells or tissues of the subject. As described above, among other things, such doses will vary depending on the gene product and the disease being treated. However, given the production of the campstatin analogs of the present disclosure, the dose issues of these peptides can thus be addressed.

[0141] The pharmaceutical compositions useful in practicing the present invention can be administered to deliver a carrier in an amount sufficient to produce a dose of a campstatin analog between about 0.0005 mg / kg and 50 mg / kg body weight. In certain embodiments, the dose comprises at least 0.0005 mg / kg, or at least 0.001 mg / kg, or at least 0.002 mg / kg, or at least 0.005 mg / kg, or at least 0.01 mg / kg, or at least 0.02 mg / kg, or at least 0.04 mg / kg, or at least 0.05 mg / kg, or at least 0.1 mg / kg, or at least 0.2 mg / kg, or at least 0.3 mg / kg, or at least 0.4 mg / kg, or at least 0.5 mg / kg, or at least 0.6 mg / kg, or at least 0.7 mg / kg, or at least 0.8 mg / kg, or at least 0.9 mg / kg, or at least 1 mg / kg, or at least 2 mg / kg, or at least 3 mg / kg, or at least 4 mg / kg (14.9 uM), or at least 5 mg / kg, or at least 6 mg / kg, or at least 7 mg / kg, or at least 8 mg / kg, or at least 9 mg / kg, or at least 10 mg / kg, or at least 15 mg / kg, or at least 20 mg / kg, or at least 25 mg / kg, or at least 30 mg / kg, or at least 35 mg / kg, or at least 40 mg / kg, or at least 45 mg / kg or at least 50 mg / kg, on a daily basis or using another suitable periodic regimen.

[0142] In one embodiment, the present invention contemplates the intravenous or subcutaneous administration of a vector containing a campstatin analog polynucleotide as described herein to produce a dose of campstatin analog in the target cell or tissue between about 0.0005 mg / kg and about 10 mg / kg, such as 0.0005 mg / kg, 0.001 mg / kg, 0.002 mg / kg, 0.005 mg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.125 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 1.25 mg / kg, 1.5 mg / kg, 1.75 mg / kg, 2 mg / kg (14.9 uM), 2.25 mg / kg, 2.5 mg / kg, 2.75 mg / kg, 3 mg / kg, 3.25 mg / kg, 3.5 mg / kg, 3.75 mg / kg, 4 mg / kg, 4.25 mg / kg, 4.5 mg / kg, 4.75 mg / kg, 5 mg / kg, 5.25 mg / kg, 5.5 mg / kg, 5.75 mg / kg, 6 mg / kg, 6.25 mg / kg, 6.5 mg / kg, 6.75 mg / kg, 7 mg / kg, 7.25 mg / kg, 7.5 mg / kg, 7.75 mg / kg, 8 mg / kg, 8.25 mg / kg, 8.5 mg / kg, 8.75 mg / kg, 9 mg / kg, 9.25 mg / kg, 9.5 mg / kg, 9.75 mg / kg or 10 mg / kg. In certain embodiments, the vector is administered by intravenous or subcutaneous delivery (e.g., injection or infusion) to produce a dose of campstatin analog between about 0.25 mg / kg and about 5 mg / kg. In another embodiment, the dose is between about 0.5 mg / kg and about 5 mg / kg. In yet another embodiment, the dose is between about 0.5 mg / kg and 4 mg / kg or between about 0.5 mg / kg and about 3 mg / kg.

[0143] In another embodiment, the present invention contemplates intramuscular administration of a vector containing a calpain inhibitor analog polynucleotide to produce a dose between about 0.0005 mg / kg and about 50 mg / kg, such as 0.0005 mg / kg, 0.001 mg / kg, 0.002 mg / kg, 0.005 mg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.125 mg / Kg, 0.25 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / kg, 10.5 mg / kg, 11 mg / kg, 11.5 mg / kg, 12 mg / kg, 12.5 mg / kg, 13 mg / kg, 13.5 mg / kg, 14 mg / kg, 14.5 mg / kg, 15 mg / kg, 15.5 mg / kg, 16 mg / kg, 16.5 mg / kg, 17 mg / kg, 17.5 mg / kg, 18 mg / kg, 18.5 mg / kg, 19 mg / kg, 19.5 mg / kg, 20 mg / kg, 20.5 mg / kg, 21 mg / kg, 21.5 mg / kg, 22 mg / kg, 22.5 mg / kg, 23 mg / kg, 23.5 mg / kg, 24 mg / kg, 24.5 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg or 50 mg / kg in a target cell or tissue. In a preferred embodiment, the vector is administered by intramuscular delivery (e.g., injection) in an amount sufficient to produce a dose of the calpain inhibitor analog between about 0.25 mg / kg and about 35 mg / kg. In another embodiment, the dose is between about 0.25 mg / kg and 30 mg / kg, or between about 0.25 mg / kg and 10 mg / kg, or between about 0.25 mg / kg and 5 mg / kg. For example, in one particular embodiment, the expected dose of the calpain inhibitor analog is about 2.5 mg / kg.

[0144] In yet another embodiment, the vector is introduced by intravitreal administration to produce a dose of the campastatin analog in the vitreous of the eye between about 1 μg and about 10 mg (931 uM), such as 1 μg, 1.25 μg, 1.5 μg, 1.75 μg, 2 μg, 2.25 μg, 2.5 μg, 2.75 μg, 3 μg, 3.25 μg, 3.5 μg, 3.75 μg, 4 μg, 4.25 μg, 4.5 μg, 4.75 μg, 5 μg, 5.25 μg, 5.5 μg, 5.75 μg, 6 μg, 6.25 μg, 6.5 μg, 6.75 μg, 7 μg, 7.25 μg, 7.5 μg, 7.75 μg, 8 μg, 8.25 μg, 8.5 μg, 8.75 μg, 9 μg, 9.25 μg, 9.5 μg, 9.75 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg or 10 mg; preferably, the dose is between about 1 μg and about 2000 μg, such as between about 10 μg and about 1800 μg, or between about 100 μg and about 1500 μg, or between about 500 μg and about 1200 μg, or between about 500 μg and about 1000 μg. In some embodiments, the therapeutically effective dose of the campastatin analog in the vitreous is at least about 0.02 mg, such as at least about 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg or 1 mg. Similar doses can be used for subretinal delivery.

[0145] In another embodiment, a pharmaceutical composition containing campstatin or its encoded polypeptide can be delivered to the central nervous system using a dosing range within the scope described herein. For example, studies have shown that intrasubstance injection of rAAV can result in local distribution of rAAV, which is suitable for treating CNS diseases affecting specific regions of the brain (such as the putamen in Parkinson's disease). As another example, more extensive CNS distribution can be achieved by intrathecal injection into the cerebrospinal fluid space (see Nat Rev Drug Discov. 2019 May; 18(5):358-378).

[0146] In another embodiment, the present invention contemplates oral administration of the pharmaceutical compositions described herein. Although the oral route is not a typical route for vector-based gene therapy, it can be suitable for combination therapies involving administration of a peptide by one route and a vector by another route. Here, the dose range of the peptide is between about 1 mg / kg and about 20 mg / kg, such as 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / kg, 10.5 mg / kg, 11 mg / kg, 11.5 mg / kg, 12 mg / kg, 12.5 mg / kg, 13 mg / kg, 13.5 mg / kg, 14 mg / kg, 14.5 mg / kg, 15 mg / kg, 15.5 mg / kg, 16 mg / kg, 16.5 mg / kg, 17 mg / kg, 17.5 mg / kg, 18 mg / kg, 18.5 mg / kg, 19 mg / kg, 19.5 mg / kg or 20 mg / kg. In a preferred embodiment, the campstatin analog peptide is administered orally at a therapeutically effective dose between about 1 mg / kg and about 10 mg / kg. For example, in a particular embodiment, the peptide is orally delivered to humans at a dose of about 1 to 5 mg / kg. In some embodiments, the oral doses described herein are administered once. In other embodiments, it is administered regularly, such as daily.

[0147] In another embodiment, the present invention contemplates the periodontal administration of the vector encoding the campstatin analogs described herein, such as infiltration within the interdental papilla, in a dose between about 1 μg and about 1000 μg, such as 1 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, 300 μg, 310 μg, 320 μg, 330 μg, 340 μg, 350 μg, 360 μg, 370 μg, 380 μg, 390 μg, 400 μg, 410 μg, 420 μg, 430 μg, 440 μg, 450 μg, 460 μg, 470 μg, 480 μg, 490 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg or 1000 μg. For example, the vector can be periodontally administered to a human to produce a campstatin analog in a dose between about 5 μg and about 500 μg. In certain embodiments, the vector is periodontally administered to a human to produce a campstatin analog in a dose between about 10 μg / interdental papilla and about 200 μg / interdental papilla or in a dose between about 20 μg / interdental papilla and about 100 μg / interdental papilla.

[0148] In one embodiment, the dosage of the vehicle results in a serum concentration of the campstatin analog in an individual between about 0.01 nM and about 30 μM. In certain embodiments, the dosage and regimen of the combination will result in a serum concentration or average serum concentration over time of the campstatin analog of at least about 0.01 nM, or at least about 0.02 nM, or at least about 0.03 nM, or at least about 0.04 nM, or at least about 0.05 nM, or at least about 0.06 nM, or at least about 0.07 nM, or at least about 0.08 nM, or at least about 0.09 nM, or at least about 0.1 nM, 0.11 nM, or at least about 0.12 nM, or at least about 0.13 nM, or at least about 0.14 nM, or at least about 0.15 nM, or at least about 0.16 nM, or at least about 0.17 nM, or at least about 0.18 nM, or at least about 0.19 nM, or at least about 0.2 nM, or at least about 0.3 nM, or at least about 0.4 nM, or at least about 0.5 nM, or at least about 0.6 nM, or at least about 0.7 nM, or at least about 0.8 nM, or at least about 0.9 nM, or at least about 1 nM or at least about 1.5 nM, or at least about 2 nM, or at least about 2.5 nM, or at least about 3 nM, or at least about 3.5 nM, or at least about 4 nM, or at least about 4.5 nM, or at least about 5 nM, or at least about 5.5 nM, or at least about 6 nM, or at least about 6.5 nM, or at least about 7 nM, or at least about 7.5 nM, or at least about 8 nM, or at least about 8.5 nM, or at least about 9 nM, or at least about 9.5 nM, or at least about 10 nM (0.01 μM).

[0149] In certain embodiments, the combined dosage and regimen will result in a serum concentration or average serum concentration over time of the campstatin analog of at least about 0.01 μM, or at least about 0.02 μM, or at least about 0.03 μM, or at least about 0.04 μM, or at least about 0.05 μM, or at least about 0.06 μM, or at least about 0.07 μM, or at least about 0.08 μM, or at least about 0.09 μM, or at least about 0.1 μM, 0.11 μM, or at least about 0.12 μM, or at least about 0.13 μM, or at least about 0.14 μM, or at least about 0.15 μM, or at least about 0.16 μM, or at least about 0.17 μM, or at least about 0.18 μM, or at least about 0.19 μM, or at least about 0.2 μM, or at least about 0.3 μM, or at least about 0.4 μM, or at least about 0.5 μM, or at least about 0.6 μM, or at least about 0.7 μM, or at least about 0.8 μM, or at least about 0.9 μM, or at least about 1 μM, or at least about 1.5 μM, or at least about 2 μM, or at least about 2.5 μM, or at least about 3 μM, or at least about 3.5 μM, or at least about 4 μM, or at least about 4.5 μM, or at least about 5 μM, or at least about 5.5 μM, or at least about 6 μM, or at least about 6.5 μM, or at least about 7 μM, or at least about 7.5 μM, or at least about 8 μM, or at least about 8.5 μM, or at least about 9 μM, or at least about 9.5 μM, or at least about 10 μM, or at least about 10.5 μM, or at least about 11 μM, or at least about 11.5 μM, or at least about 12 μM, or at least about 12.5 μM, or at least about 13 μM, or at least about 13.5 μM, or at least about 14 μM, or at least about 14.5 μM, or at least about 15 μM, or at least about 15.5 μM, or at least about 16 μM, or at least about 16.5 μM, or at least about 17 μM, or at least about 17.5 μM, or at least about 18 μM, or at least about 18.5 μM, or at least about 19 μM, or at least about 19.5 μM, or at least about 20 μM, or at least about 20.5 μM, or at least about 21 μM or at least about 21.5 μM, or at least about 22 μM, or at least about 22.5 μM, or at least about 23 μM, or at least about 23.5 μM, or at least about 24 μM, or at least about 24.5 μM, or at least about 25 μM, or at least about 25.5 μM, or at least about 26 μM, or at least about 26.5 μM, or at least about 27 μM, or at least about 27.5 μM, or at least about 28 μM, or at least about 28.5 μM, or at least about 29 μM, or at least about 29.5 μM, or at least about 30 μM.In certain embodiments, the combined dosage and regimen will result in a serum concentration or an average serum concentration over time of the campstatin analog of at most about 0.1 μM, or about 0.11 μM, or at most about 0.12 μM, or at most about 0.13 μM, or at most about 0.14 μM, or at most about 0.15 μM, or at most about 0.16 μM, or at most about 0.17 μM, or at most about 0.18 μM, or at most about 0.19 μM, or at most about 0.2 μM, or at most about 0.3 μM, or at most about 0.4 μM, or at most about 0.5 μM, or at most about 0.6 μM, or at most about 0.7 μM, or at most about 0.8 μM, or at most about 0.9 μM, or at most about 1 μM or at most about 1.5 μM, or at most about 2 μM, or at most about 2.5 μM, or at most about 3 μM, or at most about 3.5 μM, or at most about 4 μM, or at most about 4.5 μM, or at most about 5 μM, or at most about 5.5 μM, or at most about 6 μM, or at most about 6.5 μM, or at most about 7 μM, or at most about 7.5 μM, or at most about 8 μM, or at most about 8.5 μM, or at most about 9 μM, or at most about 9.5 μM, or at most about 10 μM, or at most about 10.5 μM, or at most about 11 μM, or at most about 11.5 μM, or at most about 12 μM, or at most about 12.5 μM, or at most about 13 μM, or at most about 13.5 μM, or at most about 14 μM, or at most about 14.5 μM, or at most about 15 μM, or at most about 15.5 μM, or at most about 16 μM, or at most about 16.5 μM, or at most about 17 μM, or at most about 17.5 μM, or at most about 18 μM, or at most about 18.5 μM, or at most about 19 μM, or at most about 19.5 μM, or at most about 20 μM, or at most about 20.5 μM, or at most about 21 μM, or at most about 21.5 μM, or at most about 22 μM, or at most about 22.5 μM, or at most about 23 μM, or at most about 23.5 μM, or at most about 24 μM, or at most about 24.5 μM, or at most about 25 μM, or at most about 25.5 μM, or at most about 26 μM, or at most about 26.5 μM, or at most about 27 μM, or at most about 27.5 μM, or at most about 28 μM, or at most about 28.5 μM, or at most about 29 μM, or at most about 29.5 μM, or at most about 30 μM.

[0150] Suitable ranges include from about 0.1 to about 30 μM, or from about 1 to about 29 μM, or from about 2 to about 28 μM, or from about 3 to about 27 μM, or from about 4 to about 26 μM, or from about 5 to about 25 μM, or from about 6 to about 24 μM, or from about 7 to about 23 μM, or from about 8 to about 22 μM, or from about 9 to about 21 μM, or from about 10 to about 20 μM, or from about 11 to about 19 μM, or from about 12 to about 18 μM, or from about 13 to about 17 μM, or from about 1 to about 5 μM, or from about 5 to about 10 μM, or from about 10 to about 15 μM, or from about 15 to about 20 μM, or from about 20 to about 25 μM, or from about 25 to about 30 μM. Although the exact dosage administered will vary depending on many factors including, but not limited to, the type of patient and the type of disease state being treated, the age of the patient, and the route of administration, such dosages can be readily determined by one of ordinary skill in the art.

[0151] Accordingly, in certain embodiments, a pharmaceutical composition comprising a kampstatine analog transgene and / or other transgenes and / or a kampstatine and derivative containing natural amino acids produced by other means and other therapeutic molecules is administered by subcutaneous, intradermal, intravenous, intraocular (including intravitreal, subretinal), intracerebral, intraperitoneal, intramuscular injection, periodontal administration (including gingival administration or infiltration injection within the interdental papilla), intranasal, epidural, oral, sublingual, intrathecal, intravaginal, transdermal, rectal, inhalation, or topical administration.

[0152] The formulations of the pharmaceutical compositions can be prepared by any method known or hereafter developed in the pharmaceutical arts. In general, such preparation methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single-dose or multi-dose unit.

[0153] The pharmaceutical compositions described herein can be administered to a patient several times per day or less frequently, such as once per day, once per week, once every two weeks, once per month, or even less frequently, such as once every few months or even once per year or less. The dosing frequency will be apparent to the skilled artisan and will depend on a variety of factors as described above, including but not limited to the type and severity of the disease being treated, the type and age of the patient. However, as described above, the vectors producing the kampstatine analogs of the present disclosure can be administered at less frequent intervals compared to previously known kampstatine analogs.

[0154] For example, in some embodiments, administration of a pharmaceutical composition containing a vector described herein, by intravenous, intramuscular, intraocular (including intravitreal), subcutaneous, periodontal (including gingival administration or infiltration within the interdental papilla), or topical routes is effected by a single injection. Additionally, in view of the extended residence time of the campstatin analogs described herein, certain embodiments contemplate long-term systemic administration of these campstatin analog polynucleotides, such as by intravenous, intraocular (including intravitreal and subretinal), subcutaneous, intramuscular, periodontal (including gingival administration or infiltration within the interdental papilla), or topical routes, with multiple deliveries over time at the above-described therapeutic doses, in order to provide a therapeutically effective maintenance dose of the campstatin analogs and other gene therapies, depending on the type and age of the patient and the type and severity of the disease being treated. Thus, in some embodiments, the vector is delivered at a frequency of from about once every 12 hours to once every three months or even once every 5 - 6 months, such as once every 12 hours, once every 24 hours, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 2 weeks, once every 3 weeks, once a month, once every 2 months, once every 3 months, or once every 5 - 6 months. In other embodiments, the vector is delivered at a frequency of from about once every 12 hours to about once every three months, such as once every 12 hours, once every 24 hours, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 2 weeks, once every 3 weeks, once a month, once every 2 months, once every 3 months.

[0155] As described above, pharmaceutical compositions containing the campstatin analogs and / or other gene therapy encoding vectors and / or naturally occurring amino acid-containing campstatins and derivatives and other therapeutic molecules produced by any other means can be formulated for administration by a variety of routes. Such pharmaceutical compositions can contain pharmaceutically acceptable carriers and other ingredients known to enhance and facilitate drug administration. Other formulations such as nanoparticles, liposomes, resealed erythrocytes, and immune-based systems can also be used to administer gene therapy vectors in accordance with the methods of the present invention.

[0156] Pharmaceutical compositions suitable for injectable use generally include sterile aqueous solutions (when water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiologic saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), phosphate buffered saline (PBS), or Ringer's solution.

[0157] Sterile non-volatile oils are commonly used as solvents or suspension media. For this purpose, any mild non-volatile oil can be used, including synthetic glycerol monoesters or glycerol diesters. Fatty acids such as oleic acid and their glyceride derivatives can be used in the preparation of injectables, as can natural pharmaceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants commonly used in formulating pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants such as Tweens, Spans, and other emulsifiers or bioavailability enhancers commonly used in the preparation of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for formulation purposes.

[0158] Generally speaking, the composition should be sterile and should be fluid so as to be easily injectable. Preferred pharmaceutical formulations are stable under the conditions of preparation and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. Usually, the relevant carrier can be a solvent or dispersion medium that contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and their suitable mixtures. For example, by using coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants, appropriate fluidity can be maintained. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars, polyols like mannitol, sorbitol, sodium chloride, in the composition. Prolonged absorption of injectable compositions can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0159] Sterile injectable solutions can be prepared by incorporating the required amount of the active pharmaceutical agent (the carrier) into a suitable solvent (containing, as required, a combination of one or more of the above ingredients) and then filtering to sterilize. Preferably, the injectable solution is endotoxin-free. Usually, dispersions are prepared by incorporating the active pharmaceutical agent into a sterile vehicle that contains a basic dispersion medium and the other required ingredients mentioned above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional required ingredients from a previously sterile-filtered solution.

[0160] For topical application, the pharmaceutical composition can be formulated into a suitable ointment containing the pharmaceutically active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the pharmaceutical composition can be formulated into a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0161] For topical delivery to the eye, the pharmaceutical composition provided herein can be appropriately formulated in, for example (but not limited to), isotonic, pH-adjusted sterile saline or water, which may or may not contain a preservative such as benzalkonium chloride. Alternatively, for ophthalmic use, the pharmaceutical composition can be formulated into an ointment, such as petrolatum, or eye drops.

[0162] Methods of topical administration to the eye include, for example, choroidal injection, transscleral injection or placement of a scleral patch, selective arterial catheterization, eye drops or eye ointments, intraocular administration (including transretinal, subconjunctival, intravitreal injection, suprachoroidal injection, subtenon injection, scleral pocket and scleral incision injection), via an osmotic pump, etc. The carrier or the natural amino acid-containing camptastatin peptide generated by other means can also optionally be administered intravascularly, such as intravenously (IV) or intraarterially. In choroidal injection and placement of a scleral patch, the clinician or operator performs local treatment on the eye after initiating appropriate anesthesia (including analgesics and mydriatics). A needle containing the pharmaceutical composition is introduced into and inserted into the choroid or sclera of the subject under aseptic conditions. When the needle is correctly positioned, the composition is injected into either or both of the choroid or sclera. When using any of these methods, the clinician or operator can select a sustained-release or longer-acting formulation. Thus, the procedure can be repeated only every few months or years, depending on the subject's tolerance and response to the treatment.

[0163] Intraocular administration of drugs is well known in the art. See, for example, U.S. Patent Nos. 5,632,984 and 5,770,589 and U.S. Publication No. 2016 / 0060297A1. U.S. Patent No. 6,378,526 provides a method of injecting a therapeutic or diagnostic material into the sclera at a location covering the retina, which provides a minimally invasive technique for delivering an agent to the posterior segment of the eye.

[0164] In certain embodiments, a pharmaceutical composition containing a vector comprising a transgene described herein is delivered near the eye, such as very near the posterior segment of the eye. "Near the eye" refers to a location within the orbit, which is the cavity within the skull in which the eye and its appendages are located. Generally, the composition will be delivered to a location within the eye near its intended target, such as near (within a few millimeters) the portion of the sclera covering the posterior segment of the eye or adjacent to the outer surface of the sclera. In a preferred embodiment, the pharmaceutical composition of the invention is delivered into the vitreous cavity of the eye (i.e., intravitreally).

[0165] A variety of polymeric delivery vehicles for providing controlled release have been used in the ocular environment and can be used to administer the pharmaceutical compositions of the invention. A variety of polymers can be used, such as biodegradable biocompatible polymers. For example, U.S. Patent No. 6,692,759 describes methods for making implantable devices for providing controlled release of therapeutic agents in the eye. Other useful polymers and delivery systems for ocular administration of therapeutic agents have been described. The active agent can be released as the polymer degrades. Polymers that have been used for drug delivery include, but are not limited to, poly(lactic-co-glycolic acid), polyanhydrides, ethylene-vinyl acetate, polyglycolic acid, chitosan, polyorthoesters, polyethers, polylactic acid, and poly-β-amino esters. Peptides, proteins such as collagen and albumin, and dendrimers (e.g., PAMAM dendrimers) have also been used. Any of these can be used in various embodiments of the invention.

[0166] Polyorthoesters have been introduced into the eye and have been shown to have favorable properties for sustained-release ocular drug delivery (see Einmahl, S., 2002, Invest. Ophthalmol. Vis. Sci. 43(5)). Polylactic acid particles have been used to target agents to the retina and RPE after intravitreal injection of a suspension of such particles (Bourges et al., 2003, Invest. Ophthalmol. Vis. Sci. 44(8)). Macroscopic implantable devices suitable for introduction into the posterior or anterior segment of the eye are referred to herein as ocular implants (see Jaffe, G., 2000, Invest. Ophthalmol. Hs. Sci., 41(11)). Accordingly, provided herein is an ocular implant comprising a vector and / or a campstatin analog polypeptide described herein, and optionally other suitable transgenes and other therapeutic agents for an individual. Such devices can be macroscopic implants containing the pharmaceutical composition or can consist of multiple nanoparticles or microparticles impregnated with or encapsulated with the agent. In one embodiment, the ocular implant is any ocular implant known in the art. For example, exemplary implants and methods for making them are described in US 2009 / 0220572A1. Other implants known in the art can also be used.

[0167] Other embodiments include compositions that form gels and contain soluble collagen, which can be used to deliver therapeutic agents to the posterior segment of the eye. The collagen is initially soluble and forms a low-viscosity solution, but is capable of rapidly forming a gel under appropriate conditions (such as those encountered after administration to a mammalian subject). Thus, the present invention provides a system for delivering a pharmaceutically active agent to the posterior segment of the eye. The system is designed to localize such molecules at a sufficient concentration to provide sustained delivery and, at the same time, allow the macromolecules to be released in sufficient amounts. In addition, the collagen gel can protect the carrier and the proteins generated therefrom from degradation.

[0168] The composition forms a gel after being introduced into the body, for example, after contact with physiological fluids. The composition can also form a gel after contact with a fluid such as phosphate buffered saline or other fluids containing appropriate ions. Thus, the composition can be injected at an appropriate location, such as near the posterior segment of the eye, where it forms a gel. Alternatively, a preformed gel implant can be prepared, for example, by introducing the solution into a mold or cavity of a desired shape and allowing gel formation to occur in the presence of an appropriate concentration of salt. The salt can be added before or after introducing the solution into the mold or cavity. The mold or cavity can be, for example, any structure containing a hollow space or a concave depression into which the solution can be introduced. In another embodiment, a film or membrane is formed from the collagen solution containing the therapeutic agent.

[0169] For the treatment of chronic or acute pulmonary conditions involving complement activation or that can benefit from other gene therapies, the preferred route of administration of the pharmaceutical composition is pulmonary administration. Thus, the pharmaceutical compositions of the present invention can be prepared, packaged, or sold in a formulation suitable for buccal pulmonary administration. Such a formulation can comprise dry particles that contain the active ingredient and have a diameter in the range of about 0.5 to about 7 nanometers, preferably about 1 to about 6 nanometers.

[0170] The pharmaceutical compositions of the present invention formulated for pulmonary delivery can also provide the active ingredient in the form of solution or suspension droplets. Such formulations can be prepared, packaged, or sold as an aqueous or dilute alcoholic solution or suspension (optionally sterile) containing the active ingredient and can be conveniently administered using any vaporization or atomization device. Such formulations can also contain one or more additional ingredients, including but not limited to flavoring agents such as sodium saccharin, volatile oils, buffering agents, surfactants (including alternative lung surfactants), or preservatives such as methylparaben. The droplets provided by this route of administration preferably have an average diameter in the range of about 0.1 to about 200 nanometers.

[0171] The formulations described herein that are useful for pulmonary delivery can also be used for intranasal delivery of the pharmaceutical compositions of the present invention. Another formulation suitable for intranasal administration is a coarse powder formulation that comprises the active ingredient and has an average particle size of from about 0.2 to 500 microns. This formulation is administered in the manner of taking snuff, i.e., by rapid inhalation through the nasal passages from a powder container held close to the nostrils. Formulations suitable for nasal administration can, for example, comprise from as little as about 0.1% (w / w) to as much as 100% (w / w) of the active ingredient and can also comprise one or more of the additional ingredients described herein.

[0172] As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by causing a physical breach in the tissue of a subject and administering the pharmaceutical composition through the breach in the tissue. Thus, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injection of the composition, by surgical incision, by non-surgical wound penetrating the tissue, etc. Specifically, parenteral administration is contemplated to include, but is not limited to, intravenous, subcutaneous, intraperitoneal, intramuscular, intra-articular, intravitreal, intrasternal injection and renal dialysis infusion techniques.

[0173] Formulations of pharmaceutical compositions suitable for parenteral administration comprise the active ingredient in combination with a pharmaceutically acceptable carrier (such as sterile water or sterile isotonic saline). Such formulations can be prepared, packaged or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations can be prepared, packaged or sold in unit dosage form, for example, in an ampoule or in a multi-dose container containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes and implantable sustained release or biodegradable formulations. Such formulations can also comprise one or more additional ingredients including, but not limited to, suspending, stabilizing or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (such as sterile pyrogen-free water) and then parenteral administration of the reconstituted composition.

[0174] The pharmaceutical composition can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. Such suspension or solution can be formulated according to known techniques and, in addition to the active ingredient, can contain additional ingredients described herein, such as dispersing agents, wetting agents, or suspending agents. Such sterile injectable preparations can be prepared using non-toxic parenterally acceptable diluents or solvents, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and non-volatile oils such as synthetic mono- or di-glycerides of fatty acids. Other useful parenterally administrable preparations include those containing the active ingredient in microcrystalline form, in liposomal formulations, in microbubbles for ultrasound-triggered delivery, or as components of biodegradable polymer systems. Compositions for sustained release or implantation can contain pharmaceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, slightly soluble polymers, or slightly soluble salts.

[0175] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants, including alternative pulmonary surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions, such as gelatin; aqueous media and solvents; oily media and solvents; suspending agents; dispersing or wetting agents; emulsifying agents; demulcents; buffering agents; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymers or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed., 1985, Remington's pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

[0176] Use and therapeutic administration of campstatin analog transgenes and campstatins containing natural amino acids:

[0177] The Campastatin analogs, transgenes or peptides containing natural amino acids described herein have practical utility for many purposes. In one embodiment, they can be used for any purpose for which Campastatin and its analogs are used, provided that such use is suitable for the production of said analogs from the transgene. Thus, in certain embodiments, the methods of modulating complement activation are applicable to a living patient or subject and include part or all of a method of treating a patient for a disease or disorder associated with complement activation, particularly alternative pathway (AP)-mediated complement activation, which can amplify complement effector responses and exacerbate inflammatory damage in tissues and cells, regardless of the triggering mechanism of complement activation. Many such pathological conditions are known in the art and include, but are not limited to, atypical hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); C3 glomerulonephritis (C3GN); C3 glomerulopathy; other complement-mediated kidney diseases and glomerular inflammatory diseases; age-related macular degeneration (AMD); any ocular disorder characterized by macular degeneration, choroidal neovascularization (CNV), retinal neovascularization (RNV), proliferative vitreoretinopathy, glaucoma, uveitis, ocular inflammation, or any combination of these diseases; paroxysmal nocturnal hemoglobinuria (PNH); cold agglutinin disease (CAD); warm antibody autoimmune hemolytic anemia (wAIHA); sickle cell disease; transplant-associated thrombotic microangiopathy; rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); several autoimmune and autoinflammatory kidney diseases; autoimmune myocarditis; multiple sclerosis; traumatic brain and spinal cord injury; brain, intestine, and kidney ischemia-reperfusion (IR) injury; spontaneous and recurrent miscarriage; antiphospholipid syndrome (APS); Parkinson's disease; Alzheimer's disease; neurodegenerative inflammatory disorders based on abnormal synaptic remodeling, microglial hyperactivity, and cognitive decline; asthma; antinuclear cytoplasmic antigen-associated pauci-immune vasculitis (Wegener's syndrome); non-lupus autoimmune skin diseases such as pemphigus, bullous pemphigoid, and epidermolysis bullosa; post-traumatic shock; cancer; periodontitis; gingivitis; and atherosclerosis. In certain embodiments, the pathological condition is related to mutations and polymorphisms in genes encoding FH and / or CD46, including, but not limited to: AMD, aHUS, and membranoproliferative glomerulonephritis type II (MPGN-II, also known as dense deposit disease (DDD)). In other embodiments, the Campastatin analogs produced by expressing the polynucleotides described herein are suitable as alternatives to eculizumab or pegcetacoplan for treating diseases for which these agents are currently prescribed or are being developed in preclinical and clinical studies.These diseases include, but are not limited to, aHUS, PNH, C3G (DDD / C3GN), CAD, AMD, NMOSD (neuromyelitis optica spectrum disorder), generalized myasthenia gravis, and amyotrophic lateral sclerosis (ALS).

[0178] Blood-related and vessel-related disorders

[0179] In certain embodiments, a campastatin analog gene transfer is administered to a subject having or at risk of having a complement-mediated blood-related disorder, such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, chronic cold agglutinin disease, HELLP syndrome, and / or warm autoimmune hemolytic anemia. In some embodiments, a campastatin analog gene transfer is administered to a subject having or at risk of having a complement-mediated disorder affecting the circulatory system. For example, in some embodiments, the disorder is thrombotic microangiopathy (TMA) or vasculitis (such as IgA vasculitis), or other disorders associated with vascular inflammation such as vascular and / or lymphatic vessel inflammation. In some embodiments, the vasculitis is polyarteritis nodosa, hypocomplementemic urticarial vasculitis, pulmonary vasculitis, Wegener's granulomatosis, giant cell arteritis, Churg-Strauss syndrome, microscopic polyangiitis, pauci-immune vasculitis, Henoch-Schonlein purpura, Takayasu arteritis, Kawasaki disease, or Behcet's disease. In some embodiments, the disorder is TMA secondary to atypical hemolytic uremic syndrome. In some embodiments, the subject is positive for anti-neutrophil cytoplasmic antibody (ANCA).

[0180] Ocular disorders

[0181] In some embodiments, a campastatin analog gene transfer is administered to a subject to treat a complement-mediated ocular disorder, such as macular degeneration (such as age-related macular degeneration (AMD) or Stargardt macular dystrophy), diabetic retinopathy, glaucoma, or uveitis (such as posterior uveitis or anterior uveitis). In some embodiments, the subject has AMD or is at risk of having AMD. In some embodiments, the AMD is neovascular (wet) AMD. In some embodiments, the AMD is dry AMD. As will be understood by one of ordinary skill in the art, dry AMD encompasses geographic atrophy (GA), intermediate AMD, and early AMD. In some embodiments, subjects with GA are treated to slow or halt disease progression.

[0182] Nervous system disorders

[0183] In some embodiments, a compstatin analog transgene is used to treat a subject having or at risk of having a complement-mediated disorder affecting the nervous system (e.g., the central nervous system (CNS) and / or the peripheral nervous system (PNS)). Examples of such disorders include, for example, neurodegenerative disorders such as multiple sclerosis, other demyelinating diseases (such as neuromyelitis optica or chronic inflammatory demyelinating polyneuropathy (CIDP)), amyotrophic lateral sclerosis, chronic pain, fibromyalgia, stroke, intracerebral hemorrhage, allergic neuritis, acute or recurrent optic neuritis with myelin oligodendrocyte glycoprotein (MOG) or aquaporin (AQP)-4 antibodies, diabetic neuropathy, Huntington's disease, schizophrenia, Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, dementia with Lewy bodies (i.e., Lewy body dementia or Parkinson's disease dementia), frontotemporal dementia, progressive supranuclear palsy, corticobasal syndrome, Pick's disease, mild cognitive impairment, traumatic brain injury, traumatic spinal cord injury, multiple system atrophy, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, Guillain-Barré syndrome, glioblastoma, and leptomeningeal metastases. In some embodiments, the subject has neuropathic pain, for example, caused by lesions involving the somatosensory pathway and damage to small fibers in the peripheral nerves and / or the spinothalamocortical system in the CNS.

[0184] Renal disorders

[0185] In some embodiments, a compstatin analog transgene is used to treat a subject having or at risk of having a complement-mediated renal disorder. Such disorders include, for example, nephritis, such as glomerulonephritis, such as membranoproliferative glomerulonephritis (MPGN) (e.g., MPGN type I, MPGN type II, or MPGN type III), such as immune complex membranoproliferative glomerulonephritis (IC-MPGN). In some embodiments, the disorder is IgA nephropathy (IgAN), primary membranous nephropathy, or diabetic nephropathy. In some embodiments, the disorder is polycystic kidney disease (PKD). In some embodiments, the disorder is C3 glomerulopathy. In some embodiments, the disorder is characterized by glomerular deposits in the kidney containing one or more complement activation products, such as C3b. In some embodiments, treatment as described herein reduces the level of such deposits. In some embodiments, a subject having a complement-mediated renal disorder has proteinuria (abnormally high levels of protein in the urine) and / or an abnormally low glomerular filtration rate (GFR). In some embodiments, treatment as described herein results in a reduction in proteinuria and / or an increase or stabilization of the GFR.

[0186] In these embodiments, the treatment method generally comprises (1) identifying a subject having a disease or disorder that can be treated by complement activation modulation as described above (“complement-mediated” disease, disorder or condition), (2) measuring parameters of the disease or disorder that can be treated by complement activation modulation using standard techniques of the prior art that are well within the skill of the artisan (e.g., biopsy, histology, MRI, bone scan, X-ray, pain tolerance, posture, etc.), (3) administering to the subject an effective amount of the kampstatine analogue transgene of the present invention using a treatment regimen and duration suitable for the disorder being treated, and (4) measuring the parameters of the disease or disorder as an indication that the disease or disorder has been improved or treated. As described herein, delivery of the transgene can be effected by any suitable route of administration known in the art. Development of appropriate dosages and treatment regimens will vary with a variety of factors including, but not limited to, the type of patient and type of disease state being treated, the age of the patient, and the route of administration. The skilled artisan is familiar with the design of dosage regimens that take such variables into account.

[0187] In some embodiments, the transgene encoding the kampstatine analogue can be administered systemically, such as intravenously or subcutaneously, for treating the complement-mediated disorders described herein. In some embodiments, a local route of administration can be used, such as where the disorder primarily affects a particular body system, organ or tissue. For example, in some embodiments, the transgene encoding the kampstatine analogue can be administered intravitreally (e.g., intravitreally) for treating ocular disorders. In some embodiments, the transgene encoding the kampstatine analogue is administered via the pulmonary route (e.g., for treating disorders affecting the respiratory system). In some embodiments, the transgene encoding the kampstatine analogue is administered intrathecally for treating complement-mediated disorders affecting the central nervous system as described herein. In some embodiments, intracisternal or intraventricular administration can be used to treat complement-mediated disorders affecting the central nervous system.

[0188] In another embodiment, a camptastatin analogue transfer gene is introduced into cells of a living host in combination with other gene therapies to enhance viral vector delivery efficacy and performance. Examples of such diseases or disorders include, but are not limited to, lysosomal storage diseases / disorders such as Santavuori-Haltia disease (infantile neuronal ceroid lipofuscinosis type 1), Jansky-Beilschowsky disease (late infantile neuronal ceroid lipofuscinosis type 2), Batten disease (juvenile neuronal ceroid lipofuscinosis type 3), Kufs disease (neuronal ceroid lipofuscinosis type 4), Von Gierke disease (glycogen storage disease type 1a), glycogen storage disease type 1b, Pompe disease (glycogen storage disease type II), Forbes disease or Cori disease (glycogen storage disease type III), mucolipidosis II (I-Cell disease), mucolipidosis III (pseudo-Hurler polydystrophy), mucolipidosis IV (sialolipidosis), cystinosis (adult non-nephropathic), cystinosis (infantile nephropathic), cystinosis (juvenile nephropathic), Salla disease / infantile sialic acid storage disease, and saposin deficiency; disorders of lipid and sphingolipid degradation such as GM1 gangliosidosis (infantile, late infantile / juvenile, and adult / chronic), Tay-Sachs disease, Sandhoff disease, GM2 gangliosidosis, Ab variant, Fabry disease, Gaucher disease types I, II, and III, metachromatic leukodystrophy, Krabbe disease (early and late onset), Niemann-Pick disease types A, B, C1, and C2, Farber disease, and Wolman disease (cholesterol ester storage disease); disorders of mucopolysaccharide degradation such as Hurler syndrome (MPSI), Scheie syndrome (MPSIS), Hurler-Scheie syndrome (MPSIH / S), Hunter syndrome (MPSII), Sanfilippo A syndrome (MPSIIIA), Sanfilippo B syndrome (MPSIIIB), Sanfilippo C syndrome (MPSIIIC), Sanfilippo D syndrome (MPSIIID), Morquio A syndrome (MPSIVA), Morquio B syndrome (MPSIVB), Maroteaux-Lamy syndrome (MPS VI), and Sly syndrome (MPS VII); disorders of glycoprotein degradation such as alpha-mannosidosis, beta-mannosidosis, fucosidosis, aspartylglucosaminuria, mucolipidosis I (sialidosis), galactosialidosis, Schindler disease, and Schindler disease type II / Kanzaki disease; and leukodystrophy diseases / disorders such as abetalipoproteinemia, neonatal adrenoleukodystrophy, Canavan disease, cerebrotendinous xanthomatosis, Pelizaeus-Merzbacher disease, Tangier disease, infantile Refsum disease, and classic Refsum disease.

[0189] Other examples of such diseases / disorders in the subjects described herein include, but are not limited to, acid maltase deficiency (e.g., Pompe disease, glycogen storage disease type II, lysosomal storage disease); carnitine deficiency; carnitine palmitoyltransferase deficiency; debranching enzyme deficiency (e.g., Cori disease or Forbes disease, glycogen storage disease type III); lactate dehydrogenase deficiency (e.g., glycogen storage disease type XI); myoadenylate deaminase deficiency; phosphofructokinase deficiency (e.g., Tarui disease, glycogen storage disease type VII); phosphoglycerate kinase deficiency (e.g., glycogen storage disease type IX); phosphoglycerate mutase deficiency (e.g., glycogen storage disease type X); phosphorylase deficiency (e.g., McArdle disease, myophosphorylase deficiency, glycogen storage disease type V); Gaucher disease (e.g., affected glucocerebrosidase on chromosome 1); achondroplasia (e.g., affected fibroblast growth factor receptor 3 on chromosome 4); Huntington's disease (e.g., huntingtin on chromosome 4); hemochromatosis (e.g., HFE protein on chromosome 6); cystic fibrosis (e.g., CFTR on chromosome 7); Friedreich's ataxia (ataxin on chromosome 9); Best disease (VMD2 on chromosome 11); sickle cell disease (hemoglobin on chromosome 11); phenylketonuria (phenylalanine hydroxylase on chromosome 12); Marfan syndrome (fibrillin on chromosome 15); myotonic dystrophy (myotonic dystrophy protein kinase on chromosome 19); adrenoleukodystrophy (x-linked lignoceroyl-CoA ligase in peroxisomes); Duchenne muscular dystrophy (dystrophin on the x chromosome); Rett syndrome (methyl-CpG-binding protein 2 on the x chromosome); Leber hereditary optic neuropathy (mitochondrial respiratory protein); mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS) (mitochondria, transfer RNA); and enzyme deficiencies of the urea cycle.

[0190] Other examples of such diseases or disorders include, but are not limited to, sickle cell anemia, myotubular myopathy, hemophilia B, lipoprotein lipase deficiency, ornithine transcarbamylase deficiency, Crigler-Najjar syndrome, mucolipidosis IV, Niemann-Pick A, Schaffer lipo A, Schaffer lipo B, Schaffer lipo C, Schaffer lipo D, b-thalassemia, and Duchenne muscular dystrophy. Further examples of diseases or disorders include those caused by defects in lipid and sphingolipid degradation, mucopolysaccharide degradation, glycoprotein degradation, leukodystrophy, and the like.

[0191] The improvement in the efficacy of virus vector-mediated gene therapy can be observed in many ways by co-administering with a Campastatin analogue transgene of the present invention. For example, in some embodiments, compared to an effective dosing regimen used alone for the gene therapy, the co-administration allows the gene therapy to be administered at a reduced dose, reduced number of administrations, and / or reduced dose frequency, and these differences are readily measurable. In this regard, the efficacy of the gene therapy can be evaluated, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, or longer after administration of the therapy.

[0192] In some embodiments, the efficacy of the gene therapy is measured or indicated by the period of no recurrence of disease signs or symptoms.

[0193] In some embodiments, the humoral response and / or cellular response to the virus vector administration can be measured. For example, a decrease in the humoral response is measured or indicated by a decrease in the response magnitude or a decrease in the fold of the antibody (e.g., neutralizing antibody) level compared to the baseline. In some embodiments, the antibody level is the level of antibodies against the virus vector (e.g., capsid protein). In some embodiments, the baseline is a value, level, amount, or quantity measured or indicated in a subject before administration of the gene therapy or in a control subject not receiving the gene therapy. In some embodiments, a decrease in the humoral response is indicated by a decrease in the antibody titer of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% from the baseline.

[0194] The cellular response is indicated or measured by the secretion of granzyme B (GrB) and / or IFNγ. In some embodiments, a decrease in the cellular response is measured or indicated by a decrease in the response magnitude or a decrease in the fold of the GrB and / or IFNγ level compared to the baseline. In some embodiments, a decrease in the cellular response is indicated by a decrease in the GrB and / or IFNγ level of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% from the baseline. In some embodiments, the baseline is a value, level, amount, or quantity measured or indicated in a subject before administration of the gene therapy or in a control subject not receiving the gene therapy.

[0195] In some embodiments, the efficacy of gene therapy is measured by the presence or expression level of the campastatin analog transgene or other transgenes described herein and / or the level or activity of the protein encoded by the transgene. For example, co-delivery of the campastatin analog transgene and other gene therapies results in a level of the transgene in a subject (e.g., in the cells or tissues of the subject) that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200% or more higher than the corresponding level of the transgene in a subject to whom the campastatin analog transgene has not been administered, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months or longer after the combination therapy.

[0196] In some embodiments, the efficacy of gene therapy is measured by the stability of the expression level of the transgene in the subject relative to the corresponding expression level of the transgene in a control subject (e.g., a control subject who has received the gene therapy but has not been administered the campastatin analog transgene) over a period of, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months or longer. In some embodiments, a stable expression level is an expression level that varies by no more than 30%, 25%, 20%, 15%, 10% or 5% over a specified period of time.

[0197] In some embodiments, the efficacy of gene therapy using a transgene encoding an inhibitor of a target gene or polypeptide is measured by the expression level of the target gene and / or the expression and / or activity level of the target polypeptide. In some embodiments, co-therapy with the campastatin analog transgene and other gene therapies results in a level of expression and / or activity of the target gene and / or target polypeptide in a subject (e.g., in the cells or tissues of the subject) that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% lower than the corresponding expression and / or activity level in a subject to whom the campastatin analog transgene has not been administered, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months or longer after the combination therapy.

[0198] In another embodiment, the campastatin analog transgene is used in combination with other transgenes for the combined treatment of a disease or disorder in which both complement activation and other dysfunctions treatable by gene therapy contribute.

[0199] In some embodiments, the combined administration of a campastatin analog transgene and other gene therapies has a greater therapeutic effect on the disease or disorder or its symptoms than the effect produced by the gene therapy or the campastatin analog alone. The difference between the combined effect and the effect of the gene therapy alone or the campastatin analog alone may be a statistically significant difference. In some embodiments, the combined result is synergistic.

[0200] These diseases or disorders can be any one or combination of the diseases and disorders listed herein, where complement activation plays a role and may benefit from one or more of the gene therapies listed above.

[0201] As a specific example, growth factors of the vascular endothelial growth factor (VEGF) family control pathological angiogenesis and increased vascular permeability in important eye diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Complement activation also plays an important role in the pathogenesis of these diseases. Thus, a vector encoding a campastatin analog transgene in combination with a transgene for controlling VEGF can provide a dual mechanism therapy.

[0202] In certain embodiments, a transgene for controlling VEGF, such as those mentioned elsewhere herein, together with the campastatin analog transgene of the present invention, is administered by a viral vector as described herein. The campastatin analogs produced by the expression of the transgene in target cells and tissues have a dual function: (1) they reduce the immunogenicity of the viral vector, thereby increasing the survival rate of the vector and the production of the gene product for controlling VEGF; and (2) they directly treat the underlying disease or disorder.

[0203] The transgene for controlling VEGF and the campastatin analog transgene can be incorporated into one or more than one viral vector. In one embodiment, the two transgenes are contained on a single viral vector. In another embodiment, the campastatin analog transgene and the transgene for controlling VEGF are arranged to produce a fusion protein, where the campastatin analog is linked to the gene product for controlling VEGF. The linkage can be direct, or there may be a spacer or linker between the campastatin analog and the gene product for controlling VEGF.

[0204] In some embodiments, the transgenes encoding the campastatin analogs and VEGF inhibitors described herein can be used to treat a subject having or at risk of having a complement-mediated disorder characterized by pathologic angiogenesis and / or pathologic vascular permeability, or associated with an increased risk of developing pathologic angiogenesis and / or pathologic vascular permeability. In some embodiments, the protein is a modified VEGF-inhibitory Fab as described herein having a campastatin analog fused to the heavy or light chain of the Fab. Pathologic angiogenesis and / or pathologic vascular permeability refers to angiogenesis or vascular permeability that is abnormal, excessive, and / or otherwise harmful to the subject experiencing it. In some embodiments, the disorder is an ocular disorder. In some embodiments, the ocular disorder is AMD. In some embodiments, the protein is administered to an eye having GA, wherein the eye has not been diagnosed with neovascular AMD. In some embodiments, the protein is administered to an eye having neovascular AMD, wherein the eye has not been diagnosed with GA. In some embodiments, the protein is administered to an eye having both neovascular AMD and GA. In some embodiments, the protein is administered to an eye having neovascular AMD and intermediate AMD. In some embodiments, the protein is administered to an eye having neovascular AMD, wherein the eye has not been diagnosed with GA. In some embodiments, the ocular disorder is choroidal neovascularization (CNV), which may be a manifestation of AMD or may be caused by other reasons. In some embodiments, the ocular disorder is proliferative diabetic retinopathy, neovascular glaucoma, diabetic macular edema, retinopathy of prematurity, or macular edema secondary to retinal vein occlusion. In some embodiments, the disorder is cancer.

[0205] In yet another embodiment, campastatin analog peptides themselves, rather than their transgenes, are used to enhance the efficacy of gene therapy and / or provide a synergistic effect with the gene therapy. These methods involve administering one or more campastatin analog peptides to a subject who is receiving, has previously received, or will receive gene therapy as described herein.

[0206] In certain embodiments, the campastatin analog is administered to a subject who has received or is receiving simultaneously or sequentially one or more doses of gene therapy as described herein. In some embodiments, the subject has received gene therapy 1 day, 1 week, 2 weeks, 4 weeks, 2 months, 4 months, 6 months, or longer prior to administration of the campastatin analog. In other embodiments, the campastatin analog is administered to a subject who has not received gene therapy but may receive gene therapy in the future.

[0207] In some embodiments, the campastatin analog and the gene therapy are administered simultaneously (e.g., within about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, or 2 hours of each other). In some embodiments, the campastatin analog and the gene therapy are administered sequentially (e.g., separated by more than 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 4 weeks, or longer). In some embodiments, the subject is pre-treated with the campastatin analog prior to receiving the gene therapy, e.g., from a few minutes to 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 24 hours or longer before administration of the gene therapy.

[0208] The pre-treatment, co-treatment, or post-treatment may include a single dose or multiple doses of the campastatin analog, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. If multiple doses are administered, they may be administered within a few minutes or hours or days before, during, or after the gene therapy. Administration of the campastatin analog may be interspersed with multiple courses of the gene therapy.

[0209] In some embodiments, the campastatin analog is administered for a period of time for the purpose of inhibiting complement activation until the viral vector of the gene therapy is taken up by one or more target cells (e.g., taken up by the target cells at a certain level or extent). In a subject, cellular uptake is indicated or measured using an assay that detects a decrease in the level of the viral vector in a sample obtained from the subject (e.g., a serum sample) and / or an increase in the level of the viral vector in one or more target cells. In some embodiments, the campastatin analog is administered in combination with an immunosuppressive therapy.

[0210] Embodiments that utilize a pre-formed campastatin analog (instead of or in addition to a transgene) may involve delivery methods that include combinations of administration routes and doses. For example, as described above, the composition containing the peptide may be administered orally, subcutaneously, intravenously, ophthalmically, or intramuscularly, and the vector containing the transgene may be administered by the same or a different route, simultaneously or at different times.

[0211] In some embodiments, the combination therapy results in an improvement in the gene therapy in the subject over a specified period of time (e.g., an improvement in the disease or disorder or its symptoms described herein), as discussed in the embodiments above involving the use of a campastatin analog transgene.

[0212] The following examples are provided to describe the invention in more detail. The examples are intended to illustrate and not limit the invention.

[0213] Efficacy of Campastatin Analog Cp50 on Laser-Induced Choroidal Neovascularization in a Non-Human Primate Model of Wet Age-Related Macular Degeneration

[0214] In a non-human primate model of wet age-related macular degeneration (AMD), the effects of the campastatin analog Cp50 (described above) and a vehicle were compared. In this model, laser-induced choroidal neovascularization (CNV) was performed in the eyes of macaques.

[0215] Study Design

[0216] As described below, laser-induced CNV was performed in both eyes of macaques. Two weeks (day 14) after laser photocoagulation, the laser lesions were graded (grades 1 - 4) by fluorescein angiography (FA). Eyes with a high ratio (percentage) of grade 4 lesions were selected and randomly assigned to treatment groups.

[0217] Under ketamine (15 mg / kg, i.m.) and xylazine (2 mg / kg, i.m.), on day 15, the eyes were injected intravitreally (IVT) with either the vehicle or campastatin Cp50. FA measurements were performed at weeks 4, 6, 8, 10, and 12 after CNV (a total of 5 times).

[0218] Methods

[0219] Laser-induced choroidal neovascularization (CNV)

[0220] Briefly, under light ketamine (10 mg / kg, i.m.) anesthesia, the pupils were dilated with tropicamide / phenylephrine ( ophthalmic solution; Santen Pharmaceutical Co., Osaka, Japan). For laser treatment, the macaques were anesthetized with a combination of ketamine (15 mg / kg, i.m.; DaiiChi Sankyo Propharma Co., Tokyo, Japan) and xylazine (2 mg / kg, i.m.; Bayer Yakuhin, Osaka, Japan). A green laser (900 mW output; green scanning laser photocoagulator, GYC - 500, Nidek, Co., Gamagori, Japan) was applied in a grid pattern to eight points around the macula (approx. 75 μm, for 0.1 s). The degree of CNV lesions was evaluated using fluorescein angiography (FA) at predetermined time points.

[0221] On the day of lesion grading, under light ketamine (10 mg / kg, i.m.) anesthesia, the pupils were dilated with tropicamide / phenylephrine ( The ophthalmic solution dilates the pupil, and then the macaques are anesthetized with a combination of ketamine (15 mg / kg, i.m.) and xylazine (2 mg / kg, i.m.).

[0222] Fluorescein angiography (FA)

[0223] Fluorescein angiography (FA) was used to examine the extent of CNV in the posterior segment of the eye. 10% sodium fluorescein ( 10 mg / kg, i.v.; Novartis, Tokyo, Japan) was injected, and photographs of the posterior segment of the eye were taken with a fundus camera at approximately 1, 3, and 5 min after the i.v. injection. The lesions were graded as follows:

[0224] Grade 1: No hyperfluorescence.

[0225] Grade 2: The lesion showed hyperfluorescence without leakage.

[0226] Grade 3: The lesion showed hyperfluorescence in the early or mid-phase transit images and late leakage.

[0227] Grade 4: The lesion showed bright hyperfluorescence during transit and late leakage beyond the treatment area.

[0228] After measuring FA, the vitreous humor was removed.

[0229] Intravitreal injection

[0230] After randomization, at day 15 under ketamine (15 mg / kg, i.m.) and xylazine (2 mg / kg, i.m.), the eyes were injected intravitreally (IVT) with 50 μl of vehicle or Cp50. Before the IVT injection, the eyes were treated twice daily with levofloxacin hydrate (0.5%; Santen Pharmaceutical Co.) for three days.

[0231] Under anesthesia, a solution of tropicamide / phenylephrine hydrochloride levofloxacin hydrate (0.5%; Santen Pharmaceutical Co.) and oxybuprocaine hydrochloride (0.4%; Santen Pharmaceutical Co.) was instilled before administration.

[0232] Results

[0233] At weeks 4, 6, 8, and 12, compared with the eyes treated with vehicle, the mean grade 4 lesions in the eyes treated with campastatin Cp50 decreased ( Figure 1)。Two eyes treated with Cp50 showed a reduction in the size of CNV lesions, which was evident as early as the 4th week after laser-induced CNV. Starting from the 6th week until the end of the CNV lesion monitoring experimental window (the 12th week), Cp50 treatment led to a complete (100%) reduction in the ratio of grade 4 lesions. The third Cp50-treated animal (K-2434) showed a continuous trend of reduction in the size of CNV lesions, which decreased to 50% of the baseline at the 6th week and continued to decrease to 25% at the 12th week. In contrast, the eyes treated with the vehicle retained a grade 4 score for the CNV lesions.

[0234] The present invention is not limited to the embodiments described and exemplified herein, but can be varied and modified within the scope of the claims.

Claims

1. A campstatin analog, which comprises a peptide having the amino acid sequence Xaa1-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Xaa2-His-Arg-Cys]-Xaa3-Xaa4 (SEQ ID NO: 4); wherein Xaa1 is absent or comprises the dipeptide Tyr-Ile, Xaa2 is Ala or Glu, Xaa3 is absent or is Ile, Xaa4 is absent or represents one, two or three Lys residues, and wherein the Cys residues form a disulfide bond to form a cyclic peptide comprising the sequence within the square brackets.

2. The campstatin analog according to claim 1, which has the amino acid sequence Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Xaa2-His-Arg-Cys]-Ile-Xaa4 (SEQ ID NO: 5); wherein Xaa2 is Ala or Glu, and Xaa4 represents two or three Lys residues.

3. The campstatin analog according to claim 1 or claim 2, which comprises Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys]-Ile-Lys-Lys-Lys (SEQ ID NO: 6) or Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Glu-His-Arg-Cys]-Ile-Lys-Lys-Lys (SEQ ID NO: 7).

4. A peptide which consists essentially of SEQ ID NO: 6 or SEQ ID NO:

7.

5. A peptide which consists of SEQ ID NO: 6 or SEQ ID NO:

7.

6. The campstatin analog according to claim 1, which is selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:

16.

7. A polynucleotide which comprises a sequence encoding the campstatin analog or peptide according to any one of claims 1-6.

8. The polynucleotide according to claim 7, which encodes SEQ ID NO: 6 or SEQ ID NO:

7.

9. The polynucleotide according to claim 8, which is selected from SEQ ID NO: 17 and SEQ ID NO:

18.

10. The polynucleotide according to claim 7, which is configured in an expression cassette or a vector.

11. The polynucleotide according to claim 10, wherein the vector is an expression vector.

12. The polynucleotide according to claim 11, wherein the vector is suitable for expression in a prokaryotic or eukaryotic expression system, and / or for gene therapy.

13. The polynucleotide according to claim 12, wherein the vector is for gene therapy and is selected from retroviruses, adenoviruses, adeno-associated viruses (AAV), and herpes simplex virus-1.

14. The polynucleotide according to claim 13, wherein the vector is an adeno-associated virus (AAV) vector.

15. The polynucleotide according to claim 14, wherein the AAV vector is an AAVI, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector or any variant thereof.

16. A vector comprising a combretastatin analog polynucleotide encoding a combretastatin analog comprising SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

7.

17. The vector according to claim 16, comprising at least one insertion site for at least one transgene for delivering gene therapy.

18. The vector according to claim 16, wherein the combretastatin analog polynucleotide has the sequence of SEQ ID NO: 17 or SEQ ID NO:

18.

19. The vector according to claim 16, which is selected from retroviruses, adenoviruses, adeno-associated viruses (AAV), and herpes simplex virus-1.

20. The vector according to claim 19, which is an adeno-associated virus (AAV) vector.

21. The vector according to claim 20, which is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector or any variant or combination thereof.

22. The vector according to claim 16, comprising at least one other transgene for gene therapy.

23. The vector according to claim 22, wherein the transgene encodes a therapeutic protein, enzyme, hormone, clotting factor, cytokine, or growth factor.

24. The vector according to claim 22, wherein the gene therapy is for treating blood disorders, ocular disorders, autoimmune diseases, muscle disorders, neurological disorders, or cancer.

25. The vector according to any one of claims 16 to 24, wherein the combretastatin analog polynucleotide and / or the transgene is / are adapted for tissue- or organ-specific expression.

26. The vector according to claim 22, wherein the combretastatin analog polynucleotide and the transgene are arranged on the vector to produce a fusion protein comprising the combretastatin analog and the transgene product.

27. The vector according to claim 26, wherein the fusion protein comprises the combretastatin analog directly linked to the transgene product.

28. The vector according to claim 26, wherein the fusion protein comprises the combretastatin analog linked to the transgene product via a linker or spacer.

29. The vector according to claim 26, wherein the transgene encodes a VEGF inhibitor.

30. The carrier according to claim 29, wherein the VEGF inhibitor comprises an extracellular domain of a VEGF receptor.

31. The carrier according to claim 29, wherein the VEGF inhibitor comprises an antibody fragment selected from Fab, F(ab’)2, Fv, scFv or a single-domain antibody.

32. The carrier according to claim 31, wherein the antibody fragment is a Fab comprising a VL domain, a CL domain, a VH domain and a CH1 domain.

33. The carrier according to claim 32, which encodes a fusion protein comprising the campstatin analog directly or indirectly fused to one, two, three or four of the following: (i) the VL of the Fab; (ii) the CL of the Fab; (iii) the VH of the Fab; or (iv) the CH1 of the Fab.

34. The carrier according to claim 33, which encodes a fusion protein comprising the campstatin analog directly or indirectly fused to the N-terminus of the VL of the Fab, the C-terminus of the CL of the Fab, the N-terminus of the VH of the Fab or the C-terminus of the CH1 of the Fab.

35. A pharmaceutical composition comprising at least one carrier as claimed in any one of claims 16 - 34 and a pharmaceutically acceptable carrier.

36. The pharmaceutical composition according to claim 35, which is formulated for administration by a route selected from subcutaneous, intradermal, intravenous, intraocular (including intravitreal and subretinal), intracerebral, intraperitoneal, intramuscular injection, periodontal administration (including gingival administration or infiltration injection into the interdental papilla), intranasal, epidural, oral, sublingual, intrathecal, intravaginal, transdermal, rectal, inhalation or topical.

37. The pharmaceutical composition according to claim 35, which is formulated for systemic administration.

38. The pharmaceutical composition according to claim 35, which is formulated for topical administration.

39. The pharmaceutical composition according to claim 38, wherein the topical administration is directed to the brain and / or central nervous system, eye, lung and / or respiratory system, heart and / or vascular system, lymphatic system, kidney, spleen, pancreas, liver, gastrointestinal system, periodontal tissue, skin, bone, joint or synovial fluid or any combination thereof.

40. The pharmaceutical composition according to claim 35, which further comprises a campstatin analog having a sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO:

7.

41. A pharmaceutical composition comprising a campstatin analog having a sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 in a pharmaceutically acceptable carrier.

42. The pharmaceutical composition according to claim 41, which is formulated for administration by a route selected from subcutaneous, intradermal, intravenous, intraocular (including intravitreal, subretinal), intracerebral, intraperitoneal, intramuscular injection, periodontal administration (including gingival administration or infiltration injection into the interdental papilla), intranasal, epidural, oral, sublingual, intrathecal, intravaginal, transdermal, rectal, inhalation or topical.

43. The pharmaceutical composition according to claim 41, which is formulated for systemic administration.

44. The pharmaceutical composition according to claim 41, which is formulated for topical administration.

45. The pharmaceutical composition according to claim 44, wherein the topical administration is directed to the brain and / or central nervous system, eye, lung and / or respiratory system, heart and / or vascular system, lymphatic system, kidney, spleen, pancreas, liver, gastrointestinal system, periodontal tissue, skin, bone, joint or synovial fluid or any combination thereof.

46. The pharmaceutical composition according to claim 41, which further comprises at least one carrier as claimed in any one of claims 16 - 34.

47. A kit, which comprises a plurality of pharmaceutical compositions, wherein at least one of the pharmaceutical compositions is a peptide-containing composition, the peptide-containing composition comprises a camptastatin analog having a sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 in a pharmaceutically acceptable carrier, and at least one of the pharmaceutical compositions is a carrier-containing composition, the carrier-containing composition comprises a carrier as claimed in any one of claims 16 - 34 in a pharmaceutically acceptable carrier.

48. The kit according to claim 47, wherein at least one of the peptide-containing composition and the carrier-containing composition is formulated for systemic administration.

49. The kit according to claim 47, wherein at least one of the peptide-containing composition and the carrier-containing composition is formulated for topical administration.

50. The kit according to claim 47, wherein the peptide-containing composition is formulated for a selected route of administration, the carrier-containing composition is formulated for a route of administration different from the route of administration selected for the peptide-containing composition, optionally comprising instructions for administering the peptide-containing composition according to a schedule different from the schedule for administering the carrier-containing composition.

51. A kit, which comprises a plurality of pharmaceutical compositions, each of the pharmaceutical compositions being a carrier-containing composition, the carrier-containing composition comprises a carrier as claimed in any one of claims 16 - 34 in a pharmaceutically acceptable carrier.

52. The kit according to claim 51, wherein at least one of the pharmaceutical compositions is formulated for systemic administration.

53. The kit according to claim 51, wherein at least one of the pharmaceutical compositions is formulated for topical administration.

54. The kit according to claim 51, wherein the pharmaceutical composition is formulated for different routes of administration and optionally comprises instructions for administering another composition according to a schedule different from the schedule for administering one composition.

55. A method of treating a subject having or at risk of having a complement-mediated disorder, the method comprising administering to the subject a composition comprising at least one vector, the vector comprising a campstatin analog polynucleotide encoding a campstatin analog of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

7.

56. The method according to claim 55, wherein after administering the composition, the level of complement activity in the subject or a biological sample from the subject is reduced relative to the level before administering the composition or in an equivalent subject who has not received the composition.

57. The method according to claim 56, wherein the level of complement activity is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, 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%, or at least 90% relative to the level before the administration or in an equivalent subject who has not received the composition.

58. The method according to claim 55, wherein the composition is administered systemically to the subject.

59. The method according to claim 55, wherein the composition is administered locally to a tissue or organ of the subject.

60. The method according to claim 55, wherein the vector is an adeno-associated virus (AAV) vector.

61. The method according to claim 60, wherein the AAV vector is an AAV1, AAV2, AAV3 (such as AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector.

62. The method according to claim 55, wherein the complement-mediated disorder is a chronic disorder.

63. The method according to claim 55, wherein the complement-mediated disorder involves complement-mediated damage to red blood cells, optionally wherein the disorder is paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome.

64. The method according to claim 55, wherein the complement-mediated disorder is an autoimmune disease, optionally wherein the disorder is multiple sclerosis.

65. The method according to claim 55, wherein the complement-mediated disorder involves the kidney, optionally wherein the disorder is membranoproliferative glomerulonephritis, lupus nephritis, IgA nephropathy (IgAN), primary membranous nephropathy (primary MN), C3 glomerulopathy (C3G), or acute kidney injury.

66. The method according to claim 55, wherein the complement-mediated disorder involves the central or peripheral nervous system or the neuromuscular junction, optionally wherein the disorder is neuromyelitis optica, Guillain-Barré syndrome, amyotrophic lateral sclerosis, multifocal motor neuropathy or myasthenia gravis.

67. The method according to claim 55, wherein the complement-mediated disorder involves the respiratory system, optionally wherein the disorder is characterized by pulmonary fibrosis.

68. The method according to claim 55, wherein the complement-mediated disorder involves the vascular system, optionally wherein the disorder is characterized by vasculitis.

69. The method according to claim 55, wherein the composition is administered to the eye of a subject having an eye disorder.

70. The method according to claim 69, wherein the composition is administered intravitreally.

71. The method according to claim 69, wherein the eye disorder is age-related macular degeneration (AMD).

72. The method according to claim 71, wherein the eye has one or more of the following: (i) geographic atrophy, (ii) wet AMD, (iii) geographic atrophy and wet AMD or (iv) intermediate AMD.

73. The method according to claim 55, which comprises the steps of: (1) providing the subject; (2) administering the composition to the subject so as to produce the campastatin analogue in the subject; and (3) measuring one or more parameters of the complement-mediated disorder.

74. The method according to claim 73, wherein the measuring is carried out before, during and / or after administering the composition.

75. The method according to claim 73, wherein the measuring is carried out on equivalent subjects not administered the composition.

76. The method according to claim 55, wherein the complement-mediated disorder is selected from atypical hemolytic uremic syndrome (aHUS), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), C3 glomerulopathy, complement-mediated nephropathy and glomerular inflammatory disease, age-related macular degeneration (AMD), an ocular disorder characterized by macular degeneration, choroidal neovascularization (CNV), retinal neovascularization (RNV), proliferative vitreoretinopathy, glaucoma, uveitis, ocular inflammation or any combination of these diseases, paroxysmal nocturnal hemoglobinuria (PNH), cold agglutinin disease (CAD), warm antibody autoimmune hemolytic anemia (wAIHA), sickle cell disease, transplantation-associated thrombotic microangiopathy, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), autoimmune and autoinflammatory kidney diseases, autoimmune myocarditis, multiple sclerosis, traumatic brain and spinal cord injury, cerebral, intestinal and renal ischemia-reperfusion (IR) injury, spontaneous and recurrent miscarriage, antiphospholipid syndrome (APS), Parkinson's disease, Alzheimer's disease, a neurodegenerative inflammatory disorder based on abnormal synaptic remodeling, microglial activity and cognitive decline, asthma, antinuclear cytoplasmic antigen-related pauci-immune vasculitis (Wegener's syndrome), non-lupus autoimmune skin diseases such as pemphigus, bullous pemphigoid and epidermolysis bullosa, post-traumatic shock, cancer, periodontitis, gingivitis and atherosclerosis.

77. The method according to any one of claims 55-76, wherein the subject is a non-human primate or a human.

78. The method according to any one of claims 55-77, which comprises administering more than one dose of the composition to the subject.

79. The method according to claim 78, which comprises administering multiple doses of the composition to the subject at a predetermined time interval.

80. The method according to claim 79, wherein the time intervals are within hours, days, weeks or months of each other.

81. A method of treating a subject having or at risk of having a disease or disorder having a complement-mediated component and one or more other components, the method comprising administering to the subject a composition comprising at least one vector, the vector comprising: a) a compstatin analog polynucleotide encoding a compstatin analog of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, and b) at least one transgene encoding a gene product for treating the one or more other components.

82. The method according to claim 81, wherein after administering the composition, the level of complement activation and / or the level of the one or more other components in the subject or a biological sample from the subject is reduced or improved relative to the level before administering the composition or in an equivalent subject that has not received the composition.

83. The method according to claim 82, wherein the complement activation and / or the level of the other component is reduced or improved by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, 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% or at least 90% relative to the level in an equivalent subject before or without administration of the composition.

84. The method according to claim 81, wherein the other component is associated with a neovascular phenotype.

85. The method according to claim 81, wherein the subject is a non-human primate or a human.

86. The method according to claim 81, wherein the composition is administered to the eye of a subject suffering from an eye disorder.

87. The method according to claim 85, wherein the eye disorder is age-related macular degeneration (AMD).

88. The method according to claim 86, wherein the transgene encodes a VEGF inhibitor.

89. The method according to claim 81, wherein the vector is an adeno-associated virus (AAV) vector.

90. The method according to claim 88, wherein the AAV vector is an AAV1, AAV2, AAV3 (such as AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 vector.

91. The method according to claim 81, wherein the transgene encodes a therapeutic protein, enzyme, hormone, clotting factor, cytokine or growth factor.

92. The method according to claim 81, wherein the campastatin analogue polynucleotide and the transgene are arranged on a single vector to produce a fusion protein comprising the campastatin analogue and the transgene product.

93. The method according to claim 91, wherein the fusion protein comprises the campastatin analogue directly or connected to the transgene product by a linker or spacer.

94. The method according to claim 91, wherein the transgene encodes a VEGF inhibitor.

95. The method according to claim 81, wherein the campastatin analogue polypeptide and at least one other transgene are configured on two or more separate vectors.

96. The method according to claim 94, wherein the vector comprising the campastatin analogue and one or more vectors comprising at least one other transgene are administered by different routes.

97. The method according to claim 95, wherein the vector comprising the campastatin analogue is administered systemically and one or more vectors comprising at least one other transgene are administered locally.

98. The method according to claim 94, wherein the vector comprising the campastatin analogue is administered before, during or after administration of one or more vectors comprising at least one other transgene.

99. The method according to claim 81, which comprises the following steps: (1) Provide the subject; (2) Administer the composition to the subject, thereby producing the campastatin analog and the transgene in the subject; and (3) Measure one or more parameters of the complement-mediated disorder or other components.

100. The method according to claim 98, wherein the measurement is performed before, during, and / or after administration of the composition and / or on equivalent subjects not administered the composition.

101. The method according to any one of claims 81-99, which comprises replacing the campastatin analog polynucleotide or the vector comprising the campastatin analog polynucleotide with the campastatin analog peptide itself.

102. A method of enhancing the efficacy of gene therapy in a subject who has received, is receiving, or will receive gene therapy, the method comprising administering to the subject a vector comprising a campastatin analog polynucleotide, the vector producing in the subject a campastatin analog having SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, thereby enhancing the efficacy of the gene therapy.

103. The method according to claim 101, wherein in the subject, the efficacy of the gene therapy is improved over a selected period of time.

104. The method according to claim 102, wherein the selected period of time is at least about 1 week, 2 weeks, 4 weeks, 2 months, 3 months, 6 months, or 1 year.

105. The method according to claim 101, wherein the efficacy is evaluated by observing or measuring a reduction in the immune response to the gene therapy.

106. The method according to claim 101, wherein the efficacy is evaluated by observing or measuring an improvement in the transduction of the viral vector carrying the transgene.

107. The method according to claim 101, wherein the efficacy is evaluated by observing or measuring a reduction in the complement-mediated clearance of the viral vector carrying the transgene.

108. The method according to any one of claims 101-106, wherein the efficacy is measured by: (i) comparing a subject who has received the vector comprising the campastatin analog polynucleotide with a control subject who has not received the vector comprising the campastatin analog polynucleotide, and / or (ii) comparing a subject during or after receiving the vector comprising the campastatin analog polynucleotide with the same subject before receiving the vector comprising the campastatin analog polynucleotide.

109. The method according to claim 101, wherein the vector comprising the campastatin analog polynucleotide comprises at least one insertion site for at least one transgene for delivering gene therapy.

110. The method according to claim 101, wherein the vector comprising the campastatin analog polynucleotide is selected from retroviruses, adenoviruses, adeno-associated viruses (AAV), and herpes simplex virus-1.

111. The method according to claim 109, wherein the vector comprising the campastatin analog polynucleotide is an adeno-associated virus (AAV) vector.

112. The method according to claim 110, wherein the AAV vector is an AAVI, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 vector or any variant or combination thereof.

113. The method according to claim 101, wherein the vector comprising the campastatin analog polynucleotide comprises at least one other transgene for gene therapy.

114. The method according to claim 101, wherein the vector comprising the campastatin analog polynucleotide is different from another vector comprising other transgenes for gene therapy, and wherein the vectors are administered together.

115. The method according to claim 101, wherein the vector comprising the campastatin analog polynucleotide is different from another vector comprising other transgenes for gene therapy, and wherein the vectors are administered separately.

116. The method according to any one of claims 101-114, which comprises replacing the campastatin analog polynucleotide or the vector comprising the campastatin analog polynucleotide with the campastatin analog peptide itself.

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