Compstatin Analogs for Vector-Based Therapy

Abstract

Disclosed is a compound comprising a peptide that can bind to C3 protein and inhibit complement activation. This compound can be used as a sole treatment for vector-mediated therapy, such as viral vector-mediated gene therapy, or in combination with other viral vector-mediated gene therapy to reduce vector-induced complement activation, and when combined with other drug modalities in the same vector or multiple vectors, provides additive or synergistic therapeutic benefits. Nucleic acids encoding compstatin analogs are also disclosed, as are vectors, pharmaceutical compositions, and methods of using compstatin analogs.

Description

[Technical Field]

[0001] The present invention relates to the activation of the complement cascade in the body. In particular, the present invention provides compstatin analogs that can be vectorized for vector-mediated therapy, e.g., viral vector-mediated gene therapy, as a sole treatment for diseases associated with abnormal complement activation, or in combination with other viral 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 vector or multiple vectors. These novel compstatin natural amino acid-containing molecules can be used for therapeutic C3 modulation in systemic or local applications. [Background technology]

[0002] Various publications, including patents, published applications, technical articles, and scholarly articles, are cited throughout this application, each of which is incorporated herein by reference in its entirety.

[0003] Viral vector gene therapy uses modified viruses as drug delivery vehicles to introduce specific DNA sequences into cells. This technology has long attracted interest for its potential advantages over traditional modalities. Many types of therapeutic agents (e.g., therapeutic peptides, enzymes, antibodies, and regulatory RNA) can be encoded into DNA sequences and rapidly designed and synthesized once a target is identified. These vector therapies can be delivered once or much less frequently than traditional treatment modalities, thereby improving patient compliance.

[0004] Nearly all currently available gene therapies use one of three types of vectors: adeno-associated viral (AAV) vectors, adenoviral vectors, or lentiviral (retroviral) vectors. AAV and adenoviral vectors are typically used in gene therapies administered directly to patients via infusion or local administration (in vivo), with AAV being the most popular vector in fields other than oncology and vaccines. While the scope of viral vector-based gene therapy is expanding into new disease areas and clinical conditions, there remains an unmet clinical need to develop effective countermeasures to mitigate vector-induced immune-related adverse events.

[0005] For example, a maladaptive host immune response to the AAV capsid or transgene, which involves both innate and adaptive immune pathways, can limit the efficacy and therapeutic effectiveness of AAV-based therapies. Complement activation has been implicated as a major 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 potently induce 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 cytotoxicity via MAC). C3 activation is a central step in this cascade of events that limits the transduction efficiency of AAV particles and impairs the clinical efficacy of AAV therapy. C3 activation can lead to macrophage activation, increased AAV uptake via CR3-dependent phagocytosis, and direct AAV toxicity via activation of downstream lytic pathways. C3-derived fragments also modulate antigen presentation pathways and B cell responses, affecting the generation of anti-AAV antibodies and promoting AAV-directed cytotoxic T cell responses. Given the multifaceted role of C3 in these processes, which affect both AAV viability and effective tissue homing, C3 inhibition appears to be a central therapeutic modality that will enhance the clinical potential of systemic AAV-based therapy.

[0006] The human complement system is involved in a variety of pathologies, from autoimmune, aging, and inflammatory disorders to transplant and biomaterial-induced complications, making it a major target for therapeutic intervention. Despite growing interest, development of complement-targeting drugs has been slow, and two related anti-C5 antibodies (eculizumab and ravulizumab), approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and other indications, have long remained the only clinical options. The FDA and EMA approval of pegcetacoplan (Empaveli® / Aspaveli®, Apellis) in 2021 marked the availability of a second class of complement inhibitor with a distinct mechanism of action. Compared to existing therapies, pegcetacoplan acts upstream in the complement cascade, inhibiting activation of the central component C3, thereby providing broader control of complement effectors. Given the diverse involvement of complement in pathologies, expanding therapeutic intervention points within the cascade is highly anticipated. Recently, two more complement inhibitors, an anti-C1s mAb (stimulimab, Enjeimo) and a small molecule C5aR1 antagonist (avacopan, Tabneos), have been approved for complement-mediated diseases such as cold agglutinin disease and ANCA-associated vasculitis, respectively.

[0007] Complement primarily acts as a rapid host defense system, eliminating invading microorganisms and apoptotic cells. After being initiated by various means, including immune complexes (classical pathway) or microbial signatures (lectin pathway), the cascade converges on the activation of the plasma protein C3 by convertases. C3 cleavage releases the anaphylatoxin C3a, generating an opsonic fragment (C3b), which covalently binds to target cell surfaces. The concerted binding of proteases factor B (FB) and factor D (FD) to C3b generates the major C3 convertase (i.e., C3bBb), activating more C3. In the absence of regulatory factors, this process leads to an amplification loop (alternative pathway), rapidly opsonizing surfaces with C3b. While C3b and its degradation fragments are directly involved in phagocytosis and adaptive immune signaling, C3b also provides a platform for the formation of C5 convertases. C5 cleavage generates the inflammatory mediator C5a, which then generates the membrane attack complex (MAC), which lyses or damages susceptible cells. While these potent effector functions provide an important layer of antibacterial defense, excessive or misactivated complement activation can induce tissue damage, inflammation, and harmful immune responses, driving clinical complications. While pathway- or effector-specific inhibition may be sufficient for some diseases, other conditions require a more broad approach to inhibit complement activity.

[0008] The compstatin family of C3 inhibitors is particularly suited for broad-spectrum complement inhibition, as it largely inhibits convertase-mediated C3 activation by all pathways and blocks most effector generation. 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, potency, and pharmacokinetic properties. Substitution of residues in the cyclic core yielded compstatin analogs with significantly improved target affinity. Compstatin Cp05 (SEQ ID NO: 2) builds on the basis of pegcetacoplan (Empaveri® / Aspaveri®, Apellis), with two Cp05 units crosslinked with a 40 kDa PEG moiety to reduce renal excretion. Finally, N-methylation of the backbone and addition of a D-Tyr to the N-terminus yielded the analog Cp40 (SEQ ID NO: 3), which featured picomolar affinity and an improved pharmacokinetic profile compared to the unpegylated form.

[0009] In recent years, new analogs of Cp40 have been developed with improved solubility and pharmacokinetic profiles. PEGylation with small PEG moieties or the addition of Lys residues has been shown to increase Cp40 solubility at physiological pH (approximately 7.4) without affecting its favorable C3 inhibitory activity. Furthermore, new Cp40-based compstatin derivatives exhibited longer plasma C3 saturation times (the period during which the molar concentration of the Cp40 analog is equal to or greater than that of plasma C3) and comparable or extended half-lives compared to Cp40 after subcutaneous (sc) administration to NHPs. These improved properties facilitate Sc administration of Cp40-based derivatives, thereby improving patient compliance during chronic C3-targeted interventions and expanding the potential routes for Cp40 delivery, broadening its potential use in various indications. A recent study in patients with severe COVID-19 demonstrated that administration of Cp40 resulted in complete and sustained systemic C3 inhibition, leading to saturation of plasma C3 levels throughout the treatment period. C3 inhibition by Cp40 resulted in broad anti-inflammatory effects and a marked attenuation of 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). Furthermore, local (intragingival) administration of Cp40 to adult patients with periodontal disease significantly reduced key clinical indicators of gingival inflammation and markers of inflammatory tissue injury, with the therapeutic effect persisting up to 90 days after treatment cessation (Hasturk H et al, J Clin Invest, 2021, Dec 1;131(23):e152973. doi: 10.1172 / JCI152973. PMID: 34618684).

[0010] While compstatin itself is a peptide composed of naturally occurring amino acids, the above-mentioned compstatin analogs contain non-naturally occurring components that improve potency hundreds of times over compstatin and confer numerous pharmacokinetic benefits. These advantages, along with their small size, make Cp40 and its derivatives particularly attractive for pharmaceutical development. However, the presence of non-naturally occurring components in these analogs precludes their expression and production from DNA sequences in viral or other expression vectors. Thus, developing novel analogs that are vectorizable yet have activity and pharmacokinetic profiles comparable to those of the highly potent Cp40 and its derivatives would be an advancement in the art. Summary of the Invention

[0011] The present invention provides analogs of the complement inhibitory peptide, compstatin, that can be vectorized as a single therapeutic modality or as a fusion moiety in combination with other therapeutic modalities (such as anti-VEGF agents) and produced by expression, and that have robust complement inhibitory activity and desirable pharmacokinetic properties.

[0012] One aspect of the invention features 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 Ile, and Xaa4 is absent or represents 1, 2, or 3 Lys residues, and the Cys residues form a disulfide bond to form a cyclic peptide comprising the sequence within the brackets. In particular, the compstatin analog can 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 SEQ ID NO: 6 and / or SEQ ID NO: 7, and any one of SEQ ID NOs: 8-16.

[0013] One aspect of the present invention features a polynucleotide comprising a sequence encoding the above-described 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 some embodiments, the compstatin analog polynucleotide is placed in an expression cassette or vector. The vector can be an expression vector. It can be adapted for expression in a prokaryotic or eukaryotic expression system. In some embodiments, the vector is used for gene therapy and is selected from retroviruses, adenoviruses, adeno-associated viruses (AAV), and herpes simplex virus type 1. In some 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 of their variants.

[0015] Another aspect of the present invention features a vector or vectors containing at least one polynucleotide encoding a compstatin analog comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or any of SEQ ID NOs:8-16. The compstatin analog polynucleotide can have the sequence of SEQ ID NO:17 or SEQ ID NO:18. The vector can also contain at least one insertion site for at least one transgene for gene therapy delivery. The vector can be selected from retrovirus, adenovirus, adeno-associated virus (AAV), or herpes simplex virus type 1. In one embodiment, 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 vector, or a variant or combination thereof.

[0016] In some embodiments, the vectors described above contain at least one other transgene for gene therapy. The transgene may encode, for example, a therapeutic protein, enzyme, hormone, blood clotting factor, cytokine, or growth factor. In various embodiments, the gene therapy is for the treatment of a blood disorder, an eye disorder, an autoimmune disease, a muscle disorder, a nerve disorder, or cancer. In some embodiments, the compstatin analog polynucleotide and / or transgene are adapted for tissue- or organ-specific expression.

[0017] In some embodiments, the compstatin analog polynucleotide and the transgene are placed on a vector to generate a fusion protein comprising the compstatin analog and the transgene product. The fusion protein may comprise the compstatin analog linked directly to the transgene product or may comprise the compstatin analog linked to the transgene product via a linker or spacer.

[0018] In one embodiment, 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 the group consisting of a Fab, F(ab')2, Fv, scFv, or single-domain antibody. The antibody fragment may be a Fab comprising a VL domain, a CL domain, a VH domain, and a CHI domain. In one embodiment, the vector encodes a fusion protein comprising a compstatin analog fused directly or indirectly to one, two, three, or four of (i) the VL of the Fab, (ii) the CL of the Fab, (iii) the VH of the Fab, or (iv) the CHI of the Fab. The vector may encode a fusion protein comprising a compstatin analog fused directly or indirectly 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 CHI of the Fab.

[0019] Another aspect of the invention features a pharmaceutical composition comprising at least one vector as 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 (including gingival or intracapillary infiltration injection), intranasal, epidural, oral, sublingual, intrathecal, intravaginal, transdermal, rectal, inhalation, or topical.

[0020] The pharmaceutical composition can be formulated for systemic administration or for local administration. Local administration can be to the brain and / or central nervous system, eyes, lungs and / or respiratory system, heart and / or vascular system, lymphatic system, kidneys, spleen, pancreas, liver, gastrointestinal system, periodontal tissue, skin, bone, joints or synovial fluid, or any combination thereof. In addition to the aforementioned vector, the pharmaceutical composition can include a compstatin analog peptide described herein, which can 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 features a pharmaceutical composition comprising a compstatin 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. The composition is useful for specific therapeutic applications as described herein. The pharmaceutical composition can 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 intracapillary infiltration injection), intranasal, epidural, oral, sublingual, intrathecal, intravaginal, transdermal, rectal, inhalation, or topical. The composition can be formulated for systemic or local administration. Topical administration can be 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 compositions comprising a compstatin analog can further comprise at least one vector described herein.

[0022] Another aspect of the invention features a kit including a plurality of pharmaceutical compositions, wherein at least one pharmaceutical composition is a peptide-containing composition comprising a compstatin 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 pharmaceutical composition is a vector-containing composition comprising a vector for gene therapy described herein in a pharmaceutically acceptable carrier. In one embodiment, at least one of the peptide-containing composition and the vector-containing composition is formulated for systemic administration. In another embodiment, at least one of the peptide-containing composition and the vector-containing composition is formulated for local administration. In another embodiment, the vector-containing composition is formulated for a selected route of administration, and is formulated for a route of administration that is different from the route of administration selected for the peptide-containing composition. The kit may also include instructions for administering the peptide-containing composition on a schedule that is different from the schedule for administering the vector-containing composition.

[0023] In another embodiment of the present invention, a kit includes multiple pharmaceutical compositions, each of which is a vector-containing composition comprising a vector described herein in a pharmaceutically acceptable carrier. The kits may include pharmaceutical compositions formulated for systemic administration, local administration, or both. The kits may include pharmaceutical compositions formulated for different routes of administration. The kits may also include instructions for administering one composition on a schedule different from the schedule for administering the other composition.

[0024] Another aspect of the invention features a method of treating a subject having or at risk for a complement-mediated disorder, the method comprising administering to the subject a composition comprising at least one 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 primate or a human. In certain embodiments, after administration of the composition, the level of complement activity in the subject or in a biological sample from the subject is reduced compared to the level before administration of the composition or compared to the level in a comparable subject not administered 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% compared to the level before administration of the composition or compared to the level in a comparable subject not administered the composition.

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

[0026] In one embodiment, 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 some embodiments, the complement-mediated disorder is a chronic disorder. In some embodiments, the complement-mediated disorder involves complement-mediated red blood cell damage, and optionally is paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome. In some embodiments, the complement-mediated disorder is an autoimmune disease, and optionally is multiple sclerosis. In some embodiments, the complement-mediated disorder involves the kidney. These diseases may include membranoproliferative glomerulonephritis, lupus nephritis, IgA nephropathy (IgAN), primary membranous nephropathy (primary MN), C3 glomerulopathy (C3G), or acute kidney injury. In some embodiments, the complement-mediated disorder involves the central or peripheral nervous system, or the neuromuscular junction. For example, neuromyelitis optica, Guillain-Barré syndrome, amyotrophic lateral sclerosis, multifocal motor neuropathy, or myasthenia gravis. In some embodiments, the complement-mediated disorder involves the respiratory system, and the respiratory system disorder may be characterized by pulmonary fibrosis. In some embodiments, the complement-mediated disorder involves the vascular system, and the vascular system disorder may be characterized by vasculitis.

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

[0029] The method can include the steps of: (1) providing a subject, (2) administering a composition to the subject, thereby producing a compstatin analog in the subject, and (3) measuring one or more parameters of a complement-mediated disorder. Measurements can be performed before, during, and / or after administration of the composition, or alternatively, on a comparable subject who has not received the composition.

[0030] In certain embodiments, the complement-mediated disorder is 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); any ocular disorder characterized by macular degeneration, choroidal neovascularization (CNV), retinal neovascularization (RNV), proliferative vitreoretinopathy, glaucoma, uveitis, ocular inflammation, or any combination thereof; 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 and atherosclerosis. The disease is selected from the group consisting of systemic lupus erythematosus (SLE), autoimmune and autoinflammatory kidney disease, autoimmune myocarditis, multiple sclerosis, traumatic brain injury and spinal cord injury; ischemia-reperfusion (IR) injury of the brain, intestine, and kidney, spontaneous and recurrent abortion, antiphospholipid syndrome (APS); Parkinson's disease; Alzheimer's disease; abnormal synaptic remodeling; neurodegenerative inflammatory diseases with microglial activity and cognitive decline; asthma; antinuclear cytoplasmic antigen-associated microimmune 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 can include administering two or more doses of the composition to a subject. The method can include administering the doses of the composition to the subject at predetermined time intervals. Such time intervals can be within hours, days, weeks, or months of each other.

[0032] Another aspect of the invention features a method for treating a subject having or at risk for a disease or disorder having a complement-mediated component and one or more other components. The method includes administering to the subject a composition comprising at least one vector containing (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 that treats the one or more other components. The subject can be a primate or a human.

[0033] In certain embodiments, after administration of the composition, the level of complement activity and / or other components in the subject or in a biological sample from the subject is reduced or improved compared to the level before administration of the composition or compared to the level in a comparable subject who did not receive the composition. The level of complement activity and / or other components 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% compared to the level before administration or compared to the level in a comparable subject who did not receive 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 suffering from an ocular disorder. In one embodiment, the ocular disorder is age-related macular degeneration (AMD). In one embodiment for treating an ocular disorder, the transgene encodes a VEGF inhibitor.

[0035] In one embodiment, the method includes the use of 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. In one embodiment, the transgene encodes a therapeutic protein, enzyme, hormone, blood clotting factor, cytokine, or growth factor.

[0036] In some embodiments, the compstatin analog polynucleotide and transgene are placed in a single vector to generate a fusion protein containing the compstatin analog and the transgene product. The fusion protein may contain the compstatin analog linked to the transgene product directly or via a linker or spacer. The fusion protein may contain a VEGF inhibitor. In other embodiments, the compstatin analog polypeptide and at least one other transgene are placed in two or more separate vectors.

[0037] In some embodiments, the vector containing the compstatin analog and the vector containing at least one other transgene are administered by different routes. In some embodiments, the vector containing the compstatin analog is administered systemically, and the vector containing at least one other transgene is administered locally. In some embodiments, the vector containing the compstatin analog is administered before, during, or after the administration of the vector containing at least one other transgene.

[0038] In one embodiment, the method comprises the steps of: (1) providing a subject; (2) administering a composition to the subject, thereby producing a compstatin analog and a transgene in the subject; and (3) measuring one or more parameters or other components of a complement-mediated disorder. Measurements can be performed before, during, and / or after administration of the composition, and / or in a comparable subject who has not received the composition. In one embodiment of this method, the compstatin analog polynucleotide or a vector containing the compstatin analog polynucleotide is replaced with the compstatin analog peptide itself.

[0039] Another aspect of the invention features a method for improving the efficacy of gene therapy in a subject undergoing, undergoing, or who has undergone gene therapy. The method includes administering to the subject a vector containing a compstatin analog polynucleotide that produces a compstatin analog having SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 in the subject, thereby improving the efficacy of the gene therapy. In some embodiments, the efficacy of gene therapy is improved in the subject over a selected period of time. The selected period can be, for example, at least about 1 week, 2 weeks, 4 weeks, 2 months, 3 months, 6 months, or 1 year. In some embodiments, efficacy is assessed by observing or measuring a decrease in the immune response to the gene therapy. In some embodiments, efficacy is measured by observing or measuring an improvement in transduction of a viral vector carrying the transgene. In some embodiments, efficacy is assessed by observing or measuring a decrease in complement-mediated clearance of a viral vector carrying the transgene. In one embodiment, the efficacy of gene therapy is measured by (i) comparing a subject who received a vector containing a compstatin analog polynucleotide with a control subject who did not receive a vector containing a compstatin analog polynucleotide, and / or (ii) comparing a subject during or after receiving a vector containing a compstatin analog polynucleotide with the same subject before receiving a vector containing a compstatin analog polynucleotide.

[0040] In one embodiment of this method, the vector containing the compstatin analog polynucleotide contains at least one insertion site for at least one transgene for gene therapy delivery. The vector can be selected from retrovirus, adenovirus, adeno-associated virus (AAV), and herpes simplex virus type 1. In one embodiment, the vector containing the compstatin 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 a variant or combination thereof.

[0041] In some embodiments, the vector containing the compstatin analog polynucleotide contains at least one other transgene for gene therapy. In some embodiments, the vector containing the compstatin analog polynucleotide is distinct from another vector containing another transgene for gene therapy, and the vectors are administered together. In other embodiments, the vector containing the compstatin analog polynucleotide is distinct from another vector containing another transgene for gene therapy, and the vectors are administered separately. Another embodiment of this aspect of the invention involves replacing the compstatin analog polynucleotide or the vector containing the compstatin analog polynucleotide with the compstatin analog peptide itself.

[0042] Other features and advantages of the present invention will be understood by reference to the following detailed description. [Brief explanation of the drawings]

[0043] [Figure 1]Graph showing the rate of individual grade 4 lesions (expressed as % of grade 4 lesions at week 2) over time after laser-induced choroidal neovascularization (CNV) in a macaque primate model. Arrows indicate intravitreal treatment (day 15). Three eyes (K-2434, K-2437, and K-2485) were treated with the compstatin analog CP50, and three eyes (K-2089, K-2482, and K-2468) were treated with vehicle. DETAILED DESCRIPTION OF THE INVENTION

[0044] definition Various terms relating to the methods and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise specified. Otherwise, specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0045] The following abbreviations may be used herein: Ac, acetyl group; 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, reversed-phase high-performance liquid chromatography; Sar, N-methylglycine; sc, subcutaneous; SPR, surface plasmon resonance; TFA, trifluoroacetic acid; UPLC-ESI-MS, ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry; VBS, veronal-buffered saline; WFI, water for injection.

[0046] Unless the context clearly indicates otherwise, the singular form of a word includes the plural and vice versa. References to "a," "an," and "the" generally include the plural of the respective term. For example, a reference to "a compound" or "a method" includes a plurality of such "compounds" or "methods." Similarly, the words "comprise," "comprises," and "comprising" are to be interpreted inclusively rather than exclusively. Similarly, the words "include," "including," and "or" are all to be interpreted inclusively unless the context clearly prohibits otherwise.

[0047] The terms "comprising" or "including" are intended to include embodiments encompassed by the terms "consisting essentially of" and "consisting of." Similarly, the term "consisting essentially of" is intended to include embodiments encompassed by the term "consisting of." Furthermore, the term "consisting essentially of" limits the scope of the embodiment to the specified elements or steps and to elements or steps that do not materially affect the basic and novel characteristics of the embodiment.

[0048] As used herein, the term "about" when referring to a measurable value such as an amount, temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in some embodiments ±5%, in some embodiments ±1%, and in some embodiments ±0.1% from the specified value, such variations being appropriate for making and using the disclosed compounds and compositions.

[0049] As used herein, the term "compstatin" refers to a peptide comprising SEQ ID NO: 1, I[CVVQDWGHHRC]T (cyclic C2-C12 with disulfide bond manner shown in brackets). The term "compstatin analog" refers to a modified compstatin containing amino acid substitutions that allow for expression from DNA, as described in more detail herein. These analogs are sometimes referred to herein as being "proteinogenic." When referring to the position of a particular amino acid or analog within compstatin or a compstatin analog, those positions are sometimes referred to as "positions" within the peptide, and positions are numbered from 1 (Ile in compstatin) to 13 (Thr in compstatin). For example, a Gly residue occupies "position 8."

[0050] The terms "pharmacologically active" and "biologically active" refer to the ability of a compstatin analog of the present 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 art-recognized assays.

[0051] As used herein, the term "gene therapy" encompasses the treatment of diseases or conditions treated by the introduction of exogenous DNA into the cells of an individual with the disease or pathological condition. Gene therapy includes, for example, (1) replacing a dysfunctional or non-functioning gene with a copy of a healthy gene, (2) inactivating a disease-causing gene that malfunctions and leads to the disease, and / or (3) introducing heterologous nucleic acid into cells, tissues, or organs in the body to produce a therapeutic agent that helps treat the disease.

[0052] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to all forms of nucleic acid, including DNA and RNA, oligonucleotide.Nucleic acid includes genomic DNA, cDNA, antisense DNA / RNA, plasmid DNA, linear DNA, (poly and oligonucleotide), chromosomal DNA, spliced ​​or unspliced ​​mRNA, rRNA, tRNA inhibitory DNA or RNA (e.g., RNAi, such as small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA, or antisense RNA), locked nucleic acid analog (LNA), single-stranded and double-stranded oligonucleotide DNA (ODN), immune stimulating sequence (ISS), riboswitch and ribozyme.Nucleic acid includes naturally occurring, synthetic, and intentionally modified or altered polynucleotides.

[0053] The term "transgene" refers to a nucleic acid that is or has been introduced into a cell or organ. Transgenes include any nucleic acid, such as a nucleic acid encoding a compstatin analog of the invention, a "compstatin analog transgene," as well as any other heterologous nucleic acid that encodes a protein, peptide, or nucleic acid (e.g., miRNA, etc.). As used herein, the terms transgene and heterologous nucleic acid / polynucleotide sequence are used interchangeably.

[0054] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been 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, in which additional DNA segments may be ligated into the viral genome. Some vectors are capable of expressing genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Those skilled in the art will understand that the "viral vectors" described herein include viral components in addition to the transgene.

[0055] "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." Typically, such elements are included to facilitate proper transcription and proper translation of the heterologous polynucleotide (e.g., promoters, enhancers, intron splicing signals, maintaining the correct reading frame of the gene to allow in-frame translation of mRNA, stop codons).

[0056] As used herein, "operably linked" refers to the relationship between two or more nucleic acid sequences in which one nucleic acid sequence (e.g., a regulatory element) affects the properties of another nucleotide sequence (e.g., affects the expression of a transgene). Operably linked sequences include both expression control elements contained in or contiguous with a transgene and expression control elements acting in trans or at a distance to control transgene expression. 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, if desired, sequences that enhance secretion of the encoded product. For example, as used herein, a nucleic acid sequence (e.g., a compstatin coding sequence or other transgene) and a regulatory sequence are considered operably linked if they are covalently linked in a manner that places the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequence.

[0057] As used herein, the term "pharmaceutically acceptable salt" or "pharmaceutically acceptable ester" refers to a derivative of the disclosed compounds, in which the parent compound is modified by making an ester or acid or base salt form that is compatible with the other components of the pharmaceutical composition and not harmful to the subject to which the composition is administered. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic acid 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 conventional salts or quaternary ammonium salts of the parent compound formed, for example, from inorganic or organic acids. For example, such conventional salts include, but are not limited to, those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; 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, isethionic acid, and the like. 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 considered within the scope of the present invention.

[0058] As used herein, the terms "local administration" or "local delivery," with respect to delivery of a compstatin analog peptide or polynucleotide encoding same as described herein, refer to delivery that may rely on the peptide or polynucleotide being transported via the vascular system to its intended target tissue or site.

[0059] As used herein, "intraocular administration" or "ocular administration" of a pharmaceutical composition includes any route of administration characterized by introduction into the eye, including subretinal 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 maintain the shape of the eye.

[0060] As used herein, "intramuscular administration" of a pharmaceutical composition includes any route of administration characterized by introduction into muscle.

[0061] As used herein, "periodontal administration" of a pharmaceutical composition refers to administration into the tissues surrounding and / or around a tooth (e.g., by injection, topical application, or biodegradable implant), and includes "gingival administration" and "intracapillary infiltration." As used herein, "gingival administration" of a pharmaceutical composition includes any route of administration characterized by introduction into the gingiva or gum. "Intracapillary infiltration" or "intracapillary infiltration injection," which is a type of gingival administration, refers to administration of a pharmaceutical composition to the interdental papilla, which is the gingival tissue located coronally at the free gingival margin on the buccal and lingual surfaces of the teeth.

[0062] As used herein, "oral administration" or "enteral administration" of a pharmaceutical composition includes any route of administration characterized by introduction into the digestive tract. "Oral administration" includes oral administration as well as uptake by oral or intragastric tube. "Oral administration" or "enteral administration" also includes other routes known in the art, such as sublingual, buccal, intranasal, pulmonary, or rectal administration.

[0063] The term "treating" refers to any indication of successful treatment or amelioration of a disease or condition. Treating can include, for example, reducing or alleviating the severity of one or more symptoms of a disease or condition, or reducing the frequency with which a symptom of a disease, defect, disorder, or adverse condition, etc., is experienced by an individual, such as a human patient.

[0064] The term "preventing" refers to the prevention of a disease or condition in an individual, such as a human patient. For example, if an individual at risk of developing an inflammatory disease is treated with the compounds and / or methods of the invention and does not subsequently develop the disease or condition, the disease has been prevented in that individual.

[0065] As used herein, the term "treatment or prevention" may be used to refer to a method that results in some level of treatment or improvement of a disease or condition, and is intended to encompass a range of outcomes directed toward that end, including, but not limited to, complete prevention of the condition.

[0066] The term "parameter" as used herein refers to any measurement of bodily function that can be observed or measured using appropriate measurement techniques available in the art. As those skilled in the art will understand, measuring one or more "parameters" of bodily function can be used to detect specific dysfunctions compared to average normal parameters and can also be used to determine whether the bodily function has improved after or during treatment. Such parameters can be general, such as body temperature, blood pressure, pulse (heart rate), and breathing rate (respiratory rate), or can be specific to a particular organ, tissue, or disease or condition, such as functional test results from blood or other organs / tissues.

[0067] By "effective amount" or "sufficient amount" is meant an amount, in single or multiple doses, that alone or in combination, in combination with one or more other compositions (therapeutic agents such as drugs or immunosuppressants), procedures, protocols, or therapies, provides a detectable response of any duration (long-term or short-term), an expected or desired result in a subject, or a benefit to a subject of any measurable degree or detectable duration (e.g., minutes, hours, days, months, years, or a lifetime).

[0068] The term "therapeutically effective amount" or "therapeutically effective dose" refers to 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 routinely in connection with the stated purpose. For example, in vitro assays can optionally be employed to identify optimal dosage ranges. Selection of a specific effective dose can be determined by one of ordinary skill in the art (e.g., through clinical trials) based on consideration of several factors, including the disease to be treated or prevented, the symptoms involved, the patient's size, the patient's immune status, and other factors known to those of skill in the art. The precise dose to be employed in a formulation will also depend on the route of administration and the severity of the disease, and should be determined according to the judgment of the clinician and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0069] explanation Complement activation is essential for a robust immune system. However, in some cases, excessive or uncontrolled complement activation is a central component of many pathologies. Furthermore, viral vector-based gene therapy is substantially hindered by undesirable immunological side effects involving complement activation. These immune responses may include antibody, B cell, and / or T cell responses, and may be specific to viral antigens of the viral vector, such as viral capsid or coat proteins or peptides thereof (see, for example, Colella et al., 2018, Molecular Therapy: Methods & Clinical Development, 8:87-104).

[0070] The present invention arises from the inventors' development of proteogenic compstatin analogs with robust complement inhibitory activity, the encoding nucleic acids of which can be incorporated into nucleic acid constructs, such as viral vectors, and expressed intracellularly. These constructs can be inserted into viral vectors as stand-alone agents to provide long-acting gene therapy for the treatment of complement activation-mediated diseases, or in combination with sequences encoding other therapeutic modalities (e.g., anti-VEGF drugs). Combining such proteogenic compstatin molecules with other therapeutic modalities may provide additive or synergistic therapeutic benefits in diseases driven by abnormal complement activation or angiogenic responses, such as those typified by elevated VEGF levels or enhanced VEGF receptor signaling. Such combinations can improve viral vector transduction, prevent C3 opsonization of the capsid, and / or reduce or prevent immune responses (e.g., antibody, B cell, and / or T cell responses).

[0071] Peptides, transgenes, and vectors: One embodiment of the present invention features protein derivatives of the compstatin analog Cp40 (SEQ ID NO: 3; described in WO 2013 / 036778). The peptides of the disclosure are represented by the sequences shown below. Xaa1-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Xaa2-His-Arg-Cys]-Xaa3-Xaa4 (SEQ ID NO: 4)

[0072] In the above sequences, Xaa1 is absent or contains the dipeptide Tyr-Ile, Xaa2 is Ala or Glu, Xaa3 is absent or 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 in parentheses.

[0073] One embodiment is represented by the following sequence: Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Xaa2-His-Arg-Cys]-Ile-Xaa4 (SEQ ID NO: 5)

[0074] Exemplary sequences of the present disclosure include Cp50 and Cp51, shown below. Cp50: Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys]-Ile-Lys-Lys-Lys (SEQ ID NO: 6) Cp51: Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Glu-His-Arg-Cys]-Ile-Lys-Lys-Lys (SEQ ID NO: 7)

[0075] Other exemplary sequences include: Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile-Lys-Lys (SEQ ID NO: 8) Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile-Lys (SEQ ID NO: 9) Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile (SEQ ID NO: 10) Tyr-Ile-[Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys] (SEQ ID NO: 11) [Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile-Lys-Lys-Lys (SEQ ID NO: 12) [Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile-Lys-Lys (SEQ ID NO: 13) [Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile-Lys (SEQ ID NO: 14) [Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys]-Ile (SEQ ID NO: 15) [Cys-Ile-Trp-Gln-Asp-Trp-Gly-Ala / Glu-His-Arg-Cys] (SEQ ID NO: 16)

[0076] Cp40-KK and Cp40-KKK have previously been described to exhibit not only increased solubility compared to unmodified Cp40, but also increased plasma and vitreous retention, as well as enhanced C3 binding, compared to Cp40 and other Cp40-based analogs (see WO 2019 / 195712). Indeed, Cp40-KKK has been shown to exhibit an in vivo retention time equal to or exceeding three months after intravitreal administration. Notably, both Cp40-KK and Cp40-KKK analogs exhibit significantly improved pharmacokinetic properties compared to Cp40.

[0077] The exemplary proteogenic peptides Cp50 and Cp51 were found to have in vitro and in vivo properties similar to those of Cp40-KK and Cp40-KKK. This is surprising given the absence of unnatural residues within the Cp40 backbone and at its termini. Table 1 below shows some of these properties compared to Cp40 and other compstatin analogs. [Table 1]

[0078] The nucleic acid encoding Cp50 (YICIWQDWGAHRCIKKK (SEQ ID NO: 6)) is shown below, with all possible codons for each residue indicated. TAT / TAC-ATA / ATT / ATC-TGT / TGC-ATA / ATT / ATC-TGG-CAG / CAA-GAT / GAC-TGG-GGG / GGA / GGT / GGC-GCG / GCA / GCT / GCC-CAT / CAC-CGG / CGA / CGT / CGC-TGT / TGC-ATA / ATT / ATC-AAG / AAA-AAG / AAA-AAG / AAA (SEQ ID NO: 17)

[0079] The nucleic acid encoding Cp51 (YICIWQDWGEHRCIKKK (SEQ ID NO: 7)) is shown below, with all possible codons for each residue indicated. TAT / TAC-ATA / ATT / ATC- / TGT / TGC-ATA / ATT / ATC-TGG-CAG / CAA-GAT / GAC-TGG-GGG / GGA / GGT / GGC-GAG / GAA-CAT / CAC-CGG / CGA / CGT / CGC-TGT / TGC-ATA / ATT / ATC-AAG / AAA-AAG / AAA-AAG / AAA (SEQ ID NO: 18)

[0080] The compstatin analogs of the present invention are produced by inserting a nucleic acid encoding the peptide into a vector and expressing it. The vector can be non-viral or viral. In some embodiments, the vector is a viral vector, as described below. The vector contains one or more polypeptides encoding the compstatin analogs disclosed herein.

[0081] In certain embodiments, the compstatin analogs described herein are produced in advance (i.e., not within a patient's cells or tissues) and incorporated into pharmaceutical compositions for use in various combination therapies, as described herein. These compstatin 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.

[0082] Alternatively, because the compstatin analogs disclosed herein are composed of naturally occurring amino acids, they can be produced by expressing encoding polynucleotides in appropriate prokaryotic or eukaryotic systems. For example, the DNA construct can be inserted into a plasmid vector adapted for expression in bacterial cells (e.g., E. coli) or yeast cells (e.g., Saccharomyces cerevisiae), or a baculovirus vector for expression in insect cells, or a viral vector for expression in mammalian cells. Such vectors contain regulatory elements necessary for DNA expression in host cells, arranged in a manner that allows DNA expression in the host cells. Such regulatory elements necessary 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 according to methods known in the art.

[0083] The above-described compstatin analog polynucleotides and polypeptides are advantageous in several respects. For example, as described above, these compstatin analogs exhibit complement inhibitory activity comparable to that of the most potent compstatin analogs. Therefore, complement inhibition can be achieved at lower doses. Furthermore, these compstatin analogs are expected to have the same or similar pharmacokinetic profiles as their analogous counterparts, Cp40-KK and Cp40-KKK. Furthermore, the small size of the encoding polynucleotides allows them to be added to any vector without occupying the space that would be required for one or more transgenes of interest. This is particularly important for vectors with limited insert space, such as AAV vectors. Thus, vector strains containing compstatin analog polynucleotides as standard components can be constructed and advantageously used to improve the efficacy of vectors in the delivery of any gene therapy.

[0084] Thus, in one embodiment, the vector contains at least one insertion site for the compstatin analog peptide and another polypeptide, for example, for gene therapy. As described in the definition, such polypeptides may be referred to herein as "transgenes." In some embodiments, the compstatin polypeptide and the other polypeptide are contained within a single vector, while in other embodiments, they are contained on multiple vectors. When contained on multiple vectors, the vectors may be combined into a single pharmaceutical composition and administered together. Alternatively, the multiple vectors can be administered separately.

[0085] In certain embodiments, a compstatin polynucleotide is placed with one or more other transgenes to generate a fusion protein, in which the compstatin analog is linked to the other gene product. This arrangement is advantageous in certain cases where the linkage confers a benefit to the compstatin analog, such as targeting to a particular tissue, cell, or subcellular location, or improving one or more other pharmacokinetic parameters.

[0086] In some embodiments, the compstatin 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 includes a linker (e.g., a flexible linker) between the compstatin analog and the heterologous polypeptide. In some embodiments, the fusion protein or protein conjugate lacks a linker, and the compstatin analog is fused or conjugated directly to the heterologous polypeptide. The linker can be of any sequence and length that allows each polypeptide to maintain its biological activity, e.g., does not cause steric hindrance.

[0087] In some embodiments, a fusion protein comprises a compstatin analog of the invention and a VEGF-regulated gene product. Such a fusion protein can specifically bind to C3 and / or C3b in addition to vascular endothelial growth factor A (e.g., VEGF-A). In some examples, the fusion protein reduces or inhibits angiogenesis, e.g., compared to a reference polypeptide. In some examples, the reference polypeptide comprises the antibody (or fragment thereof) known as ranibizumab (sold as Lucentis®, Genentech).

[0088] The vehicle for expressing the compstatin polynucleotide of the present invention comprises the regulatory elements necessary for the expression of the coding sequence of the DNA in host cells, arranged in a manner that allows the expression of the DNA in host cells.Expression control elements or sequences 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 increase translation efficiency (i.e., Kozak consensus sequences); sequences that increase protein stability; and, in some cases, sequences that increase the secretion of the encoded product.In this specification, the term "expression cassette" may be used to describe a compstatin polynucleotide or other transgene together with expression control elements that promote and control expression in host cells.

[0089] In certain embodiments, the compstatin polynucleotide and / or other transgene of the present invention is carried on a viral vector, including, but not limited to, retroviral vectors (e.g., Moloney murine leukemia virus (MMLV), Harvey murine sarcoma virus, mouse mammary tumor virus, and Rus sarcoma virus), adenoviral vectors, adeno-associated viral (AAV) vectors, SV40 virus vectors, polyoma virus vectors, Epstein-Barr virus vectors, papilloma virus vectors, herpes virus vectors, vaccinia virus vectors, and polio virus vectors.

[0090] In one embodiment, the selected vector is a viral vector primarily used in gene therapy. The compstatin polynucleotide and / or other transgene can be incorporated into any type of viral vector used in gene therapy, such as recombinant retrovirus, adenovirus, adeno-associated virus (AAV), and herpes simplex virus type 1.

[0091] Retroviruses are enveloped viruses that belong to the Retroviridae family. Once inside a host cell, the virus replicates by transcribing RNA into DNA using viral reverse transcriptase. Retroviral DNA replicates as part of the host genome and is called a provirus. A nucleic acid of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art. Protocols for producing replication-deficient retroviruses are known in the art.

[0092] In some embodiments, the retrovirus is a lentivirus. Lentiviruses include human immunodeficiency viruses (HIV-1 and HIV-2), simian immunodeficiency viruses (SIV), feline immunodeficiency viruses (FIV), equine infectious anemia (EIA), and visna viruses. Lentivirus-derived vectors 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).

[0093] Herpes simplex virus (HSV)-based viral vectors are also suitable for use as provided herein. Many replication-deficient HSV vectors contain deletions that remove one or more intermediate-early genes to prevent replication. Advantages of herpes vectors include their ability to enter a latent phase, allowing long-term DNA expression, and their large viral DNA genome, which can accommodate up to 25 kb of foreign DNA.

[0094] In some embodiments, the vector is an adenovirus vector. Adenoviruses are a large family of viruses with double-stranded DNA. Adenoviruses replicate in the nucleus of host cells and use the host cell's machinery to synthesize viral RNA, DNA, and proteins. Adenoviruses are known in the art to affect both replicating and non-replicating cells, accommodate large transgenes, and encode proteins without integrating into the host cell genome. Viral replication can be rendered defective by deleting some genes required for viral replication. To allow for additional room for larger DNA insertions, the unnecessary E3 region, which is not essential for replication, is often deleted. The adenoviruses that serve as the basis for viral vectors can be of any origin, subgroup, subtype, subtype mixture, or serotype. For example, the adenovirus can be of 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 serogroup (e.g., serotypes 49 and 51), or any other adenovirus serotype. Adenovirus serotypes 1-51 are available from the American Type Culture Collection (Manassas, VA). Non-group C adenoviruses, as well as non-human adenoviruses, can be used to prepare replication-deficient adenoviral vectors.

[0095] In one embodiment, recombinant AAV (rAAV) is the vector of choice. AAV particles contain a linear, single-stranded AAV nucleic acid genome bound to an AAV capsid protein envelope. AAV cannot replicate without a helper virus, which can be adenovirus, vaccinia virus, or herpesvirus. In the absence of a helper virus, AAV inserts its genome into the host cell chromosome and becomes latent. Subsequent infection with the helper virus rescues the latent integrated copy, which then replicates and generates infectious viral progeny.

[0096] Recombinant AAV (rAAV) vectors consist of a recombinant viral genome and capsid proteins. The rAAV genome is assembled from polynucleotides encoding a transgene, regulatory elements, and viral elements required for packaging the rAAV genome. Methods for constructing rAAV genomes are known in the art. AAV expression vectors can be constructed with AAV inverted terminal repeats (ITRs) flanking restriction sites for transgene insertion, either directly using available restriction sites or by excising the transgene with a restriction enzyme, polishing the ends, and ligating it into the AAV expression vector, optionally with a linker. The transgene is incorporated into the AAV expression vector along with one or more expression control elements, as described above, including, for example, an enhancer, promoter, and / or posttranscriptional regulatory element (PRE) flanked by the AAV ITRs.

[0097] Methods for producing rAAV vectors with specific capsid proteins are known in the art. Briefly, viral particles are produced by supplying the components necessary for packaging the rAAV genome into capsids in trans, or the necessary components can be supplied by recombinant host cells. Some or all of the necessary elements can be under the control of an inducible or constitutive promoter. The recombinant AAV genome, rep sequence, cap sequence, and helper functions for generating rAAV can be delivered to packaging host cells using any appropriate genetic element (vector). Typically, recombinant AAV is produced by transfecting host cells with the recombinant AAV genome (including the transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. The AAV helper function vector encodes AAV helper function sequences (i.e., rep and cap) that function in trans for productive AAV replication and encapsidation. Accessory function vectors typically encode nucleotide sequences for non-AV-derived viral and / or cellular functions required for AAV replication, including elements involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and assembly of the AAV capsid.

[0098] The AAV vectors described herein generally comprise a rAAV genome encoding one or more transgenes operably linked to one or more regulatory elements in a manner that allows transcription, translation, and / or expression of the transgenes in target cells or tissues, flanked by 5' and 3' ITRs. The ITR sequences are usually about 145 bp in length. AAV ITR sequences can be modified using standard molecular biology techniques, for example, by inserting, deleting, or substituting one or more nucleotides, as long as the modification of the ITR sequence does not interfere with the function of the AAV vector. AAV ITRs are derived from any of several AAV serotypes. The AAV 3' and 5' ITR sequences may be derived from the same or different AAV serotypes.

[0099] 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 their variants.In some embodiments, the AAV vector is an AAV2 / 6, AAV2 / 8, or AAV2 / 9 vector (e.g., AAV6, AAV8, or AAV9 serotypes with AAV2 ITRs).Other AAV vectors are described, for example, in Sharma et al., Brain Res Bull. 2010 Feb 15;81(2-3):273.In general, any AAV serotype can be used to deliver the transgenes described herein.However, serotypes have different tropisms, for example, preferentially infecting different tissues. In some embodiments, AAV serotypes can be used that target a desired cell type or organ. For example, serotypes that target the central nervous system (including other targets) include, but are not limited to, AAV1, AAV2, AAV4, AAVVS, AAV6, AAV7, AAV8, AAV9, and AAV10. As another example, successful retinal gene transfer has been observed with serotypes AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, and AAV9. AAV7 and AAV8 have been shown to provide excellent, long-term delivery of genes to 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).

[0100] 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 exogenous conditions or compounds. Constitutive promoters are non-regulated promoters that allow for constitutive transcription of the associated gene. Inducible promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone-inducible promoter system, and the tetracycline repressor-inducible system. Constitutive promoters include the chicken beta-actin promoter, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, and the 3-actin promoter. In certain embodiments, when transgene expression that mimics native expression is preferred, the native promoter for the selected transgene, or a fragment thereof, can be used.

[0101] In some embodiments, the regulatory sequence confers tissue-specific gene expression. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are known in the art. For example, promoters active in the liver include the transthyretin (TTR) gene promoter, human alpha-1-antitrypsin (hAAT) promoter, lipoprotein AI promoter, hepatitis B virus core promoter, alpha-fetoprotein (AFP), human factor IX promoter, thyroxine-binding globulin (TBG) promoter, TTR minimal enhancer / promoter, alpha-antitrypsin promoter, and LSP1 promoter. Examples of enhancers active in the liver include the hepatic control regions 1 (HCR-1) and 2 (HCR-2) of apolipoprotein E (ApoE). As another example, suitable neuron-specific promoters include, but are not limited to, neuron-specific enolase (NSE) (GenBank Accession No: X51956) and human neurofilament light chain promoter (NEFL) (GenBank Accession No: L04147). Glial-specific promoters include, but are not limited to, the glial fibrillary acidic protein (GFAP) promoter (GenBank Accession No: M65210), the S100 promoter (GenBank Accession No: M65210), and the glutamine synthetase promoter (GenBank Accession No: X59834).

[0102] The transgenes of the gene therapy viral vectors described herein can encode a functional version of a protein that, through some deficiency in the endogenous version in the subject (including a deficiency in expression of the endogenous version), causes a disease or disorder in the subject.

[0103] Therapeutic proteins include myophosphorylase, glucocerebrosidase, fibroblast growth factor receptor 3, huntingtin, HFE protein, CFTR, frataxin, VMD2, hemoglobin, phenylalanine hydroxylase, fibrillin, dystopia myotonica protein kinase, lignoceroyl-CoA ligase, dystrophin, methyl-CpG-binding protein 2, beta-hemoglobin, myotubularin, cathepsin A, factor IX, lipoprotein lipase, beta-galactosidase, ornithine transcarbamylase, iduronate-2-sulfatase, acid alpha-glucosidase, UDP-glucuronosyltransferase 1-1, Glc Also included are NAc-1-phosphotransferase, GlcNAc-1-phosphotransferase, mucopilin-1, microsomal triglyceride transfer protein, sphingomyelinase, acid ceramidase, lysosomal acid lipase, alpha-L-iduronidase, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-1-CoA alpha-glucosaminide acetyl-1-transferase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine 6-sulfatase, alpha-mannosidase, alpha-galactosidase A, cystic fibrosis transmembrane regulator, and respiratory proteins.

[0104] By way of further example, therapeutic proteins may also be functional versions of proteins associated with disorders of lipid and sphingolipid degradation (e.g., beta-galactosidase-1, beta-hexosaminidase A and B, GM2 activator protein, 8-galactosidase A, glucocerebrosidase, glucocerebrosidase, glucocerebrosidase, arylsulfatase A, galactosylceramidase, sphingomyelinase, NPCI, HE). I protein (cholesterol transport disorders), acid ceramidase, lysosomal acid lipase); mucopolysaccharide degradation disorders (e.g., L-iduronidase, L-iduronidase, L-iduronidase, iduronate sulfatase, heparan N-sulfatase, N-acetylglucosaminidase, acetyl-1-CoA-glucosaminidase, acetyltransferase, acetylglucosamine-6-SulFAtase, galactosamine-6-SulFAtase, arylsulfatase, glycoprotein degradation disorders (e.g., mannosidase B, glucuronidase); glycoprotein degradation disorders (e.g., mannosidase, l-fucosidase, aspartylglycosaminidase, neuraminidase, lysosomal protective protein, lysosomal 8-N-acetylgalactosaminidase, lysosomal 8-N-acetylgalactosaminidase); lysosomal storage disorders (e.g., palmitoylprotein thioesterase, at least four subtypes, lysosomal membrane protein, glucose-6-phosphate dehydrogenase, phatase, glucose-6-phosphate translocase, acid maltase, debranching enzyme amylo-1,6 glucosidase, N-acetylglucosamine-1-phosphotransferase, N-acetylglucosamine-1-phosphotransferase, ganglioside sialidase (neuraminidase), lysosomal cystine transport protein, lysosomal cystine transport protein, lysosomal cystine transport protein, sialic acid transport protein saposin, A, B, C, D);and leukodystrophies (e.g., microsomal triglyceride transfer protein / apolipoprotein B, peroxisomal membrane transfer protein, peroxin, aspartoacylase, sterol-27-hydroxylase, proteolipid protein, ABCI transporter, peroxisomal membrane protein 3, or peroxisome biogenesis factor 1, phytanic acid oxidase);

[0105] 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 drug or component involved in the gene editing process. Generally, such a process results in long-term or permanent modifications to genomic DNA, such as the insertion, replacement, mutagenesis, or removal of target DNA. Gene editing involves delivering a nucleic acid encoding a desired DNA sequence and inserting the desired sequence into a target site in genomic DNA using an endonuclease. Thus, the gene editing transgene can include these nucleic acids encoding the desired DNA sequence 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 nucleic acids encoding one or more components capable of carrying out the gene editing process, alone or in combination with other components, as known in the art.

[0106] The viral vectors described herein can be used for gene expression regulation. In such embodiments, the transgene of the viral vector is a gene expression regulation transgene. Such a transgene encodes a gene expression regulator that can enhance, suppress (e.g., silence), or regulate the expression of one or more endogenous genes. The endogenous gene can encode any of the proteins described herein, provided that the protein is an endogenous protein of the subject. Thus, the subject may have any of the diseases or disorders described herein that would benefit from gene expression regulation.

[0107] Gene expression modulators include DNA-binding proteins (e.g., artificial transcription factors or transcriptional silencer proteins such as NRF) and therapeutic RNAs, including inhibitors of mRNA translation (antisense), RNA interference drugs (RNAi), catalytic RNA molecules (ribozymes), and RNAs that bind to proteins or other molecular ligands (aptamers).

[0108] Exemplary transgenes encode interfering RNA, antisense RNA, ribozymes, and aptamers that reduce the level of intracellular angiogenic factors. For example, RNAi can be miRNA, shRNA, or siRNA that reduces intracellular vascular endothelial growth factor (VEGF) levels. For example, RNAi can be shRNA or siRNA that reduces intracellular VEGF or VEGF receptor (VEGFR) levels. RNAi agents that target VEGF include, for example, the RNAi described in U.S. Patent Publication No. 2011 / 0224282. For example, VEGF-A, VEGFR1, or VEGFR2-specific siRNAs are suitable. Suitable nucleic acid gene products also include VEGF-A, VEGFR1, or VEGFR2-specific ribozymes; VEGF-A, VEGFR1, or VEGFR2-specific antisense; VEGF-A, VEGFR1, or VEGFR2-specific siRNA; and the like. Also suitable as gene product is miRNA, for example, by regulating the expression of VEGF gene through post-transcriptional repression or mRNA degradation, thereby reducing the level of VEGF.Examples of suitable miRNA include, for example, miR-15b, miR-16, miR-20a and miR-20b.See, for example, Hua et al. (2006) PLoS ONE 1:el16.Anti-VEGF aptamers (for example, EYEOOI) are also suitable.For anti-VEGF aptamers, see, for example, Ng et al. (2006) Nature Reviews Drug Discovery 5:123; and U.S. Patent Nos. 6,426,335; 6,168,778; 6,147,204; 6,051,620; and 6,011,020. See Nos. 6,426,335; 6,168,778; 6,147,204; 6,051,698; and 6,011,020. For example, aptamers can be directed to VEGF16s, the isoform primarily responsible for pathological ocular neovascularization and vascular permeability.

[0109] In some embodiments, the transgene encodes a polypeptide (e.g., an antibody or a fusion protein) that inhibits or reduces the activity of a disease-causing 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, VEGFR1 tyrosine kinase activity inhibitors; VEGFR2 tyrosine kinase activity inhibitors; antibodies against VEGF; antibodies against VEGFRI; antibodies against VEGFR2; soluble VEGFR; and the like. Antibodies specific to VEGF include, for example, bevacizumab (AVASTIN). (商標) ) and ranibizumab (also known as rhuFAb V2). Anti-angiogenic polypeptides such as endostatin, PEDF, and angiostatin are also suitable for use.

[0110] Anti-angiogenic polypeptides include, for example, recombinant polypeptides containing VEGF receptors. For example, a suitable anti-angiogenic polypeptide is the soluble form of VEGFR-1 known as sFlt-1 (Kendall et al. (1996) Biochem. Biophys. Res Commun. 226:324). Suitable anti-angiogenic polypeptides also include immunoglobulin-like (Ig) domain 2 of a first VEGF receptor (e.g., Fltl) alone or in combination with Ig domain 3 of a second VEGF receptor (e.g., Flkl or Flt4); anti-angiogenic polypeptides may also include stabilizing and / or multimerizing components. Such recombinant anti-angiogenic polypeptides are described, for example, in U.S. Pat. No. 7,521,049. Suitable anti-VEGF antibodies as heterologous gene products include single-chain Fv (scFv) antibodies. For anti-VEGF antibodies, see, for example, U.S. Pat. Nos. 7,758,859 and 7,740,844. Additional transgenes are described, for example, in Bordet et al., Drug Discovery 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.

[0111] In certain embodiments, the compstatin analogs described herein are produced in advance (i.e., not in a patient's cells or tissues) and incorporated into pharmaceutical compositions for use in various combination therapies, as described herein. These compstatin analogs can be prepared by various synthetic methods of peptide synthesis via condensation of one or more amino acid residues according to conventional peptide synthesis methods.

[0112] Alternatively, because the compstatin analogs disclosed herein are composed of naturally occurring amino acids, they can be produced by expressing encoding polynucleotides in suitable prokaryotic or eukaryotic systems. For example, DNA constructs can be inserted into plasmid vectors adapted for expression in bacterial cells (e.g., Escherichia coli) or yeast cells (e.g., Saccharomyces cerevisiae), or into baculovirus vectors for expression in insect cells, or viral vectors for expression in mammalian cells. Such vectors contain regulatory elements necessary for expression of DNA in host cells, arranged in a manner that allows expression of the DNA in the host cells. Such regulatory elements necessary 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 according to methods known in the art.

[0113] Pharmaceutical Compositions and Their Administration To achieve one or more of the utilities described herein, another aspect of the present invention features pharmaceutical compositions containing a vector carrying a compstatin analog polynucleotide as described and exemplified herein. Such pharmaceutical compositions can contain an active ingredient (e.g., a viral vector containing a transgene) in a form suitable for administration to a subject, or the pharmaceutical composition can contain the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or a combination thereof. These pharmaceutical compositions can be delivered to a subject to allow production of the polypeptide and the encoded protein. In some embodiments, the pharmaceutical composition contains sufficient genetic material to allow the recipient to produce a therapeutically effective amount of the protein in the subject.

[0114] The dosage may vary and depends on the type, onset, progression, severity, frequency, duration, or probability of the disease being treated, the desired clinical endpoint, previous or concurrent treatment, the subject's general health, age, sex, race, or immunological competence, and other factors that would be understood by one skilled in the art. The dosage, number of doses, frequency of doses, or duration of doses can be proportionally increased or decreased depending on side effects of the treatment or therapy, complications, or other risk factors and the condition of the subject. Those skilled in the art will understand the factors that can affect the dosage and timing required to provide an amount sufficient to provide a therapeutic or prophylactic benefit.

[0115] The dosage to achieve a therapeutic effect, e.g., the dosage (vg / kg) of vector genome / kg body weight of a viral vector such as rAAV, varies based on several factors, including, but not limited to, the route of administration, the choice of serotype of the AAV vector, the viral transduction efficiency in the selected tissue of interest, the heterologous polynucleotide expression level required to achieve a therapeutic effect, the particular disease being treated, the host immune response to the viral vector, the host immune response to the heterologous polynucleotide or expression product (protein), and / or the stability of the expressed protein. Those skilled in the art can determine the dosage range of a vector genome or non-viral vector for treating a patient with a particular disease or disorder based on the above-mentioned factors as well as other factors.

[0116] In embodiments utilizing vectors, the viral vector dose should be at least 1 x 10 per kilogram of subject body weight to achieve a therapeutic effect. 8 in the range of vector genomes (vg / kg), or more, e.g., 1x10 per kilogram of subject body weight 9 , 1x10 10 , 1x10 11 , 1x10 12 , 1x10 13 , or 1x10 14 In particular, the dose range for recombinant AAV vg / kg is approximately 1x10 11 From about 5x10 13vg / kg and up to approximately 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 The recombinant AAV vg / kg is in the range of 1000-150 ...

[0117] As used herein, "dosage unit" refers to a physically discrete unit suitable as a unitary dosage for a subject to be treated, each unit containing a predetermined amount, optionally in association with a pharmaceutical carrier, calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect) when administered one or more times. In various embodiments, dosage units may be contained within ampoules and vials and may comprise liquid compositions, or compositions in a lyophilized or lyophilized state, for example, to which a sterile liquid carrier may be added prior to administration or in vivo delivery. Individual dosage units may be included in multi-dose kits or containers. Viral particles, non-viral vectors, and pharmaceutical compositions thereof may be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0118] In some embodiments, dosages are calculated based on the amount of therapeutic gene product produced in the target cells or tissues of a subject. As noted above, such dosages will vary depending on the particular gene product and the disease being treated. However, since the compstatin analogs of the present disclosure can be produced in various embodiments, dosages of these peptides can be accommodated.

[0119] Pharmaceutical compositions useful for practicing the present invention can be administered to deliver a sufficient amount of vector to produce a dose of compstatin analog of about 0.0005 mg / kg to 50 mg / kg of body weight. In certain embodiments, the dose is 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, per day, or another suitable regular regimen. 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.

[0120] In one embodiment, the present invention contemplates intravenous or subcutaneous administration of a vector comprising a compstatin analog polynucleotide described herein to produce a dose of about 0.0005 mg / kg to about 10 mg / kg of a compstatin analog in a target cell or tissue, e.g., 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 (14.9uM), 2.25mg / kg, 2.5mg / kg, 2.75mg / kg, 3mg / kg, 3.25mg / kg, 3.5mg / kg, 3.75mg / kg, 4m g / kg, 4.25mg / kg, 4.5mg / kg, 4.75mg / kg, 5mg / kg, 5.25mg / kg, 5.5mg / kg, 5.75mg / kg, 6mg / kg , 6.25mg / kg, 6.5mg / kg, 6.75mg / kg, 7mg / kg, 7.25mg / kg, 7.5mg / kg, 7.75mg / kg, 8mg / kg, 8.2 5mg / kg, 8.5mg / kg, 8.75mg / kg, 9mg / kg, 9.25mg / kg, 9.5mg / kg, 9.75mg / kg, or 10mg / kg. In one embodiment, the vector is administered by intravenous or subcutaneous delivery (e.g., injection or infusion) to produce a dose of about 0.25 mg / kg to about 5 mg / kg of compstatin analog. In another embodiment, the dose is about 0.5 mg / kg to about 5 mg / kg. In yet another embodiment, the dose is about 0.5 mg / kg to about 4 mg / kg, or about 0.5 mg / kg to about 3 mg / kg.

[0121] In another embodiment, the invention contemplates intramuscular administration of a vector comprising a compstatin analog polynucleotide for production in a target cell or tissue, and contemplates a dose of about 0.0005 mg / kg to about 50 mg / kg, e.g., 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, g, 0.5mg / kg, 1mg / kg, 1.5mg / kg, 2mg / kg, 2.5mg / kg, 3mg / kg, 3.5mg / kg, 4mg / kg, 4.5mg / kg, 5mg / kg, 5.5mg / kg, 6mg / kg, 6.5mg / kg, 7mg / kg, 7.5mg / kg, 8mg / kg, 8.5mg / kg, 9mg / kg, 9.5mg / kg, 10mg / kg, 10.5mg / kg, 11mg / kg, 11.5mg / kg, 12mg / kg, 12.5mg / kg, 13mg / kg, 13.5mg / kg, 14mg / kg, 14.5mg / kg, 15mg / kg, 15.5mg / kg, 16mg / kg, 16.5mg / kg, 17mg / kg, 17.5mg / kg, 18mg / kg, 18.5mg / kg, 19mg / kg, 19.5mg / kg, 20mg / kg, 20.5mg / kg, 21mg / kg, 21.5mg / kg, 22mg / kg, 22.5mg / kg, 23mg / kg, 23.5mg / kg, 24mg / kg, 24.5mg / kg, 25m g / 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 preferred embodiment, the vector is administered via intramuscular delivery (e.g., injection) in an amount sufficient to produce a dose of the compstatin analog of between about 0.25 mg / kg and about 35 mg / kg.In other embodiments, the dose is between about 0.25 mg / kg and about 30 mg / kg, or between about 0.25 mg / kg and about 10 mg / kg, or between about 0.25 mg / kg and about 5 mg / kg. For example, in one embodiment, the intended dose of the compstatin analog is about 2.5 mg / kg.

[0122] In yet another embodiment, the vector is introduced by intravitreal administration to produce a dose of about 1 μg to about 10 mg (931 μM) of compstatin analog in the vitreous of the eye, e.g., 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, 5.5μg, 5.75μg, 6μg, 6.25μg, 6.5μg, 6.75μg, 7μg, 7.25μg, 7.5μg, 7.75μg, 8μg, 8.25μg, 8.5μg, 8.75μg, 9μg, 9.25μ g, 9.5μg, 9.75μg, 10μg, 20μg, 30μg, 40μg, 50μg, 60μg, 70μg, 80μg, 90μg, 100μg, 150μg, 200μg, 250μg, 300μg, 350 μg, 400μg, 450μg, 500μg, 550μg, 600μg, 650μg, 700μg, 750μg, 800μg, 850μg, 900μg, 950μg, 1mg, 1.1mg, 1.2mg, 1.3 mg, 1.4mg, 1.5mg, 1.6mg, 1.7mg, 1.8mg, 1.9mg, 2mg, 2.1mg, 2.2mg, 2.3mg, 2.4mg, 2.5mg, 2.6mg, 2.7mg, 2.8mg, 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, and preferably the dose is from about 1 μg to about 2,000 μg, for example, from about 10 μg to about 1,800 μg, or from about 100 μg to about 1,500 μg, or from about 500 μg to about 1,200 μg, or from about 500 μg to about 1,000 μg. In some embodiments, the therapeutically effective amount of a compstatin analog in the vitreous is at least about 0.02 mg, e.g., 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.

[0123] In another embodiment, pharmaceutical compositions containing compstatin or a polypeptide encoding it can be delivered to the central nervous system using doses within the ranges described herein. For example, studies have shown that intraparenchymal injection of rAAV results in localized distribution of rAAV, making it suitable for treating CNS disorders affecting specific brain regions, such as the putamen in Parkinson's disease. As another example, intrathecal injection into the cerebrospinal fluid space can achieve broader CNS distribution. (Nat Rev Drug Discov. 2019 May; 18(5):358-378).

[0124] In another embodiment, the present invention contemplates oral administration of the pharmaceutical compositions described herein. While oral administration is not common in vector-based gene therapy, it may be suitable for combination therapies in which the peptide is administered by one route and the vector by another. In this context, the peptide dose ranges from about 1 mg / kg to about 20 mg / kg, e.g., 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, and the like. , 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 compstatin analog peptide is administered via oral delivery at a therapeutically effective dose of about 1 mg / kg to about 10 mg / kg. For example, in certain embodiments, the peptide is orally delivered to a human at a dose of about 1 to 5 mg / kg. In some embodiments, the oral doses described herein are administered once. In other embodiments, the doses are administered periodically, such as daily.

[0125] In another embodiment, the present invention contemplates periodontal administration, such as intrapapillary infiltration, of a vector encoding a compstatin analog described herein at a dose of about 1 μg to about 1,000 μg, e.g., 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, 510 μg, 520 μg, 530 μg, 540 μg, 550 μg, 560 μg, 57 00μg, 210μg, 220μg, 230μg, 240μg, 250μg, 260μg, 270μg, 280μg, 290μg, 300 μg, 310μg, 320μg, 330μg, 340μg, 350μg, 360μg, 370μg, 380μg, 390μg, 400μg, The compstatin analog may be administered in the range of 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 1,000 μg. For example, periodontal administration of the vector to a human can result in a compstatin analog dose of about 5 μg to about 500 μg. In some embodiments, periodontal administration of the vector to a human results in a compstatin analog dose of about 10 μg to about 200 μg per interdental papilla, or about 20 μg to about 100 μg per interdental papilla.

[0126] In one embodiment, the vector is administered in an amount that results in a serum concentration of the compstatin analog in the individual of between about 0.01 nM and about 30 μM. In certain embodiments, the combination administration and regimen results in a serum concentration, or average serum concentration over time, of the compstatin 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).

[0127] In certain embodiments, the combined dose and regimen provides a serum concentration, or average serum concentration over time, 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 is 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, is 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.This results in a concentration of the compstatin analog of 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 dose and regimen provides a serum concentration, or average serum concentration over time, of up to about 0.1 μM, or up to about 0.11 μM, or up to about 0.12 μM, or up to about 0.13 μM, or up to about 0.14 μM, or up to about 0.15 μM, or up to about 0.16 μM, or up to about 0.17 μM, or up to about 0.18 μM, or up to about 0.19 μM, or up to about 0.2 μM, or up to about 0.3 μM, or up to about 0.4 μM, or up to about 0.5 μM, or up to about 0.6 μM, or up to about 0.7 μM, or up to about 0.8 μM, or up to about 0.9 μM, or up to about 1 μM, or up to about 1.5 μM, or up to about 2 μM, or up to about 2.5 μM, or up to about 3 μM, or up to about 3.5 μM, or up to about 4 μM, or up to about 4.5 μM, or up to about 5 μM, or up to about 5.5 μM, or up to about 6 μM, or up to about 6.5 μM, or up to about 7 μM, or up to about 7.5 μM, or up to about 8 μM, or up to about 8.5 μM, or up to about 9 μM, or up to about 9.5 μM, or up to about 10 μM, or up to about 10.5 μM, or up to about 11 μM, or up to about 11.5 μM, or up to about 12 μM, or up to about 12.5 μM, or up to about 13 μM, or up to about 13.5 μM, or up to about 14 μM, or up to about 14.5 μM, or up to about 15 μM, or up to about 15.5 μM, or up to about 16 μM, or up to about 16.5 μM, or up to about 17 μM, or up to about 17.5 μM, or up to about 18 μM, or up to about 18.5 μM, or up to about 19 μM, or up to about 19.5 μM, or up to about 20 μM, or up to about 20.5 μM, or up to about 21 μM, or up to about 21.5 μM, or up to about 22 μM, or up to about 22.5 μM, or up to about 23 μM, or up to about 23.5 μM, or up to about 24 μM, or up to about 24.5 μM, or up to about 25 μM, or up to about 25.5 μM, or up to about 26 μM, or up to about 26.5 μM, or up to about 27 μM, or up to about 27.5 μM, or up to about 28 μM, or up to about 28.5 μM, or up to about 29 μM, or up to about 29.5 μM, or up to about 30 μM.

[0128] Suitable ranges include about 0.1 to about 30 μM, or about 1 to about 29 μM, or about 2 to about 28 μM, or about 3 to about 27 μM, or about 4 to about 26 μM, or about 5 to about 25 μM, or about 6 to about 24 μM, or about 7 to about 23 μM, or about 8 to about 22 μM, or about 9 to about 21 μM, or about 10 to about 20 μM, or about 11 to about 19 μM, or about 12 to about 18 μM, or about 13 to about 17 μM, or about 1 to about 5 μM, or about 5 to about 10 μM, or about 10 to about 15 μM, or about 15 to about 20 μM, or about 20 to about 25 μM, or about 25 to about 30 μM. The exact dose to be 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, but such doses can be readily determined by one of ordinary skill in the art.

[0129] Thus, in certain embodiments, pharmaceutical compositions comprising compstatin analog transgenes and / or other transgenes and / or derivatives of natural amino acid-containing compstatin and other therapeutic molecules produced by other means are administered via subcutaneous, intradermal, intravenous, intraocular (including intravitreal, subretinal), intracerebral, intraperitoneal, intramuscular injection, periodontal administration (including gingival or intracapillary infiltration injection), intranasal, epidural, oral, sublingual, intrathecal, intravaginal, transdermal, rectal, inhalation, or topical administration.

[0130] The formulations of pharmaceutical compositions can be prepared by any method known or hereafter developed in the pharmaceutical arts. Generally, such preparation methods include the step of bringing the active ingredient into association with the carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0131] The pharmaceutical compositions described herein can be administered to patients as frequently as several times daily, or can be administered, for example, once daily, once weekly, once every two weeks, once a month, or even less frequently, such as once every few months, or once a year or less. The frequency of administration will be readily apparent to those skilled in the art and, as noted above, will depend on any number of factors, including, but not limited to, the type and severity of the disease being treated, and the type and age of the patient. However, as noted above, vectors producing the disclosed compstatin analogs can be administered at less frequent intervals than previously known compstatin analogs.

[0132] For example, in some embodiments, intravenous, intramuscular, intraocular (including intravitreal), subcutaneous, periodontal (including gingival administration or intracapillary infiltration), or topical administration of pharmaceutical compositions comprising the vectors described herein is via a single injection. Furthermore, given the extended residence time of the compstatin analogs described herein, certain embodiments contemplate chronic systemic administration of these compstatin analog polynucleotides at the above-described therapeutic doses, e.g., via multiple deliveries over time, via intravenous, intraocular (including intravitreal and subretinal), subcutaneous, intramuscular, periodontal (including gingival administration or intracapillary infiltration), or topical routes of administration, to provide a therapeutically effective maintenance dose of compstatin 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 from about once every 12 hours to about once every 3 months, or once every 5-6 months, e.g., 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 every month, once every 2 months, once every 3 months, or once every 5-6 months. In other embodiments, the vector is delivered from about once every 12 hours to about once every 3 months, e.g., 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 every month, once every 2 months, or once every 3 months.

[0133] As described above, pharmaceutical compositions containing compstatin analogs and / or other gene therapy encoding vectors and / or derivatives of natural amino acids containing compstatin and other therapeutic molecules produced by any other means can be formulated for administration by a variety of routes. Such pharmaceutical compositions can include pharmaceutically acceptable carriers and other components known to enhance and facilitate drug administration. Other formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically-based systems, can also be used to administer gene therapy vectors according to the methods of the present invention.

[0134] Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Carriers suitable for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), phosphate buffered saline (PBS), or Ringer's solution.

[0135] Sterile fixed oils are conventionally employed as solvents or suspending media. Any brand of fixed oil can be used for this purpose, including synthetic monoglycerides and diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful for preparing injectables, as are pharmaceutically acceptable natural oils, such as olive oil or castor oil, especially those that are polyoxyethylated. These oil solutions or suspensions may contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose, or similar dispersants commonly used in formulating pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers, or bioavailability enhancers commonly used in the production of pharmaceutically acceptable solid, liquid, or other dosage forms, can also be used for formulation purposes.

[0136] Generally, compositions should be sterile and fluid for easy syringability. Preferred pharmaceutical formulations are stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. In general, relevant carriers can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. It is often preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the compositions. Prolonged absorption of injectable compositions can be achieved by including in the composition agents that delay absorption, for example, aluminum monostearate and gelatin.

[0137] Sterile injectable solutions can be prepared by incorporating the active agent (vector) in the required amount in a suitable solvent, adding one or a combination of the above-listed ingredients as needed, and then sterilizing by filtration. Preferably, the injectable solution is endotoxin-free. Generally, dispersions are prepared by incorporating the active agent into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying, which yield a powder of the active agent and any additional desired ingredients from a previously sterile-filtered solution.

[0138] 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.Pharmaceutical 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 ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0139] For topical delivery to the eye, the pharmaceutical compositions provided herein can be suitably formulated, for example (but not limited to), in isotonic, pH-adjusted sterile saline or water, with or without a preservative, such as benzalkonium chloride. Alternatively, for ophthalmic use, the pharmaceutical compositions can be formulated as an ointment, such as petrolatum, or eye drops.

[0140] Methods of local administration to the eye include, for example, choroidal injection, transscleral injection or placement of a scleral patch, selective arterial catheterization, eye drops or ophthalmic ointment, intraocular administration including transretinal, subconjunctival bulbar, intravitreal injection, suprachoroidal injection, sub-Tenon injection, scleral pocket, and scleral cut-down injection, and osmotic pumps. Natural amino acid-containing Constatin peptides produced by vectors or other means can also be administered intravascularly, such as intravenously (IV) or intraarterially. For choroidal injection and scleral patch, after initiating appropriate anesthesia, including an analgesic and an ophthalmoplegic agent, the clinician or operator performs a local approach to the eye. A needle containing the pharmaceutical composition is aimed at the subject's choroid or sclera and inserted under sterile conditions. Once the needle is properly positioned, the composition is injected into either the choroid or sclera, or both. When using any of these methods, the clinician or operator can choose a sustained-release or long-acting formulation. Therefore, the procedure only needs to be repeated every few months to years, depending on the subject's tolerance and response to the treatment.

[0141] Intraocular administration of drugs is well known in the art. See, e.g., U.S. Patent Nos. 5,632,984, 5,770,589, and U.S. Patent Application Publication No. 5,770,589. U.S. Patent No. 6,378,526 provides a method for intrascleral injection of therapeutic or diagnostic substances overlying the retina, providing a minimally invasive technique for delivering drugs to the posterior capsule segment of the eye.

[0142] In some embodiments, a pharmaceutical composition containing a vector containing a transgene described herein is delivered near the eye, for example, close to the posterior capsule segment of the eye. "Near the eye" refers to a location within the orbit, the cavity within the skull where the eyeball and its appendages are located. Typically, the composition is delivered near the intended target within the eye, for example, close to (within a few millimeters of) the portion of the sclera covering the posterior capsule segment of the eye, or immediately adjacent to the outer surface of the sclera. In a preferred embodiment, the pharmaceutical composition of the present invention is delivered into the vitreous cavity of the eye (i.e., intravitreal).

[0143] Many polymeric delivery vehicles for providing sustained release have been used in the ophthalmic context and can be used to administer the pharmaceutical compositions of the present invention. Various polymers, including biocompatible polymers that may be biodegradable, can be used. For example, U.S. Patent No. 6,692,759 describes a method for manufacturing an implantable device for the sustained release of a therapeutic agent into the eye. U.S. Patent No. 6,692,759 describes a method for manufacturing an implantable device for the sustained release of a therapeutic agent into the eye. Other polymers and delivery systems useful for intraocular 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(beta-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 present invention.

[0144] Poly(orthoesters) have been introduced into the eye and have shown advantageous properties for sustained intraocular drug delivery (see Einmahl, S., 2002, Invest. Ophthalmol. Vis. Sci. 43(5)). Polylactic acid particles have been used to target drugs to the retina and RPE after intravitreal injection of a suspension of such particles (see Bourges et al., 2003, Invest. Ophthalmol. Vis. Sci. 44(8)). Macroscopic implantable devices suitable for introduction into the posterior or anterior capsular segment of the eye are referred to herein as ocular implants (see Jaffe, G., 2000, Invest. Ophthalmol. Hs. Sci., 41(11)). Thus, provided herein are ocular implants containing the vectors and / or compstatin analog polypeptides described herein, and optionally other suitable transgenes and other therapeutic agents for an individual. Such a device may be a macroscopic implant containing a pharmaceutical composition, or may contain a plurality of nanoparticles or microparticles impregnated or encapsulated with a drug. In one embodiment, the ocular implant is any ocular implant known in the art. Exemplary implants and methods for their manufacture are described, for example, in US 2009 / 0220572 A1. Other implants known in the art may also be used.

[0145] Other embodiments include gel-forming compositions containing soluble collagen useful for delivering therapeutic agents to the posterior capsule segment of the eye. Collagen is initially soluble and low in viscosity, but forms a solution that can rapidly form a gel under appropriate conditions, such as those encountered during administration to a mammalian subject. The present invention therefore provides a system for delivering pharmaceutically active agents to the posterior capsule segment of the eye. This system is designed to localize such molecules at sufficient concentrations and provide sustained delivery, while simultaneously releasing macromolecules in sufficient quantities. Furthermore, the collagen gel can protect the vector and the proteins produced therefrom from degradation.

[0146] The composition forms a gel after introduction into the body, for example, upon contact with physiological fluids. The composition can also form a gel upon contact with phosphate-buffered saline or other fluids containing appropriate ions. Thus, the composition can be injected into an appropriate location, for example, adjacent to the posterior capsule segment of the eye, where it forms a gel. Alternatively, a pre-shaped gel implant can be made, 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 any structure containing, for example, a hollow space or a concave depression into which a solution can be introduced. In another embodiment, a film or membrane is formed from a collagen solution containing a therapeutic agent.

[0147] For the treatment of chronic or acute pulmonary conditions involving complement activation or other diseases for which gene therapy is effective, pulmonary administration is the preferred route of administration of pharmaceutical compositions. Accordingly, pharmaceutical compositions of the present invention can be prepared, packaged, or sold as formulations suitable for pulmonary administration via the oral cavity. Such formulations can comprise dry particles comprising the active ingredient and having diameters ranging from about 0.5 to about 7 nanometers, preferably from about 1 to about 6 nanometers.

[0148] Pharmaceutical compositions of the present invention formulated for pulmonary delivery can also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations can be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions containing the active ingredient, optionally sterile, and conveniently administered using any nebulizer or spray device. Such formulations can further contain one or more additional ingredients, including, but not limited to, flavoring agents such as saccharin sodium, volatile oils, buffers, surface active agents including substituted pulmonary surfactants, or preservatives such as methylhydroxybenzoates. The droplets provided by this route of administration preferably have an average diameter in the range of about 0.1 to about 200 nanometers.

[0149] The formulations described herein as useful for pulmonary delivery are also useful for nasal delivery of the pharmaceutical compositions of the present invention. Another formulation suitable for nasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 to 500 micrometers. Such a formulation is administered in the same manner as snuff is taken, i.e., by rapid inhalation through the nasal passages from a container of the powder held close to the nostrils. Formulations suitable for nasal administration can, for example, contain from about 0.1% (w / w) to 100% (w / w) of the active ingredient and can further include one or more additional ingredients described herein.

[0150] As used herein, "parenteral administration" of a pharmaceutical composition includes any administration route characterized by physically breaking the target tissue and administering the pharmaceutical composition through the break in the tissue. Thus, parenteral administration includes, but is not limited to, administration by injection of the pharmaceutical composition, administration by applying the composition through a surgical incision, administration by applying the composition through a tissue-penetrating non-surgical wound, etc. In particular, parenteral administration includes, but is not limited to, intravenous, subcutaneous, intraperitoneal, intramuscular, intraarticular, intravitreal, intrasternal injection, and kidney dialysis infusion techniques.

[0151] Pharmaceutical compositions suitable for parenteral administration include 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 continuous administration. Injectable formulations can be prepared, packaged, or sold in unit dosage forms, such as ampoules or multi-dose containers containing a preservative. Oral formulations include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0152] Pharmaceutical compositions can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. These suspensions or solutions can be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations 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 fixed oils such as synthetic mono- or diglycerides. Other useful parenteral formulations include those containing the active ingredient in microcrystalline form, in liposomal formulations, in microbubbles for ultrasound-emitted delivery, or as a component of biodegradable polymer systems. Sustained-release or implantable compositions may contain pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsifiers, ion exchange resins, poorly soluble polymers, or poorly soluble salts.

[0153] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants, including substituted pulmonary surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that can 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.

[0154] Uses and Therapeutic Administration of Compstatin Analog Transgenes and Natural Amino Acid-Containing Compstatins The compstatin analog transgenes or natural amino acid-containing peptides described herein are useful for many purposes. In one embodiment, they can be used for any purpose for which compstatin and its analogs are utilized, so long as such use is suitable for generating the analog from a transgene. Thus, in certain embodiments, methods for modulating complement activation are applied to living patients or subjects and include some or all of methods for treating patients for diseases or disorders 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 mechanism that triggers 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 glomerulopathies; other complement-mediated nephropathies and glomerular inflammatory diseases; age-related macular degeneration (AMD); ophthalmic disorders characterized by macular degeneration, choroidal neovascularization (CNV), retinal neovascularization (RNV), proliferative vitreoretinopathy, glaucoma, uveitis, ocular inflammation, or any combination thereof; paroxysmal nocturnal hemoglobinuria (PNH); cold agglutinin disease (CAD); warm antibody autoimmune hemolytic anemias (wAIHAs); sickle cell disease; transplant-associated thrombotic microangiopathy; rheumatoid arthritis (RA); systemic lupus erythema (SLE). These conditions include, but are not limited to, systemic lupus erythematosus (SLE); some autoimmune and autoinflammatory kidney diseases; autoimmune myocarditis; multiple sclerosis; traumatic brain injury and spinal cord injury; ischemia-reperfusion (IR) injury of the brain, intestine, and kidney, spontaneous and recurrent abortion, antiphospholipid syndrome (APS), Parkinson's disease, Alzheimer's disease; abnormal synaptic remodeling; other neurodegenerative inflammatory diseases with excessive microglial activity and cognitive decline; asthma; antinuclear cytoplasmic antigen-associated microimmune vasculitis (Wegener's syndrome); pemphigus; bullous pemphigoid; non-lupus autoimmune skin diseases such as epidermolysis bullosa; post-traumatic shock; cancer; periodontitis; gingivitis; and atherosclerosis.In certain embodiments, the pathological condition is associated with mutations and polymorphisms in the 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, compstatin analogs produced by expression of the polynucleotides described herein are suitable for use as an alternative to eculizumab or pegcetacoplan in the treatment of diseases for which these drugs are currently prescribed or are being developed in preclinical and clinical trials. These diseases include, but are not limited to, aHUS, PNH, C3G (DDD / C3GN), CAD, AMD, NMOSD (neuromyelitis optical spectrum disorder), myasthenia gravis, and amyotrophic lateral sclerosis (ALS).

[0155] Blood and vascular diseases In certain embodiments, a compstatin analog transgene is administered to a subject suffering from or at risk of a complement-mediated blood-related disorder, such as paroxysmal nocturnal hemoglobinuria (PH), atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, chronic cold agglutinin disease, HELLP syndrome, and / or warm autoimmune hemolytic anemia. In some embodiments, a compstatin analog transgene is administered to a subject suffering from or at risk of a complement-mediated disorder affecting the circulatory system. For example, in some embodiments, the disorder is thrombotic microangiopathy (TMA) or vasculitis (e.g., IgA vasculitis) or other disorders associated with vascular inflammation, e.g., inflammation of blood vessels and / or lymphatic vessels. In some embodiments, the vasculitis is polyarteritis nodosa, hypocomplementemic urticarial vasculitis, pulmonary vasculitis, Wegener's granulomatosis, giant cell arteritis, Churg-Strauss syndrome, microscopic polyangiitis, microimmune vasculitis, Henoch-Schonlein purpura, Takayasu's arteritis, Kawasaki disease, or Behçet's disease. In some embodiments, the disorder is TMA secondary to atypical hemolytic uremic syndrome. In some embodiments, the subject is antineutrophil cytoplasmic antibody (ANCA) positive.

[0156] eye disorders In some embodiments, the compstatin analog transgene is administered to a subject for the treatment of a complement-mediated ocular disorder, such as macular degeneration (e.g., age-related macular degeneration (AMD) or Stargardt's macular dystrophy), diabetic retinopathy, glaucoma, or uveitis (e.g., posterior capsular uveitis or anterior capsular uveitis). In some embodiments, the subject has or is at risk for AMD. In certain embodiments, the AMD is neovascular (wet) AMD. In some embodiments, the AMD is dry AMD. As will be understood by those skilled in the art, dry AMD includes geographic atrophy (GA), intermediate AMD, and early AMD. In some embodiments, a subject with GA is treated to slow or stop disease progression.

[0157] Nervous system disorders In some embodiments, a compstatin analog transgene is used to treat a subject suffering from or at risk for a complement-mediated disorder affecting the nervous system, e.g., the central nervous system (CNS) and / or peripheral nervous system (PNS). Examples of such disorders include, for example, neurodegenerative diseases such as multiple sclerosis, other demyelinating diseases (e.g., neuromyelitis optica and chronic inflammatory demyelinating polyneuropathy (CIDP)), amyotrophic lateral sclerosis, chronic pain, fibromyalgia, stroke, intracerebral hemorrhage, allergic neuritis, acute or recurrent optic neuritis associated with myelin oligodendrocyte glycoprotein (MOG) or aquaporin (AQP)-4 antibodies, diabetic neuropathy, and Huntington's disease. In some embodiments, the subject suffers from neuropathic pain, e.g., pain resulting from a lesion involving the somatosensory pathway with damage to peripheral nerve fibers and / or the spinal-cortical system of the CNS.

[0158] Kidney damage In some embodiments, a compstatin analog transgene is used to treat a subject suffering from or at risk of a complement-mediated kidney disorder. Such disorders include, for example, nephritis, e.g., glomerulonephritis, e.g., membranoproliferative glomerulonephritis (MPGN) (e.g., MPGN type I, MPGN type II, or MPGN type III), e.g., 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, e.g., C3b. In some embodiments, the treatment described herein reduces the level of such deposits. In some embodiments, subjects suffering from complement-mediated nephropathy suffer from proteinuria (abnormally high levels of protein in the urine) and / or an abnormally low glomerular filtration rate (GFR). In some embodiments, the treatments described herein result in a decrease in proteinuria and / or an increase or stabilization of the GFR.

[0159] In these embodiments, the therapeutic method generally includes the steps of: (1) identifying a subject having a disease or condition treatable by modulation of complement activation as described herein (a "complement-mediated" disease, condition, or disorder); (2) measuring a parameter of the disease or condition treatable by modulation of complement activation using standard techniques well within the skill of one of ordinary skill in the art (e.g., biopsy, histology, MRI, bone scan, x-ray, pain tolerance, posture, etc.); (3) administering to the subject an effective amount of a compstatin analog transgene of the present invention using a treatment regimen and duration appropriate for the condition being treated; and (4) measuring the parameter of the disease or condition as an indicator that the disease or condition has been ameliorated or treated. Delivery of the transgene can be by any suitable route of administration known in the art, as described herein. Development of an appropriate dosage and treatment regimen will vary depending on many factors, including, but not limited to, the type of patient and the type of condition being treated, the patient's age, and the route of administration. Those skilled in the art are familiar with designing administration regimens that take such variables into account.

[0160] In some embodiments, a transgene encoding a compstatin analog can be administered systemically, e.g., intravenously or subcutaneously, for the treatment of a complement-mediated disorder described herein. In some embodiments, a local administration route may be used, e.g., when the disorder primarily affects a particular body system, organ, or tissue. For example, in some embodiments, a transgene encoding a compstatin analog can be administered intraocularly (e.g., intravitreally) to treat an ocular disorder. In some embodiments, a transgene encoding a compstatin analog is administered via the pulmonary route (e.g., to treat a disorder affecting the respiratory system). In some embodiments, a transgene encoding a compstatin analog is administered intradermally to treat a complement-mediated disorder affecting the central nervous system, as described herein. In some embodiments, intrapleural or intraventricular administration can be used to treat a complement-mediated disorder affecting the central nervous system.

[0161] In another embodiment, the compstatin analog transgene is introduced into living host cells along with other gene therapies to improve the efficiency of viral vector delivery and performance. Examples of such diseases or disorders include, for example, Santavuori-Hartier disease (infantile neuronal ceroid lipofuscinosis type 1), Jansky-Bielshowski 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 2), Forbes disease or Cori disease (glycogen storage disease type 3), mucolipidosis II (I-cell disease), mucolipidosis III (pseudo-Hurler polydystrophy), mucolipidosis IV (sialolipidosis), cystinosis (adult non-nephrotic form ... Lysosomal storage diseases / disorders, such as cystinosis (infantile nephropathy), cystinosis (juvenile or adolescent nephropathy), Salla disease / infantile sialic acid storage disease, and saposin deficiency; disorders of lipid and sphingolipid breakdown, 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 lipid and sphingolipid breakdown, such as Hurler syndrome (MPSI), Scheie syndrome (MPS) Mucopolysaccharidase disorders such as IS), Hurler-Scheie syndrome (MPS IH / S), Hunter syndrome (MPS II), Sanfilippo A syndrome (MPS IIIA), Sanfilippo B syndrome (MPS IIIB), Sanfilippo C syndrome (MPS IIIC), Sanfilippo D syndrome (MPS IIID), Morquio A syndrome (MPS IVA), Morquio B syndrome (MPS IVB), Maroteaux-Lamy syndrome (MPS VI), and Sly syndrome (MPS VII);Glycoprotein degradation diseases, such as alpha-mannosidosis, beta-mannosidosis, fucosidosis, aspirylglucosaminuria, mucolipidosis I (sialidosis), galactosialidosis, Schindler disease, and Schindler disease type II / Kanzaki disease; and leukodystrophic diseases / disorders, such as abetalipoproteinemia, neonatal adrenoleukodystrophy, Canavan disease, cerebrotendinous xanthomatosis, Pelizaeus-Merzbach disease, Tangier disease, infantile Refum disease, and classic Refum disease.

[0162] Further examples of such diseases / disorders of interest as described herein include acid maltase deficiency (e.g., Pompe disease, glycogen storage disease type 2, lysosomal storage diseases); carnitine deficiency; carnitine palmityltransferase deficiency; debranching enzyme deficiency (e.g., Cori's disease or Forbes disease, glycogen storage disease type 3); myoadenylate deaminase deficiency; phosphofructokinase deficiency (e.g., Tarui's disease, glycogen storage disease type 7); phosphogylcerate kinase deficiency (e.g., phosphoglucose deficiency (e.g., glycogen storage disease type 9); phosphoglucose deficiency (e.g., glycogen storage disease type 10); phosphorylase deficiency (e.g., McArdle disease, myophosphorylase deficiency, glycogen storage disease type 5); Gaucher disease (e.g., chromosome 1, where the enzyme glucocerebrosidase is affected); achondroplasia (e.g., chromosome 4, where fibroblast growth factor receptor 3 is affected); Huntington's disease (e.g., chromosome 4, Huntington's); hemochromatosis (e.g., chromosome 6 , HFE protein); cystic fibrosis (e.g., chromosome 7, CFTR); Friedreich's ataxia (chromosome 9, frataxin); Best's disease (chromosome 11, VMD2); sickle cell disease (chromosome 11, hemoglobin); phenylketonuria (chromosome 12, phenylalanine hydroxylase); Marfan syndrome (chromosome 15, fibrillin); myotonic dystrophy (chromosome 19, dystrophic myotonin protein kinase); adrenoleukodystrophy (x chromosome, lignoceroyl-CoA ligase in peroxisomes); Duchenne muscular dystrophy (x chromosome, dystrophin); Rett syndrome (x chromosome, methyl-CpG-binding protein 2); Leber's hereditary optic neuropathy (mitochondrial, respiratory protein); mitochondrial encephalomyopathy, lactic acidosis, and stroke (MELAS) (mitochondrial, transfer RNA); and urea cycle enzyme deficiencies.

[0163] Further 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, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, b-thalassemia, and Duchenne muscular dystrophy. Further examples of diseases or disorders include those resulting from deficiencies in lipid and sphingolipid degradation, mucopolysaccharide degradation, glycoprotein degradation, leukodystrophies, etc.

[0164] The improved efficacy of viral vector-mediated gene therapy by combined administration with the compstatin analog transgene of the present invention can be seen in many ways. For example, in some embodiments, combined administration allows the gene therapy to be administered in a lower dose, less frequently, and / or less frequently than an effective administration regimen for gene therapy alone, and these differences are easily measured. The efficacy of gene therapy in this regard can be evaluated, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, or more after administration of the therapeutic agent.

[0165] In some embodiments, the efficacy of gene therapy is measured or indicated by the period without recurrence of disease signs or symptoms.

[0166] In some embodiments, humoral and / or cellular responses to administration of the viral vector can be measured. For example, a decrease in humoral response is measured or indicated by a decrease in the magnitude or fold decrease in response from baseline in antibody (e.g., neutralizing antibody) levels. In some embodiments, the antibody level is the level of an antibody against the viral vector, e.g., a capsid protein. In some embodiments, baseline is a value, level, amount, or quantity measured or indicated in a subject prior to administration of gene therapy or in a control subject not receiving gene therapy. In some embodiments, a decrease in humoral response is indicated by at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease in antibody titer from baseline.

[0167] The cellular response is indicated or measured by secretion of granzyme B (GrB) and / or IFNy. In some embodiments, a decrease in the cellular response is measured or indicated by a decrease in the magnitude or fold decrease in the response from baseline GrB and / or IFNy levels. In some embodiments, a decrease in the cellular response is indicated by at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease in GrB and / or IFNy levels from baseline. In some embodiments, baseline is a value, level, amount, or quantity measured or indicated in a subject prior to administration of gene therapy or in a control subject not administered gene therapy.

[0168] In some embodiments, the efficacy of gene therapy is measured by the presence or level of expression of a compstatin analog transgene or other transgene described herein, and / or the level or activity of the protein encoded by the transgene. For example, combined delivery of a compstatin analog transgene and other gene therapy results in transgene levels in a subject (e.g., in the subject's cells or tissues) that are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, or more after combined treatment, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, or more, relative to the corresponding transgene level in a subject not administered the compstatin analog transgene.

[0169] In some embodiments, the efficacy of gene therapy is measured by a stable expression level of the transgene in a subject over a period of, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, or more, relative to the corresponding expression level of the transgene over the same period in a control subject (e.g., a control subject who has undergone gene therapy and has not received a compstatin analog transgene). In some embodiments, a stable expression level is an expression level that differs by less than 30%, 25%, 20%, 15%, 10%, or 5% over a defined period of time.

[0170] In some embodiments, the efficacy of gene therapy with 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 level and / or activity of the target polypeptide. In some embodiments, combined treatment with a compstatin analog transgene and other gene therapies results in the expression level of the target gene and / or the expression level and / or activity of the target polypeptide in a subject (e.g., a cell or tissue of a subject) being reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the corresponding expression and / or activity level in a subject not administered the compstatin analog transgene, e.g., over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, or more.

[0171] In another embodiment, a compstatin analog transgene is used with other transgenes in a combination treatment of a disease or condition in which both complement activation and another gene therapy treatable dysfunction contribute to the disease or condition.

[0172] In some embodiments, the therapeutic effect of the combined administration of a compstatin analog transgene and additional gene therapy on a disease or condition, or a symptom thereof, is greater than the effect produced by either gene therapy or treatment with the compstatin analog alone. The difference between the combined effect and the effect of gene therapy alone or the compstatin analog alone can be statistically significant. In some embodiments, the combined result is synergistic.

[0173] These diseases or conditions may be any or a combination of the diseases and conditions listed herein in which complement activation plays a role and which may benefit from one or more of the gene therapies listed above.

[0174] As a specific example, the vascular endothelial growth factor (VEGF) family of growth factors regulates pathological angiogenesis and increased vascular permeability in important ocular diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Complement activation also plays an important role in the development of these diseases. Thus, combining a vector encoding a compstatin analog transgene with a transgene for VEGF regulation can provide a dual-mechanism treatment.

[0175] In one embodiment, a VEGF-regulating transgene, e.g., as referenced elsewhere herein, is administered in conjunction with a compstatin analog transgene of the invention via a viral vector as described herein. The compstatin analog produced by expression of the transgene in target cells or tissues serves a dual function: (1) reducing the immunogenicity of the viral vector, improving vector persistence and production of the VEGF-regulating gene product, and (2) directly treating the underlying disease or condition.

[0176] The VEGF-regulating transgene and the compstatin analog transgene can be incorporated into one or more viral vectors. In one embodiment, both transgenes are contained in a single viral vector. In another embodiment, the compstatin analog transgene is placed together with the VEGF-regulating transgene to generate a fusion protein, in which the compstatin analog is linked to the VEGF-regulating gene product. The linkage can be direct, or there can be a spacer or linker between the compstatin analog and the VEGF-regulating gene product.

[0177] In some embodiments, the transgenes encoding the compstatin analogs and VEGF inhibitors described herein can be used to treat subjects suffering from or at risk of a complement-mediated disorder characterized by, or associated with, pathological angiogenesis and / or pathological vascular permeability, or an increased risk of developing pathological angiogenesis and / or pathological vascular permeability. In some embodiments, the protein is a modified VEGF-inhibiting Fab having a compstatin analog fused to the heavy or light chain of the Fab, as described herein. Pathological angiogenesis and / or pathological vascular permeability refers to angiogenesis or vascular permeability that is abnormal, excessive, and / or 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 suffering from GA, but the eye has not been diagnosed with neovascular AMD. In some embodiments, the protein is administered to an eye suffering from neovascular AMD, but the eye has not been diagnosed with GA. In some embodiments, the protein is administered to an eye suffering from neovascular AMD and GA. In some embodiments, the protein is administered to an eye suffering from neovascular AMD and intermediate AMD. In some embodiments, the protein is administered to an eye suffering from neovascular AMD, but the eye has not been diagnosed with GA. In some embodiments, the eye disorder is choroidal neovascularization (CNV), which may be a symptom of AMD or may be caused by other causes. In some embodiments, the eye 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.

[0178] In yet another embodiment, the compstatin analog peptide itself is used in place of the transgene to improve the efficacy of and / or provide synergy with gene therapy. These methods involve administering one or more compstatin analog peptides to a subject who is undergoing, has previously undergone, or will undergo gene therapy as described herein.

[0179] In some embodiments, the compstatin analog is administered to a subject who has received one or more doses of gene therapy, or who is receiving gene therapy simultaneously or sequentially, as discussed 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 more before administering the compstatin analog. In other embodiments, the compstatin analog is administered to a subject who has not received gene therapy but may receive gene therapy in the future.

[0180] In some embodiments, the compstatin analog and 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 compstatin analog and gene therapy are administered sequentially (e.g., 1 hour or more, 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 2 weeks or more, 4 weeks or more, or more than a day apart). In some embodiments, the subject is pretreated with a compstatin analog prior to receiving gene therapy, e.g., from a few minutes to 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 24 hours or more prior to gene therapy administration.

[0181] Pre-treatment, co-treatment, or post-treatment can include a single dose or multiple doses of a compstatin analog, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. Multiple doses can be administered minutes, hours, or days before, during, or after gene therapy. Administration of a compstatin analog can also span multiple courses of gene therapy.

[0182] In some embodiments, a compstatin analog is administered for a period of time to inhibit complement activation until the gene therapy viral vector is taken up by one or more target cells (e.g., until it is taken up by the target cells to a certain level or extent). Cellular uptake is indicated or measured in the subject using an assay that detects a decrease in the level of the viral vector in a sample (e.g., a serum sample) obtained from the subject and / or an increase in the level of the viral vector in one or more target cells. In some embodiments, the compstatin analog is administered in combination with immunosuppressive therapy.

[0183] Embodiments utilizing a preformed compstatin analog (instead of or in addition to a transgene) can include delivery methods that include a combination of administration routes and dosages. For example, a composition containing the peptide can be administered orally, subcutaneously, intravenously, intraocularly, or intramuscularly, and a vector containing the transgene can be administered by the same route or by a different route, at the same time or at different times, as described above.

[0184] In some embodiments, the combination treatment results in an improvement in gene therapy (e.g., an improvement in a disease or disorder described herein or a symptom thereof) in the subject over a specified period of time, as discussed above for embodiments involving the use of a compstatin analog transgene.

[0185] The following examples are provided to more fully describe the present invention and are intended to be illustrative and not limiting. [Example]

[0186] Example 1. Efficacy of the Constatin Analog Cp50 on Laser-Induced Choroidal Neovascularization in a Non-Primate Model of Exudative Age-Related Macular Degeneration

[0187] The effects of the compstatin analog Cp50 (described above) were compared to vehicle in a non-human primate model of wet age-related macular degeneration (AMD), in which laser-induced choroidal neovascularization (CNV) was performed in macaque eyes.

[0188] Experimental design Laser-induced CNV was performed in both eyes of macaques as described below. Two weeks after laser photocoagulation (day 14), laser lesions were graded (grades 1-4) via fluorescein angiography (FA). Eyes with a high percentage of grade 4 lesions were selected and randomly assigned to treatment groups.

[0189] On day 15, under ketamine (15 mg / kg, im) and xylazine (2 mg / kg, im), eyes were intravitreally injected with either vehicle or compstatin Cp50 (drug or ). FA measurements were performed at weeks 4, 6, 8, 10, and 12 (a total of five times) after CNV.

[0190] method Laser-induced choroidal neovascularization (CNV) Under mild ketamine (10 mg / kg, im) anesthesia, pupils were dilated with tropicamide / phenylephrine (Midrin P® eye drops; Santen Pharmaceutical Co., Ltd., Osaka, Japan). For laser treatment, macaques were anesthetized with a combination of ketamine (15 mg / kg, im, Daiichi Sankyo Propharma Co., Ltd., Tokyo, Japan) and xylazine (2 mg / kg, im, Bayer Yakuhin, Osaka, Japan). A green laser (900 mW output, Green Scan Laser Photocoagulator, GYC-500, Nidek Co., Ltd., Gamagori, Japan) was applied in a grid pattern to eight spots (approximately 75 μm, 0.1 s) around the macula. The extent of the CNV lesion was assessed using fluorescein angiography (FA) at predetermined time points.

[0191] On the day of lesion grading, under light ketamine (10 mg / kg, im) anesthesia, the pupils were dilated with tropicamide / phenylephrine (Midrin-P® eye drops), and then the macaques were anesthetized with a combination of ketamine (15 mg / kg, im) and xylazine (2 mg / kg, im).

[0192] Fluorescein angiography (FA) The extent of CNV in the retrobulbar segment was confirmed using fluorescein angiography (FA). 10% sodium fluorescein (Fluorescein®; 10 mg / kg, iv, Novartis, Tokyo, Japan) was injected, and images of the retrobulbar segment were taken with a fundus camera approximately 1, 3, and 5 minutes after intravenous injection. Lesions are graded as follows: Grade 1: No hyperfluorescence observed. Grade 2: The lesion shows hyperfluorescence without leakage. Grade 3: The lesion shows hyperfluorescence on early or mid-stage images and exhibits delayed leakage. Grade 4: The lesion shows progressively bright hyperfluorescence and late leakage beyond the treated area. After measuring FA, the vitreous humor was removed.

[0193] Intravitreal injection After randomization, mice received a 50 μl intraocular injection (IVT) of either vehicle or Cp50 under ketamine (15 mg / kg, im) and xylazine (2 mg / kg, im) on day 15. Prior to the IVT injection, eyes were treated with levofloxacin hydrate (0.5%; Cravit®, Santen Pharmaceutical Co., Ltd.) twice daily for 3 days.

[0194] Under anesthesia, tropicamide / phenylephrine hydrochloride (Midrin® P), levofloxacin hydrate (0.5 mg / mL; Santen Pharmaceutical Co., Ltd.), and oxybuprocaine hydrochloride (0.4%; Santen Pharmaceutical Co., Ltd.) were instilled prior to administration.

[0195] result At weeks 4, 6, 8, and 12, the mean grade 4 lesions in compstatin Cp50-treated eyes were reduced compared with vehicle-treated eyes (Figure 1). In two Cp50-treated eyes, CNV lesion size decreased, becoming evident by week 4 after laser-induced CNV. Cp50 treatment led to a complete (100%) reduction in grade 4 lesion rate from week 6 onward until the end of the experimental period of CNV lesion monitoring (week 12). The third Cp50-treated animal (K-2434) showed a consistent trend toward a decrease in CNV lesion size, decreasing to 50% of baseline by week 6 and continuing to decrease to 25% by week 12. In contrast, vehicle-treated eyes maintained their grade 4 CNV lesion scores.

[0196] The present invention is not limited to the embodiments described and exemplified herein, but is capable of variation and modification within the scope of the appended claims.

Claims

1. 1. 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), Xaa1 is absent or contains the dipeptide Tyr-Ile, Xaa2 is Ala or Glu, Xaa3 is absent or He, and Xaa4 is absent or represents 1, 2, or 3 Lys residues, and A compstatin analog in which the Cys residues form a disulfide bond to form a cyclic peptide comprising the sequence in brackets.

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

3. 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), 3. The compstatin analog of claim 1 or 2, comprising:

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

7.

5. A peptide consisting of SEQ ID NO:6 or SEQ ID NO:

7.

6. 2. The compstatin analog of claim 1, selected from the group consisting of 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 comprising a sequence encoding the compstatin analog or peptide of any one of claims 1 to 6.

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

7.

9. 9. The polynucleotide of claim 8, selected from SEQ ID NO: 17 and SEQ ID NO:

18.

10. The polynucleotide of claim 7 arranged in an expression cassette or vector.

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

12. 12. The polynucleotide of claim 11, wherein the vector is adapted for expression in a prokaryotic or eukaryotic expression system and / or is used in gene therapy.

13. 13. The polynucleotide of claim 12, wherein the vector is used in gene therapy and is selected from a retrovirus, an adenovirus, an adeno-associated virus (AAV), and herpes simplex virus type 1.

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

15. 15. The polynucleotide of claim 14, wherein the AAV vector is an AAVI, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 vector, or a mutant of any of them.

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

7.

17. 17. The vector of claim 16, comprising at least one insertion site for at least one transgene for gene therapy delivery.

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

18.

19. 17. The vector of claim 16, which is selected from a retrovirus, an adenovirus, an adeno-associated virus (AAV), and a herpes simplex virus type 1.

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

21. 21. The vector of 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 of claim 16, which comprises at least one other transgene for gene therapy.

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

24. 23. The vector of claim 22, wherein the gene therapy is for the treatment of a blood disorder, an eye disorder, an autoimmune disease, a muscle disorder, a nerve disorder, or cancer.

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

26. 23. The vector of claim 22, wherein the compstatin analog polynucleotide and the transgene are positioned on the vector to produce a fusion protein comprising the compstatin analog and the transgene product.

27. 27. The vector of claim 26, wherein the fusion protein comprises a compstatin analog directly linked to a transgene product.

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

29. 27. The vector of claim 26, wherein the transgene encodes a VEGF inhibitor.

30. 30. The vector of claim 29, wherein the VEGF inhibitor comprises the extracellular domain of a VEGF receptor.

31. 30. The vector of claim 29, wherein the VEGF inhibitor comprises an antibody fragment selected from the group consisting of a Fab, F(ab')2, Fv, scFv, or single domain antibody.

32. The vector of claim 31, wherein the antibody fragment is a Fab comprising a VL domain, a CL domain, a VH domain, and a CHI domain.

33. (i) the VL of Fab, (ii) CL of Fab; (iii) the VH of a Fab, or (iv) Fab CHI 33. The vector of claim 32, encoding a fusion protein comprising a compstatin analog directly or indirectly fused to one, two, three, or four of:

34. 34. The vector of claim 33, encoding a fusion protein comprising a compstatin analog fused directly or indirectly to the N-terminus of the VL of a Fab, the C-terminus of the CL of a Fab, the N-terminus of the VH of a Fab, or the C-terminus of the CHI of a Fab.

35. A pharmaceutical composition comprising at least one vector according to any one of claims 16 to 34 and a pharmaceutically acceptable carrier.

36. 36. The pharmaceutical composition of claim 35, 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 intrapapillary infiltration injection), intranasal, epidural, oral, sublingual, intrathecal, intravaginal, transdermal, rectal, inhalation, or topical.

37. 36. The pharmaceutical composition of claim 35, formulated for systemic administration.

38. 36. The pharmaceutical composition of claim 35, formulated for topical administration.

39. 39. The pharmaceutical composition of claim 38, wherein the local administration is to the brain and / or central nervous system, eyes, lungs and / or respiratory system, heart and / or vascular system, lymphatic system, kidneys, spleen, pancreas, liver, gastrointestinal system, periodontal tissue, skin, bone, joint or synovial fluid, or any combination thereof.

40. 36. The pharmaceutical composition of claim 35, further comprising a compstatin 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 compstatin 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. 42. The pharmaceutical composition of claim 41, 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 intrapapillary infiltration injection), intranasal, epidural, oral, sublingual, intrathecal, intravaginal, transdermal, rectal, inhalation, or topical.

43. 42. The pharmaceutical composition of claim 41, formulated for systemic administration.

44. 42. The pharmaceutical composition of claim 41, formulated for topical administration.

45. 45. The pharmaceutical composition of claim 44, wherein the local administration is to the brain and / or central nervous system, eyes, lungs and / or respiratory system, heart and / or vascular system, lymphatic system, kidneys, spleen, pancreas, liver, gastrointestinal system, periodontal tissue, skin, bone, joint or synovial fluid, or any combination thereof.

46. 42. The pharmaceutical composition of claim 41, further comprising at least one vector according to any one of claims 16 to 34.

47. A kit comprising a plurality of pharmaceutical compositions, at least one pharmaceutical composition is a peptide-containing composition comprising a compstatin 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 A kit, wherein at least one pharmaceutical composition is a vector-containing composition comprising the vector according to any one of claims 16 to 34 in a pharmaceutically acceptable carrier.

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

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

50. The peptide-containing composition is formulated for a selected route of administration, and the vector-containing composition is formulated for a route of administration different from the route of administration selected for the peptide-containing composition; 48. The kit of claim 47, optionally comprising instructions for administering the peptide-containing composition on a schedule different from the schedule for administering the vector-containing composition.

51. A kit comprising a plurality of pharmaceutical compositions, each of which is a vector-containing composition comprising the vector according to any one of claims 16 to 34 in a pharmaceutically acceptable carrier.

52. 52. The kit of claim 51, wherein at least one pharmaceutical composition is formulated for systemic administration.

53. 52. The kit of claim 51, wherein at least one pharmaceutical composition is formulated for topical administration.

54. 52. The kit of claim 51, wherein the pharmaceutical compositions are formulated for different routes of administration, optionally including instructions for administering one composition on a schedule that differs from the schedule for administering the other composition.

55. 1. A method of treating a subject having or at risk for a complement-mediated disorder, comprising: A method comprising administering to a subject a composition comprising at least one 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.

56. 56. The method of claim 55, wherein after administration of the composition, the level of complement activity in the subject or in a biological sample from the subject is reduced compared to the level before administration of the composition or compared to the level in a comparable subject that has not been administered the composition.

57. 57. The method of 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% compared to the level before the administration or compared to the level in a comparable subject not administered the composition.

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

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

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

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

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

63. 56. The method of claim 55, wherein the complement-mediated disorder involves complement-mediated red blood cell damage, and optionally is paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome.

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

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

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

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

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

69. 56. The method of claim 55, wherein the composition is administered to the eye of a subject suffering from an eye disorder.

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

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

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

73. 56. The method of claim 55, (1) providing a target; (2) administering the composition to the subject, thereby producing a compstatin analog in the subject; and (3) measuring one or more parameters of complement-mediated damage; A method comprising:

74. 74. The method of claim 73, wherein the measurements are performed before, during, and / or after administration of the composition.

75. 74. The method of claim 73, wherein the measurements are performed on a comparable subject who has not been administered the composition.

76. 56. The method of claim 55, wherein the complement-mediated disorder is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS); dense deposition disease (DDD); C3 glomerulonephritis (C3GN); C3 glomerulopathy; complement-mediated nephropathy and glomerular inflammatory disease; age-related macular degeneration (AMD); macular degeneration, choroidal neovascularization (CNV), retinal neovascularization (RNV), proliferative vitreoretinopathy, glaucoma, uveitis, ocular inflammation, and any combination thereof; an eye disorder characterized by 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. the method is selected from the group consisting of systemic lupus erythematosus (SLE), autoimmune and autoinflammatory kidney disease, autoimmune myocarditis, multiple sclerosis, traumatic brain injury and spinal cord injury; ischemia-reperfusion (IR) injury of the brain, intestine, and kidney, spontaneous and recurrent abortion, antiphospholipid syndrome (APS), Parkinson's disease, Alzheimer's disease; neurodegenerative inflammatory diseases with abnormal synaptic remodeling, microglial activity, and cognitive decline; asthma; antinuclear cytoplasmic antigen-associated microimmune 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. 77. The method of any one of claims 55 to 76, wherein the subject is a non-human primate or a human.

78. 78. The method of any of claims 55 to 77, comprising administering two or more doses of the composition to the subject.

79. 79. The method of claim 78, comprising administering doses of the composition to the subject at predetermined time intervals.

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

81. 1. A method of treating a subject having or at risk for a disease or disorder having a complement-mediated component and one or more other components, 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 that treats one or more other components; Administering to a subject a composition comprising at least one vector comprising:

82. 82. The method of claim 81, wherein after administration of the composition, the levels of complement activation and / or other components in the subject or in a biological sample from the subject are reduced or improved compared to the levels before administration of the composition or compared to the levels in a comparable subject that has not been administered the composition.

83. 83. The method of claim 82, wherein the levels of complement activation and / or other components are 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% compared to levels before administration or compared to levels in a comparable subject who has not received the composition.

83. 82. The method of claim 81, wherein the other component is associated with a neovascular phenotype.

84. 82. The method of claim 81, wherein the subject is a non-human primate or a human.

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

86. 86. The method of claim 85, wherein the eye disorder is age-related macular degeneration (AMD).

87. 87. The method of claim 86, wherein the transgene encodes a VEGF inhibitor.

88. 82. The method of claim 81, wherein the vector is an adeno-associated virus (AAV) vector.

89. 89. The method of claim 88, wherein the AAV vector is an AAV1, AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector.

90. 82. The method of claim 81, wherein the transgene encodes a therapeutic protein, enzyme, hormone, blood clotting factor, cytokine, or growth factor.

91. 82. The method of claim 81, wherein the compstatin analog polynucleotide and the transgene are placed in a single vector to produce a fusion protein comprising the compstatin analog and the transgene product.

92. 92. The method of claim 91, wherein the fusion protein comprises a compstatin analog linked to a transgene product, either directly or via a linker or spacer.

93. 92. The method of claim 91, wherein the transgene encodes a VEGF inhibitor.

94. 82. The method of claim 81, wherein the compstatin analog polypeptide and at least one other transgene are located on two or more separate vectors.

95. 95. The method of claim 94, wherein the vector comprising the compstatin analog and the one or more vectors comprising at least one other transgene are administered by different routes.

96. 96. The method of claim 95, wherein the vector containing the compstatin analog is administered systemically and one or more vectors containing at least one other transgene are administered locally.

97. 95. The method of claim 94, wherein the vector comprising the compstatin analog is administered before, during, or after administration of one or more vectors comprising at least one other transgene.

98. 82. The method of claim 81 , (1) providing a target; (2) administering the composition to the subject, thereby producing the compstatin analog and the transgene in the subject; and (3) measuring one or more parameters or other components of complement-mediated injury; A method comprising:

99. 99. The method of claim 98, wherein the measurements are performed before, during, and / or after administration of the composition, and / or are performed on a comparable subject who has not received the composition.

100. 100. The method of any one of claims 81 to 99, comprising replacing the compstatin analog polynucleotide or a vector containing a compstatin analog polynucleotide with the compstatin analog peptide itself.

101. A method for improving the effectiveness of gene therapy in a subject undergoing, currently undergoing, or who has undergone gene therapy, comprising administering to the subject a vector containing a compstatin analog polynucleotide that produces a compstatin analog having SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 in the subject, thereby improving the effectiveness of the gene therapy.

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

103. 103. The method of 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.

104. 102. The method of claim 101, wherein efficacy is assessed by observing or measuring a reduction in the immune response to the gene therapy.

105. 102. The method of claim 101, wherein efficacy is measured by observing or measuring an improvement in transduction of a viral vector carrying the transgene.

106. 102. The method of claim 101, wherein efficacy is assessed by observing or measuring a reduction in complement-mediated clearance of the viral vector carrying the transgene.

107. A method according to any one of claims 101 to 106, wherein the effectiveness (i) comparing subjects who received a vector comprising a compstatin analog polynucleotide with control subjects who did not receive a vector comprising a compstatin analog polynucleotide; and / or (ii) comparing a subject during or after receiving a vector containing a compstatin analog polynucleotide with the same subject before receiving a vector containing a compstatin analog polynucleotide; Measured by,method.

108. 102. The method of claim 101, wherein the vector comprising the compstatin analog polynucleotide comprises at least one insertion site for at least one transgene for gene therapy delivery.

109. 102. The method of claim 101, wherein the vector comprising the compstatin analog polynucleotide is selected from a retrovirus, an adenovirus, an adeno-associated virus (AAV), and a herpes simplex virus type 1.

110. 110. The method of claim 109, wherein the vector comprising the compstatin analog polynucleotide is an adeno-associated virus (AAV) vector.

111. 111. The method of claim 110, wherein the AAV vector is an AAVI, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 vector, or any mutant or combination thereof.

112. 102. The method of claim 101, wherein the vector containing the compstatin analog polynucleotide contains at least one other transgene for gene therapy.

113. 102. The method of claim 101, wherein the vector containing the compstatin analog polynucleotide is different from another vector containing another transgene for gene therapy, and the vectors are administered together.

114. 102. The method of claim 101, wherein the vector containing the compstatin analog polynucleotide is distinct from and administered separately from another vector containing another transgene for gene therapy.

115. 115. The method of any of claims 101-114, comprising replacing a compstatin analog polynucleotide or a vector containing a compstatin analog polynucleotide with the compstatin analog peptide itself.

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