Patent Application
20240358855
The complement system is continuously activated at low levels and both membrane-bound and soluble intraocular complement regulatory proteins tightly regulate this spontaneous complement activation. However, dysregulation of the complement system is associated with numerous diseases and conditions, including autoimmune diseases, angioedema, ocular disease, and others. In particular, over-activation of the complement system can result in ocular inflammation, which contributes to vision loss in a number of ocular diseases such as, for example, age-related macular degeneration (AMD), and diabetic macular edema.
Three distinct pathways activate the complement cascade. The classical, lectin, and alternative pathways all converge on an amplification loop that, if over-activated, results in inflammation and cell lysis. A fourth pathway, termed the intrinsic pathway, has been suggested. The cleavage of C3 into C3b and its deposition onto surfaces, (e.g., cells or extracellular matrix) initiates the amplification loop. Complement factor B (FB) and factor D (FD) contribute to the formation of a C3-convertase (C3bBb), which drives forward the amplification loop by cleaving additional C3 into C3b. However, this cascade can be prevented through inactivation of C3b (forming iC3b) by the enzyme complement factor I (FI) and its cofactor, complement factor H (FH) or its truncated splice variant FHL-1. iC3b cannot generate C3-convertase. If these regulators fail to control the complement cascade, downstream consequences include the release of the anaphylatoxins C3a and C5a, and ultimately the production of the terminal complement complex (TCC). TCC is also commonly referred to as the membrane attack complex (MAC), which can integrate into cell membranes causing lysis and death. Thus, the deposition of TCC/MAC can be regarded as a marker of complement activation.
Complement dysregulation occurs when the activation and control mechanisms of complement that together play crucial roles in maintaining health and tissue homeostasis fail; when the extremely delicate balance between activation and control is disturbed, tissue damage and disease ensue (Ricklin et al. 2016, 2018; Zelek et al 2019; Harris et al. 2018; Morgan & Harris 2015; Thurman and Holers 2006; Barlow 2017; Gavriilaki and Brodsky 2020; Zipfel et al. 2006; Schmidt et al. 2016).
Complement-based therapies are being developed that will play a key role in the treatment of complement-mediated diseases (Zelek et al. 2019; Harris et al. 2018; Morgan and Harris 2015; Ricklin and Lambris 2007; Kassa et al 2019; Read et al. 2004; Ren et al. 2010; Dobo et al. 2018; Gavriilaki and Brodsky 2020; Huber-Lang et al. 2016). However, additional and more effective therapies are needed.
The gene therapy methods described herein can reduce or prevent C3b amplification and exert downstream effects, including MAC formation within the neural retina, retinal pigmented epithelium (RPE), choroid, choriocapillaris (CC), Bruch's membrane, and/or other ocular cells and tissues to re-establish appropriate control of the complement system.
In an aspect, disclosed herein is a gene therapy vector for treatment of conditions associated with complement dysfunction comprising a genetic cargo encoding two, three, or more than three activities selected from: a) one or more a complement proteins selected from CFH (FH), CFHT (FHL-1), oCFHT (oFHL-1), and CFI (FI); b) one of more binding proteins that specifically binds CFB (FB), CFD (FD), CFP, CFHR-1(FHR-1), CFHR-2 (FHR-2), CFHR-3 (FHR-3), CFHR-4 (FHR-4), CFHR-5 (FHR-5), C3, C4A, C4B, C5, C6, C7, C8A, C8B, C8G, or C9; c) HTRA1 protein or a transcriptional activator protein that increases expression of HTRA1; d) a binding protein that specifically binds ApoE2 or VEGFA; e) an inhibitory RNA that targets CFB, CFD, CFP, CFHR-1, CFHR-2, CFHR-3, CFHR-4, CFHR-5, C3, C4A, C4B, C5, C6, C7, C8A, C8B, C8G, or C9; and f) an inhibitory RNA that targets ApoE2 or VEGFA.
A “gene therapy vector” is a viral or nonviral vector used to deliver a polynucleotide (“cargo”) to a target cell or tissue. In some cases a gene therapy vector is a viral vector such as a recombinant adeno-associated virus vector (rAAV or AAV), adeno virus (AV), anellovirus, or lentivirus vector.
As used herein, “cargo” refers to the entire nucleic acid delivered to a cell using a gene therapy vector. The cargo may be DNA or RNA, depending on the choice of vector. In the case of certain viral vectors the cargo includes DNA between and including terminal repeats (e.g., inverted terminal repeats in the case of AAV and other vectors, or long terminal repeats in the case of lentiviral and other vectors). The cargo can include sequences that encode transcribed RNA and/or protein coding sequences, as well as regulatory elements, spacer sequences, terminal sequences, and the like. A cargo may include coding sequences for gene product (RNA or protein) one or more than one (e.g., two, three or four) gene products. Vectors encoding multiple gene products are used for multigene therapy. As used herein, a cargo is delivered by a single vector. Generally, a cargo is a single polynucleotide (e.g., a single contiguous DNA sequence encoding multiple coding sequences.
As used herein, “gene product” refers to a polypeptide or RNA encoded in a vector cargo and produced in a cell transduced by a gene therapy vector. Without limitation a gene product may be an mRNA, a polypeptide encoded by an mRNA, or an inhibitory nucleic acid (e.g., an inhibitory RNA). One, two, three, or more than three gene products can be encoded in a single cargo that is delivered by a single vector.
As used herein, “multigene cargo” refers a cargo that encodes multiple (e.g., 2, 3 or 4) gene products.
As used herein, “multigene therapy” refers to gene therapy in which a single cargo encoding two or more (e.g., two, three or four) gene products is administered.
As used herein, “transgene” refers to a portion of a cargo that encodes one protein.
As used herein, the term “activity” refers to the biological effect that results from expression of a gene product encoded in a gene therapy cargo. See Section 2.1, below.
As used herein, “delivery” (e.g., delivery of a gene therapy vector, delivery of a gene therapy cargo, delivery of an activity, delivery of a gene product, delivery of a cargo, delivery of a polynucleotide, etc.) refers to associating a viral or nonviral vector with a target cell under conditions in which the vector cargo is introduced (e.g., transduced, in the case of a viral vector) into the cell allowing the gene product(s) encoded in the cargo to be expressed in the cell. A gene therapy vector can be delivered to a subject by injection, infusion, and other methods. Generally a gene therapy vector results in delivery (directly or indirectly) to the eye. In some approaches, delivery results in transduction of RPE cells, endothelial choroidal cells or melanocyte cells.
As used herein, a “target cell” refers to the cell(s) or cell type(s) in which a gene product delivered by a therapy cargo is expressed. Importantly, a gene product expressed in a target cell may have a biological effect in neighboring cells and surrounding tissues. For example, the gene product may be secreted from the cell in which it is expressed and have effects in other cells. For example, CFHT may be expressed in and secreted by RPE cells and have effects in surrounding choroid tissue.
As used herein, the term “complement protein” refers to any protein associated with the complement system. Complement proteins may be synthesized by the liver and other tissues, and circulate in the blood and extracellular compartments as inactive precursors. When stimulated by one of several triggers, proteases in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. The end result of this complement activation or complement fixation cascade is stimulation of phagocytes to clear foreign and damaged material, inflammation to attract additional phagocytes, and activation of the cell-killing membrane attack complex. About 50 proteins and protein fragments make up the complement system, including serum proteins, and cell membrane receptors. Three biochemical pathways activate the complement system: the classical complement pathway, the alternative complement pathway, and the lectin pathway. The alternative pathway accounts for the majority of terminal pathway activation and so therapeutic efforts in disease have revolved around its inhibition. Some proteins of the alternative pathway can be classified as “early,” “intermediate,” and “late” based on where they act in the alternative complement pathway.
As used herein, the term “complement regulatory protein” refers broadly to membrane-bound and soluble intraocular complement regulatory proteins including: CFH, CFHT, CFI, CFB, CFD, CFP, CFHR1-5, C3, C4, C5, C6, C7, C8, C9, CD46, CD55, and CD59. As used herein, “complement regulatory proteins” are a subcategory of “complement proteins.”
As used herein, “exogenous protein” refers to a protein encoded in a gene therapy cargo. In some cases, the exogenous protein or a variant thereof is normally expressed in cells of the subject receiving gene therapy. For example, 402Y 621 CFH produced from a cargo is exogenous, while CFH (e.g., 402Y 621; 402Y 62V or 402H 62V) expressed from genomic sequences of the subject is an endogenous protein. In some cases, an exogenous protein is a not found in nature (e.g., a specific nanobody or trap) or a variant of a complement protein found in nature. Examples of variants include an optimized protein such as described in Section 3.4, below.
As used herein, an “endogenous protein” refers to a protein expressed by a subject prior to or in the absence of, treatment with a gene therapy vector.
As used herein, a “disease modifying protein” is a protein other than a complement protein that is associated with risk of or development of an ocular disease such as AMD. Examples of disease modifiers include HTRA1, VEGF, and ApoE2.
As used herein, “protective CFH” and “protective CFHT” refer to the CFH/T variant in which residue 62 is isoleucine (621) and residue 402 is tyrosine (402Y). See Hageman et al., 2005, Proc. Natl Acad Sci 102:7227-32 and U.S. Pat. No. 7,745,389, which is incorporated by reference in its entirety.
“Reference sequence” refers to a nucleic acid or protein sequence for which a sequence, database accession number, or published patent or literature reference is provided in this disclosure (see, e.g., Table 19). Reference sequences are provided to identify proteins and sequence elements. When an amino acid or nucleic acid sequence is provided in this disclosure for a protein, gene product, promoter, regulatory element, or the like, it will be understood that artificially or naturally occurring variants with substantially the same functional properties may be used. For example, protein or nucleic acid variants with at least about 85%, at least about 90% or at least about about 95% sequence identity to the reference sequence (i.e., variants substantially identical to a reference sequence) can be used in the methods and compositions of the invention and any reference to a protein of nucleotide sequence should be understood to refer to functionally similar variants. In addition, it will be understood that, due to codon degeneracy, a specified amino acid sequence can be encoded by a number of different DNA or RNA sequences. Provision of a particular protein-encoding nucleic acid sequence is not intended to limit the invention to that single sequence but encompasses other nucleic acid sequences encoding the protein or a variant or analog thereof. Similarly, an amino acid sequence provided as a translation of a nucleic acid sequence does not limit the nucleic acid sequence that encodes the protein to the particular nucleic acid sequence from which the protein was translated. A number of other nucleic acid sequences can also encode the protein.
As used herein, “sequence identity” in reference to similarity of two proteins (a target protein and a reference protein) or two nucleic acids (a target nucleic acid and a reference nucleic acid) is a quantification of identity of amino acids or nucleobases when the reference and target sequence are optimally aligned. Sequence identity can be determined manually by inspection, especially when the target and reference have greater than 90% identity and are easy to align. Alternatively, for nucleotide sequences, percent identity to a reference nucleic acid sequence can be determined using a BLAST or BLAST 2.0 comparison program (described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively) with default parameters. BLASTP with default parameters can be used to determine percent to a reference polypeptide sequence. The BLASTN program uses as default parameters a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). Software for BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site.
As used herein, the term a “target,” in reference to a binding agent (such as an antibody) that targets a protein means the binding agent specifically binds to the protein. The term “target,” in reference to an inhibitory RNA that targets a polynucleotide means the binding is complementary to a portion of the polynucleotide and hybridizes to the polynucleotide.
Dysregulation of complement activation is associated with development of age-related macular degeneration (AMD) and many other diseases. A variety of treatment approaches have been proposed or explored for modulating the complement system and restoring health, including administration of small molecule inhibitors, direct administration of therapeutic proteins, treatment with antibodies against complement activities, and gene therapy.
We have determined that coordinated modulation of multiple (e.g., two or three) proteins in target cells or tissues to intervene at multiple regulatory points within the alternative pathway amplification cascade can reestablish appropriate regulation of the complement system in Chromosome 1- (Chr1-) directed AMD. (See Hageman and Richards, WO2020019002A1, incorporated herein by reference, for a discussion of Chr1-directed AMD.) The mutigene therapy described herein is a superior intervention strategy for treating this multi-factoral and complex age-related disease.
In addition, patients suffering from AMD can manifest a range of pathology in the same eye. These can, for example, range from normal histology to the presence of drusen and other abnormal extracellular deposits to retinal, RPE and choroidal cell death to geographic atrophy to neovacularization.
Removal of proteins or mRNAs produced locally in the eye (e.g. RPE) or systemically (e.g. liver or endothelial cells) that target critical early alternative complement pathway steps (CFB, CFD, C3), in concert with perturbation of intermediate (e.g. CFH, CFHT, CFHR1-5) and later (e.g. C5, C6, C7, C8, C9) alternative complement pathway steps will allow for tighter control of dysfunctional complement and reduction of disease progression. Additionally, introduction of multigene constructs that contain cargo that can block disease modifying or accelerating steps (APOE, VEGFA, HTRA1) will provide added benefit. In contrast to augmentation or inhibition of a single complement protein, introduction of multigene cargo permits augmentation or diminution of complement elements active at two or more stages of activation of the alternative complement (AC) system. In one approach, administration of a multigene cargo results in augmentation or diminution of complement proteins in two, three, or four different classes as well as (optionally) with disease modifying proteins. This approach allows multiple steps during disease progression to be controlled.
Multigene therapy requires that several gene therapy elements work together, and involves challenges specific to use of a single cargo to deliver more than one gene product or more than one activity. Important factors in multigene therapy include (I) the combination of gene products or activities delivered using a gene therapy vector; (II) regulatory elements that ensure the delivered activities are expressed and biologically active at the location(s), level(s), and/or time(s) required for the desired therapeutic effect; (III) the organization of the gene therapy vector and cargo; and (IV) the selection of a delivery system such as a specific viral vector or class of viral vectors or class of viral or nonviral vectors.
Each gene product encoded on a multigene cargo and delivered to a cell or tissue is associated with an activity. As noted above, the term “activity” refers to the biological effect that results from expression of a gene product encoded in a multigene cargo. The combination of activities delivered by a multigene cargo provides therapeutic benefit. Delivery of a gene product of a multigene cargo results in a perturbation of a target or target function. In some cases, delivery of a gene product results in augmentation of a target function, e.g., introduction of an exogenous complement protein or variant thereof, or disease modifier protein or variant thereof, or introduction of an activator (e.g., CRISPRa) that increases transcription of an endogenous protein. In some cases, delivery of a gene product results in diminution of a target function (i.e., diminution of an activity characteristic of a target complement protein or a target disease modifier protein). For illustration, in one example the gene product is aflibercept and the activity is diminution (effect) of VEGFA (target function). In this case the perturbation is diminution and the target function is VEGFA activity. In a related activity, the gene product is an inhibitory anti-VEGFA nanobody, the perturbation is diminution, and the target function is VEGFA activity. Thus, two different gene products can have the same activity, and the same activity can result from administration of two multigene cargos encoding different gene products. As a third example, gene product is an shRNA that targets VEGFA RNA, the perturbation is diminution, and the target function is VEGFA activity. See Sections 5.21 (aflibercept), 5.3 (nanobodies), and 5.1.1 (shRNA).
TABLE 2 provides exemplary activities that, delivered in combination, provide therapeutic benefit. In some cases, each activity delivered by a multigene cargo relates to a different target. For example, a multigene cargo that carries two activities may deliver activities that perturb two different targets, e.g., CFHT+CFI or CFHT+C7. In some cases, a multigene cargo that carries two activities may deliver activities that perturb the same target (e.g., CFHT+CFHT). Likewise, a multigene cargo that carries three activities may deliver two activities that perturb the same target and one activity that perturbs a different target (e.g., CFHT+CFHT+HTRA1). A multigene cargo that delivers two activities that perturb the same target may deliver two copies or versions of the same gene product (e.g., two copies of a CFHT encoding sequence) or may deliver two different gene products that perturb the same target (e.g., a nanobody that targets C7 and an shRNA that targets C7).
As noted above, the perturbation resulting from administration of a gene product can be characterized as diminution or augmentation.
As used herein, “augmentation” can refer to increasing the amount, quantity or activity of a target, by, for example, introducing a cargo comprising a nucleic acid sequence encoding an exogenous protein. In an other approach, the cargo includes protein and RNA components of a CRISPR activation (CRISPRa) system to increase endogenous expression of the target (e.g., HTRA1; see Williams et al. WO2020210724A1, incorporated herein by reference). In some cases an activity delivered by the multigene cargo is an exogenous complement protein (e.g., the cargo comprises a nucleic acid encoding a complement protein), and delivery of the cargo results in expression of the exogenous protein. The exogenous protein may be a wild-type human complement protein, an optimized, variant or protective complement protein, a disease modifying protein, or the like. In this approach, augmentation of an activity is achieved by delivering a cargo to a cell, wherein a protein encoded by the cargo is expressed. Some expressed proteins are secreted from the cell in which they are synthesized. As noted in Section 1, the gene product may be secreted from the cell in which it is expressed and have effects in other cells.
As used herein, “diminution” refers broadly to reducing the quantity or activity of a target in the eye or other tissue. Diminution can refer to reducing expression of a target, reducing the amount of active target (e.g., by trapping), inactivating, altering or inhibiting the biological effect of a the target. In one approach diminution results from expression of cargo encoding an inhibitory RNA or inhibitory binding agent (e.g., trap, antibody, aptamer). In some cases an activity delivered by the single vector is a binding activity (e.g., nanobody, trap, aptamer) that, when delivered and expressed, reduces the amount or activity of an endogenous protein (e.g., complement protein) produced locally (e.g., RPE) or systemically (e.g., liver, endothelial cells) such as FHR1-5. In some cases an activity delivered by the single vector is an inhibitory RNA that, when delivered and expressed, reduces the amount or activity of an endogenous polypeptide locally in the transduced cell (e.g., C7).
TABLES 3 and 4 describe exemplary combinations of targets and categories of gene products that can be administered in a multigene cargo for coordinate expression. TABLE 3 illustrates delivery of two gene products. TABLE 4 illustrates delivery of three gene products. The labels “Activity 1,” “Activity 2,” and “Activity 3” do not indicate a particular order or 5′ to 3′ arrangement of genes in the cargo. “(P)” refers to expression of a complement protein (naturally occurring, modified, or optimized), disease modifier protein, or other protein to augment levels of the complement polypeptide in the target cell. “B” refers to a binding agent. Examples of binding activities include single chain antibodies (e.g., nanobodies), aptamers, and traps. “R” refers to an inhibitory RNA such as shRNA, siRNA, miRNA.
In some approaches the cargo encodes two activities under control of one promoter. In some approaches the cargo encodes three activities under control of two or three promoters. In some approaches at least one activity is an inhibitory RNA (e.g., shRNA). In some approaches at least one activity is an exogenous complement protein (e.g., CFH, CFHT, CFI) and at least one activity is an inhibitory RNA (e.g., shRNA).
TABLE 5 is provided for illustration and not limitation and shows various schema for organizing transgenes according to the invention. “ITR” indicates vector is AAV; “LTR” indicates the vector is Lentivirus; [NV] indicates the vector is non-viral vector.
Complement Factor H (CFH) is secreted into the bloodstream or locally by cells and tissues and acts as an inhibitor of complement activation in fluid phases (e.g. blood; extracellular compartments) and on self cell surfaces. CFH accelerates the decay of the complement alternative pathway (AP) C3 convertase C3bBb, thus preventing local formation of more C3b and, as a cofactor of the serine protease factor I, regulates proteolytic degradation of already-deposited C3b. As further explained in the following paragraph, multiple haplotypes associated with AMD risk or protection have been identified for the CFH/CFHT gene (Risk, Protective, Neutral and Protective-Deletion). For purposes of the compositions and methods described herein, CFH is preferably a protective form (I62, 402Y). Alternatively, a neutral form (62V, 402Y) may be employed. It will be recognized that the CFH protein exists in various forms in nature and that it is possible to modify the protein sequence without loss of biological activity. In some approaches, CFH variants with at least about 90% or about 95% sequence identity to a naturally occurring CFH sequence can be used.
CFHT is encoded by a CFH gene splice variant. CFHT contains the first 7 SCRs of CFH and a unique four amino acid carboxy-terminal tail. It performs many of the same functions as CFHT. Multiple haplotypes associated with AMD risk or protection have been identified for the CFH/CFHT gene (Risk, Protective, Neutral and Protective-Deletion). For purposes of the compositions and methods described herein, CFHT is preferably a protective form (I62, 402Y). See, e.g., U.S. Pat. No. 7,745,389; WO2020019002; U.S. Pat. No. 7,867,727; and Hageman et al., Proc. Nat. Acad. Sci. USA, 202(20):7227-32, 2005, all incorporated by reference. An illustrative protective CFHT polypeptide sequence, lacking the signal peptide, is provided in SEQ ID NO:253. Alternatively, a neutral form of CFHT (62V, 402Y) may be used. It will be recognized that the CFHT protein exists in various forms in nature (see, e.g., Skerka et al., Br. J. Pharmacol 178:2823-2831, 2021) and that it is possible to modify the protein sequence without loss of biological activity. In some approaches, CFHT variants with at least about 90% or about 95% sequence identity to SEQ ID NO:253 can be used.
eCFH/T is an artificial gene sequence that, when delivered to a cell, results in expression of CFH and a modified CFHT (comprising a carboxy-terminal sequence CIRVSKSFTL. eCFH/T is described in detail in WO2020019002, incorporated herein by reference.
Optimized CFHT (oCFHT) proteins are designed to increase the affinity and/or avidity of CFHT binding to cell surfaces and CFHT ligands such as glycosaminoglycans (GAGs) associated with extracellular matrices, such as basement membrane associated GAGs. oCFHTs comprise a CFHT domain along with additional copies of short consensus repeat 7 (SCR7), SCR7 variants, and/or dimerization domains. As indicated above, CFHT is preferably a protective form, but alternatively may be a neutral form. The introduction of optimized CFHT complement pathway regulator proteins will be employed to reduce the degree of risk in AMD patients—by shifting the balance at the RPE/CC interface from risk toward protection—in patients with chromosome-1-directed risk AMD. This approach should reduce visual loss associated with early, late stage geographic atrophy and late-stage neovascular forms of AMD. In addition to AMD, these agents should be useful in reducing the complications of Chr1-directed pathologies in other diseases that exhibit alternative complement pathway dysfunction (e.g. membranoproliferative glomerulonephritis, dense deposit disease, IgA nephropathy, etc.). TABLE 6 lists three forms of oCFHT, which are also discussed below.
In one form of optimized CFHT, increased avidity of modified protective or non-protective CFHT binding is accomplished by the addition of 1, 2,3 or more additional single consensus repeat 7 (SCR7) domains. SCR7 domains contain important amino acid residues for binding to endogenous ligands (including, but not limited to, GAGs, C3b, lipid and protein malondialdehyde (MDA) and malondialdehyde-acetalaldehyde (MAA) modified adducts, carbohydrates, adrenomedullin, CRP, SIBLING proteins, etc.) (Sanchez-Corral et al., 2018, Front. Immunol. 9:1607-1625) in both CFH and CFHT. An exemplary amino acid sequence of CFH SCR7 is provided as SEQ ID NO:242. Exemplary DNA sequences encoding SCR7 are provided at SEQ ID NO:252 (wild-type) and SEQ ID NOs:2-5 (human codon-optimized). As illustrated herein, e.g., Section 10.5, the in vitro functions of mCFHT (cofactor, decay accelerating activity and ligand binding activity) will better regulate complement activation than non-modified CFHT protein, as has been demonstrated for other engineered mini-FH/mini-CFH proteins (Schmidt, C. Q., et al., 2013, J Immunol 190:5712-5721).
In addition to the human SCR-7 sequence (e.g., SEQ ID NO:242), polypeptide variants with at least 85% sequence identity, preferably at least about 90% sequence identity, sometimes at least about 95% sequence identity may be used provided the SCR-7 sequence confers improved binding to C3b, MDA-LDL and CRP when incorporated into CFHT as described herein (e.g., to produce mCFHT.1).
In a second form of optimized CFHT (herein referred to dCFHT), a dimerization domain (FHR-1 SCR1-2, also referred to as “D”) sequence is included at the N- and/or C-terminal regions of CFHT to provide increased avidity for cell surfaces and to increase protein half-life and avidity (see Yang et al, 2018, “An Engineered Complement Factor H Construct For Treatment Of C3 Glomerulopathy” J Am Soc Nephrol. 29(6):1649-1661). In a preferred embodiment the dimerization domain is derived from SCRs 1 and 2 from Complement Factor H-Related 1 (CFHR-1, FHR-1). Exemplary nucleotide sequences encoding the FHR-1 SCR1 and SCR2 domains are provided at SEQ ID Nos: 52 to 55. In other embodiments dimerization sequences (SCRs 1-2) from CFHR2 or CFHR5 are used. SCRs 1-2 from CFHR1, 2 and 5 are conserved and share about 85% amino acid identity. See, e.g., Goicoechea de Jorge, 2013, Dimerization of complement factor H-related proteins modulates complement activation in vivo, PNAS, 110 (12) 4685-4690. In other approaches, dimerization domains from other proteins may be used. Exemplary amino acid sequences encoding the FHR-1, FHR-2 and FHR-5 SCR1 and SCR2 domains are available in the scientific literature and databases, and are provided at SEQ ID Nos: 1, 228 and 233. Exemplary nucleotide sequences encoding the FHR-2 and FHR-5 SCR1 and SCR2 domains are provided at SEQ ID Nos: 229-232 and 234-237. Dimerization domains for use in the invention include sequences of SCR1-2 from FHR-1, FHR-2 and FHR-5; as well as variants with at least 85% sequence identity, preferably at least about 90% sequence identity, sometimes at least about 95% sequence identity which retain the ability to homodimerize.
Modified-Dimer CFHT (mdCFHT)
A third form of enhanced CFHT protein contains both additional SCR7 domains (as found in mCFHT) and additional dimerization domain(s) (as found in dCFHT). mdCFHT will reduce complement activation and provide several potential therapeutic benefits. Without limiting the invention to specific embodiments, the following exemplary constructs are contemplated (numbers 1-7 refer to short consensus repeats (SCR) in CFHT:
As a result of these modifications, the half-life of CFHT protein on cell and extracellular surfaces will be increased, as has been demonstrated with other engineered mini-FH/mini-CFH proteins (Yang et al., J Am Soc Nephrol 29:1649-1661, 2018). CFHT is a ‘negative’ regulator of the AP system and expression of the modified CFHT protein, containing the CFHR-1 dimerization domain, will bind to monomeric CFHR1 (or CHFR2 or CFHR-5) protein in the eye, thereby preventing it from competing with CFH/CFHT for C3b, MDA, PTX3, adrenomedullin and/or CRP binding and thus reducing activation of C3b or other inflammatory molecules. Lastly, when the region of the CFHT protein dimer containing extra SCR7 domain(s) binds to self, it will bind nearby C3b protein(s), leaving the non-SCR-containing portion of the dimer to bind additional C3b protein(s) or GAG(s) on cell surfaces or extracellular matrices (e.g., BM; choroidal stroma, etc.) or other endogenous ligands (including, but not limited to, GAGs, lipid and protein modified adducts, carbohydrates, adrenomedullin, CRP, PTX, SIBLING proteins, etc.). Binding to as many C3b and GAG molecules as possible will reduce CFB binding to C3b and block generation of the C3 convertase complex that amplifies AP complement activation. Inhibition of C3 convertase generation will significantly reduce both the inflammatory anaphylatoxins C3a and C5a and membrane attack complex (MAC) that drive all stages of AMD progression.
CFB circulates in the blood as a single chain polypeptide. Upon activation of the alternative pathway, it is cleaved by complement factor D, yielding the noncatalytic chain Ba and the catalytic subunit Bb. The active subunit Bb is a serine protease which associates with C3b to form the alternative pathway C3 convertase. Polymorphisms in this gene are associated with a reduced risk of age-related macular degeneration. Human CFB cDNA and amino acid sequences are provided at Genbank accession nos. NM_001710 (reference sequences). It will be recognized that the CFB protein exists in various forms in nature and that it is possible to modify the protein sequence without loss of biological activity. In some approaches, CFB variants with at least about 90% or about 95% sequence identity to the reference sequence can be used.
CFI regulates complement activation by cleaving C3b and C4b. Complement Factor I is produced by furin cleavage of a proprotein to generate a glycoprotein heterodimer consisting of a disulfide linked heavy chain and light chain. Human CFI cDNA and amino acid sequences are provided at Genbank accession nos. NM_000204 (reference sequences). It will be recognized that the CFI protein exists in various forms in nature and that it is possible to modify the protein sequence without loss of biological activity. In some approaches, CFI variants with at least about 90% or about 95% sequence identity to the reference sequence can be used.
CFP (properdin) is a positive and sometimes negative regulator of the complement alternate pathway depending on disease context. See Chen et al., 2018, “Properdin: A Multifaceted Molecule Involved In Inflammation And Diseases” Mol Immunol. 102:58-72. It binds to and stabilizes C3- and C5-convertase enzyme complexes and inhibits CFI-CFH mediated degradation of Complement C3 beta chain (C3b). Properdin promotes the association of C3b with Factor B and provides a focal point for the assembly of C3bBb on a surface. It binds to preformed alternative pathway C3-convertases and inhibits the Factor H—mediated cleavage of C3b by Factor I.
Complement Component C3 plays a central role in the activation of the complement system. Its activation is required for both classical and alternative complement activation pathways. One form of C3-convertase, also known as C4b2a, is formed by a heterodimer of activated forms of C4 and C2. It catalyzes the proteolytic cleavage of C3 into C3a and C3b, generated during activation through the classical pathway as well as the lectin pathway. C3a is an anaphylatoxin and the precursor of some cytokines such as ASP, and C3b serves as an opsonizing agent. Factor I can cleave C3b into C3c and C3d, the latter of which plays a role in enhancing B cell responses. In the alternative complement pathway, C3 is cleaved by C3bBb, another form of C3-convertase composed of activated forms of C3 (C3b) and factor B (Bb). Once C3 is activated to C3b, it exposes a reactive thioester that allows the peptide to covalently attach to any surface that can provide a nucleophile such as a primary amine or a hydroxyl group. Activated C3 can then interact with factor B. Factor B is then activated by factor D, to form Bb. The resultant complex, C3bBb, is called the alternative pathway (AP) C3 convertase. C3bBb is deactivated in steps. First, the proteolytic component of the convertase, Bb, is removed by complement regulatory proteins having decay-accelerating factor (DAF) activity. Next, C3b is broken down progressively to first iC3b, then C3c+C3dg, and then finally C3d. Factor I is the protease that cleaves C3b but requires a cofactor (e.g. Factor H, CR1, MCP or C4BP) for activity.
Complement Component C4 serves a number of critical functions in immunity, tolerance, and autoimmunity with the other numerous components. Furthermore, it is a crucial factor in connecting the recognition pathways of the overall complement system instigated by antibody-antigen (Ab-Ag) complexes to the other effector proteins of the innate immune response. The C4 protein derives from the C4A-C4B genes, which allows for an abundant variation in the levels of their respective proteins within a population. Inhibition of C4A reduces C3 and C5 convertase activity.
The Complement Component C5 gene encodes a preproprotein that is proteolytically processed to generate multiple protein products, including the C5 alpha chain, C5 beta chain, C5a anaphylatoxin and C5b. The C5 protein is composed of the C5 alpha and beta chains, which are linked by a disulfide bridge.
The Complement Component C6 gene encodes a component of the complement cascade. The encoded protein is part of the membrane attack complex that can be incorporated into the cell membrane and cause cell lysis. Mutations in this gene are associated with complement component-6 deficiency. Transcript variants encoding the same protein have been described.
The Complement Component C7 gene encodes a serum glycoprotein that forms a membrane attack complex together with complement components C5b, C6, C8, and C9 as part of the terminal complement pathway of the innate immune system. Elevated Levels of C7 are associated with Chr1 Risk AMD. Exemplary C7 gene and cDNA sequences are provided at Genbank accession nos. NG-11692 and NM_000587 (reference sequences).
The Complement Component C8A, C8B and C8G genes encode protein components of the complement system that contains three polypeptides, alpha, beta and gamma. C8 proteins participate in the formation of the membrane attack complex.
The Complement Component C9 gene encodes the final component of the complement system. It participates in the formation of the membrane attack complex (MAC). The MAC assembles on membranes to form a pore, permitting disruption and cell death. Mutations in this gene cause C9 deficiency. Exemplary C9 gene and cDNA sequences are provided at Genbank accession nos. NG_009894 and NM_001737 (reference sequences).
The Complement Complement Factor H Related 1 gene encodes a secreted protein involved in complement regulation belonging to the complement factor H protein family. It binds to Pseudomonas aeruginosa elongation factor Tuf together with plasminogen, which is proteolytically activated. It is proposed that Tuf acts as a virulence factor by acquiring host proteins to the pathogen surface, controlling complement, and facilitating tissue invasion. Dimerized forms of CFHR1 protein have avidity for tissue-bound complement fragments and efficiently compete with the physiological complement inhibitor CFH. It can also associate with lipoproteins and may play a role in lipid metabolism.
The Complement Factor H Related 2 gene encodes a protein involved in regulation of complement. Mutations in CFHR genes have been associated with dense deposit disease and atypical haemolytic-uremic syndrome. Alternatively spliced transcript variants have been found for this gene. Its dimerized forms have avidity for tissue-bound complement fragments and efficiently compete with the physiological complement inhibitor CFH. It can also associate with lipoproteins and may play a role in lipid metabolism.
The Complement Factor H Related 3 gene encodes a secreted protein which binds to heparin, and is likely involved in complement regulation. Mutations in this gene are associated with decreased risk of age-related macular degeneration, and with an increased risk of atypical hemolytic-uremic syndrome. Alternatively spliced transcript variants encoding different isoforms have been described.
The Complement Factor H Related 4 gene encodes a protein that enhances the cofactor activity of CFH, and is involved in complement regulation. This protein can associate with lipoproteins and may play a role in lipid metabolism. Alternatively spliced transcript variants encoding different isoforms (varying in the number of SCRs) have been described.
The Complement Factor H Related 5 gene encodes a protein having nine SCRs with the first two repeats having heparin binding properties, a region within repeats 5-7 having heparin binding and C reactive protein binding properties, and the C-terminal repeats being similar to a complement component 3 b (C3b) binding domain. This protein co-localizes with C3, binds C3b in a dose-dependent manner, and is recruited to tissues damaged by C-reactive protein. Allelic variations in this gene have been associated, but not causally linked, with two different forms of kidney disease: membranoproliferative glomerulonephritis type II (MPGNII) and hemolytic uraemic syndrome (HUS).
VEGF-A is a member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) growth factor family. It is a heparin-binding protein, which exists as a disulfide-linked homodimer. This growth factor induces proliferation and migration of vascular endothelial cells and is essential for both physiological and pathological angiogenesis.
VEGF-B seems to play a role only in the maintenance of newly formed blood vessels during pathological conditions. VEGF-B plays also an important protective role on several types of neurons, including the retina.
VEGF-C is a member of the PDGF/VEGF family that mainly functions to promote the growth of lymphatic vessels (lymphangiogenesis). It acts on lymphatic endothelial cells (LECs), mainly via its primary receptor VEGFR-3, promoting survival, growth and migration. It is a ligand for the orphan receptor VEGFR-3. It can also promote the growth of blood vessels and regulate their permeability. The effect on blood vessels can be mediated via VEGFR-3 or its secondary receptor VEGFR-2.
VEGF expression in the retinal pigment epithelium (RPE) is associated with neovascular AMD. Aflibercept is a recombinant fusion protein that binds VEGF-A that is used in treatment of AMD. See Holash et al., 2002, “VEGF-Trap: a VEGF blocker with potent antitumor effects” Proc Natl Acad Sci USA 99:11393-8. Sequences of aflibercept with various signal peptides are provided (see SEQ ID NOs.: 6-8).
HTRA1 is a Serine Peptidase 1(SEQ ID NO:240). This protein is a secreted enzyme that is proposed to regulate the availability of insulin-like growth factors (IGFs) by cleaving IGF-binding proteins. HtrA levels are reduced in subjects at risk of developing AMD. Methods for increasing ocular HTRA1 levels are described in WO2020210724. These include delivery of a cargo encoding the HTRA1 protein under control of an operably linked promoter and delivery of a CRISPR activation system (including CAS and sgRNA) as described in WO2020210724.
APOE2 is an allelic isoform of APOE apolipoprotein. APOE functions in lipoprotein-mediated lipid transport between organs via the plasma and interstitial fluids. Serum APOE regulates the expression of inflammatory cytokines and vascular endothelial growth factor (VEGF) family of cytokines in retinal pigment epithelial (RPE) cells (Qureshi, et al., 2017, “Serum APOE, leptin, CFH and HTRA1 levels in Pakistani age related macular degeneration patients” JPMA J. Pakistan Med Assoc., 67:52-857). APOE2 is associated with enhanced risk of developing AMD. See Toops et al., 2016, “Apolipoprotein E Isoforms and AMD,” Adv. Exp. Med. Biol. 854: 3-9; Levy et al., 2015, “APOE Isoforms Control Pathogenic Subretinal Inflammation in Age-Related Macular Degeneration Neurosci 35(40):13568-76.
APOE4 is an allelic isoform of APOE. In the circulation, APOE is present as part of several classes of lipoprotein particles, including chylomicron remnants, VLDL, IDL, and some HDL. APOE interacts significantly with the low-density lipoprotein receptor (LDLR), which is essential for the normal processing (catabolism) of triglyceride-rich lipoproteins. In peripheral tissues, APOE is primarily produced by the liver and macrophages, and mediates cholesterol metabolism. In the central nervous system, APOE is mainly produced by astrocytes and transports cholesterol to neurons via APOE receptors, which are members of the low density lipoprotein receptor gene family. APOE is the principal cholesterol carrier in the brain. APOE is required for cholesterol transportation from astrocytes to neurons. APOE qualifies as a checkpoint inhibitor of the classical complement pathway by complex formation with activated C1q.
Expression of exogenous CFH, CFHT, and CFI is carried out using art known techniques. For example, a cDNA sequence, which is optionally codon-optimized, is linked to a promoter and other regulatory elements and delivered to a target cell as described herein. A heterologous signal peptide can be linked to a protein. Exemplary transgenes encoding CFH, CFHT, eCFH/T, oCFHT, CFI, and HTRA1 are described herein.
Diminution of complement proteins is achieved using specific binding agents or inhibitory RNA as described below. As used herein, ‘binding agent’ refers to an agent (antibody, aptamer, trap or other) that binds to and sequesters and/or reduces the quantity or activity of the protein bound. For target molecules that are synthesized locally in occular tissue (e.g., by the RPE, retina and/or choroid) such as C3, C4, C5, C6, C7, C8G, C9, CFB, CFD, an inhibitory RNA (e.g., hpRNA) strategy may be preferred over binding agents. A “binding agent” that is a polypeptide or is a complex of linked or associated polypeptides can be referred to as a “binding protein.”
As noted in TABLE 2, diminution (e.g., inhibition of activity or reduction in levels) of certain complement components and other biomolecules will provide therapeutic benefit to subjects with, or at risk of developing, AMD. There are primarily two broad categories of inhibitors: Inhibitory RNAs (described below in § 5.1) and binding agents. Binding agents include traps such as aflibercept or variants thereof (described below in § 5.2), and single chain antibodies such as nanobodies (described below in § 5.3).
Reduction of expression of targets (diminution of target) can be achieved using inhibitory RNA molecules or binding agents as described below. For target molecules that are synthesized locally by the RPE, retina and/or choroid (e.g., C3, C4, C5, C6, C7, C8G, C9, CFB, CFD) an inhibitory RNA (e.g., hpRNA) may be used to reduce target expression. Alternatively or additionally, a binding agent may be used to reduce the target biological activity.
Inhibitory RNA techniques use engineered RNA molecules to inhibit gene expression through various biological mechanisms (e.g., transcript cleavage, sequestration, inhibition of protein translation or RNA aptamer that inhibit protein activity). Suitable inhibitory RNA molecules include small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), and anti-sense RNA that target an endogenous RNA transcript. For example, administration of a vector encoding and expressing an inhibitory RNA directed against complement componant C7 will reduce endogenous C7 mRNA and/or C7 polypeptide. The reduction in C7 results in a therapeutically beneficial reduction in MAC formation.
As used herein, an “antisense strand” refers to the strand of a double stranded region of an RNAi agent (e.g., siRNA, shRNA, miRNA) that includes a region complementary or substantially complementary to a target RNA sequence or corresponding DNA (e.g., a human C7 mRNA including a 5′ UTR, exons of an open reading frame (ORF), or a 3′ UTR). The region of “complementarity” or “substantial complementarity” need not be fully complementary to the target sequence and may have sequence % identity or % similarity of least 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
A “sense strand,” as used herein, refers to the strand of a RNAi agent (siRNA, shRNa, miRNA) that includes a region that is complementary or substantially complementary to a region of the antisense strand.
shRNA, siRNA, and miRNA can be designed using well-defined principles using publicly and commercially available software. Illustrative companies that provide shRNA design algorithms and services include Thermo Fisher, InvivoGen, Biosettia, Hairpin Technologies, Horizon Discovery, Eurofins Genomics and others. For example, shRNA can be identified using the Thermo Fisher website rnaidesigner.thermofisher.com/rnaiexpress/help/shrna_enter_sequence_parameters. Publicly available software Includes software available through The Broad Institute. See, also Fakhr et al, Cancer Gene Therapy 23:73-82, 2016, which describes various parameters from different algorithms for designing functional small inhibitory RNAs. Design considerations are additionally described by Ros XB-D, Gu S. Methods 103: 157-166, 2016.
The specificity or knockdown level of a shRNA, miRNA, or siRNA can be confirmed using real-time PCR (RT-PCR) analysis for mRNA level or ELISA assay for the protein level. Experimental controls may be run in parallel to assess knockdown. Some examples of experimental controls that may be used, include but are not limited to, a mock-infected or mock-transfected sample, an empty vector, an shRNA encoding a scrambled target or seed region, an shRNA targeting another gene entirely such as, housekeeping genes GAPDH or Actin, or a GFP positive control. In some embodiments, an shRNA or siRNA results in expression levels that are reduced by at least about 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to a control, such as a mock-infected or empty vector control.
In some embodiments, the inhibitor RNA is an shRNA that induces RNA interference in a cell. The shRNA comprises a hairpin structure such that the RNA molecule comprises a double stranded region of paired antisense and sense strand connected by a loop of unpaired nucleotides. shRNAs are generally designed to bypass Drosha/DGCR8 cleavage. After export from the nucleus by exportin 5, shRNAs are cleaved by Dicer to form a duplex that can be loaded into Argonaute-RISC complexes.
In typical embodiments, the double-stranded region is about 19-25 base pairs in length. The loop region is typically from 4-11 nucleotides in length. In some embodiments, the duplex region is 19, 20, 21, or 22 base pairs in length.
In some embodiments, an shRNA targets the 5′ or 3′ untranslated region of the target RNA sequence. In other embodiments, an shRNA targets a protein-coding region.
In some embodiments, an anti-sense strand of the shRNA is 100% complementary to a target sequence. In some embodiments, the antisense stranded of the shRNA has at least 80%, at least 85%, at least 90%, or at least 99% complementarity with the target sequence.
TABLE 7 shows examples of shRNA targeting sequences (antisense) useful in multigene therapy.
A microRNA (miRNA) is a small nan-coding RNA molecule, that functions in RNA silencing and post-transcriptional regulation of gene expression. miRNAs base pair with complementary sequences within the mRNA transcript. As a result, the mRNA transcript may be silenced by one or more of the mechanisms such as cleavage of the mRNA strand, destabilization of the mRNA through shortening of its poly(A) tail, and decrease translation efficiency of the mRNA transcript into proteins by ribosomes. The term “miRNA” as used herein includes both naturally occurring and artificial miRNAs, e.g., a cellular miRNA in which the stem is modified to be partially complementary to an mRNA of interest.
Artificial miRNAs are typically designed to undergo cleavage both by Drosha/DGCR8 and Dicer. They can be transcribed from their own promoters, embedded in an intron, or located in the 3′-UTR of a protein-coding gene. Thus, in some embodiments, miRNAs resemble the siRNAs of the shRNA pathway, except that miRNAs derive from regions of RNA transcripts that fold back on themselves to form short hairpins (pri-miRNA). Once transcribed as pre-miRNA, the hairpins are cleaved out of the primary transcript in the nucleus by Drosha. The hairpins, or pre-miRNA, are then exported from the nucleus into the cytosol where the loop of the hairpin is cleaved by Dicer. The resulting product is a double strand RNA with overhangs at the 3′ end, which is then incorporated into RISC. Once in the RISC, the second strand is discarded and the miRNA that is now in the RISC is a mature miRNA, which binds to mRNAs that have complementary sequences.
In one aspect, a difference between miRNAs and siRNAs from the shRNA pathway is that base pairing with miRNAs comes from the 5′ end of the miRNA, which is also referred to as the seed sequence. Since the seed sequence is short, each miRNA may target many more mRNA transcripts.
In one embodiment, short hairpin RNA constructs are designed to be expressed as human miRNA (e.g., miR-30 or miR-21) primary transcripts. This design can add a Drosha processing site to the hairpin construct. The hairpin stem duplex of miRNA is typically 22 bp. In some embodiments, the antisense has perfect complementarity to desired target. In some embodiments, the duplex region is 22 bp and the loop region comprises a 15-19-nt loop from a human miR. Adding the miR loop and miR30 flanking sequences on either or both sides of the hairpin results in greater than 10-fold increase in Drosha and Dicer processing of the expressed hairpins when compared with conventional shRNA designs without microRNA. Increased Drosha and Dicer processing translates into greater siRNA/miRNA production and greater potency for expressed hairpins.
In some embodiments, an RNA inhibitor molecule that targets expression of a complement modulating gene, is a double stranded siRNA. In general, the guide strand of a given siRNA recognizes and binds to a target sequence that is complementary to the guide strand sequence. Stringent complementarity between guide strand and target sequence is, however, not required, as it is known in the art that a guide strand can still efficiently recognize and bind to a target sequence. Accordingly, in some embodiments, the guide strand sequence is 100% complementary to the target sequence. In some embodiments, the guide strand has as at least 80%, at least 85%, at least 90%, or at least 99% complementarity with the target sequence.
In certain embodiments, an siRNA comprises a first strand and a second strand that have the same number of nucleosides; however, the first and second strands are offset such that the two terminal nucleosides on the first and second strands are not paired with a residue on the complimentary strand. In certain instances, the two nucleosides that are not paired are thymidine resides. The siRNA should include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the siRNA, or a fragment thereof, can mediate down regulation of the target gene. Thus, as noted above, an siRNA includes a region which is at least partially complementary to the target RNA. It is not necessary that there be perfect complementarity between the siRNA and the target, but the correspondence must be sufficient to enable the siRNA, or a cleavage product thereof, to direct sequence specific silencing, such as by RNAi cleavage of the target RNA.
Although perfect complementarity, particularly in the antisense strand, is often desired, in some embodiments an siRNA has one or more, but preferably 10, 8, 6, 5, 4, 3, 2, or fewer mismatches with respect to the target RNA. The mismatches are typically most tolerated in the terminal regions. Thus, mismatches may be present in the terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5′ and/or 3′ terminus.
In some embodiments, the ds region of the siRNA can be about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments siRNAs have a duplex region of 7, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. In some embodiments, siRNAs have a duplex resin of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs of 2-3 nucleotides, preferably one or two 3′ overhangs, of 2-3 nucleotides.
An antisense oligonucleotide (ASO) is a single-stranded nucleic acid molecule, typically a deoxyribonucleotide molecule, that is complementary to the mRNA target. Not to be bound by theory, down regulation of a target polypeptide is usually achieved by induction of RNase H endonuclease activity that cleaves the RNA-DNA heteroduplex leading to a reduction in translation of the target gene. Other ASO-driven mechanisms include inhibition of 5′ cap formation, alteration of splicing process (splice-switching), and steric hindrance of ribosomal activity.
Design considerations include ensuring that an ASO does partially binds to a nontarget mRNA, as 6-7 base pairs between the ASO and nontarget mRNA can be sufficient to induce RNase activity, leading to cleavage of the wrong target. Software that analyzes RNA secondary and tertiary structure can be used to select target sequences that do not have marked secondary structure. In some embodiments, the length of an ASO is about 20 nucleotides and can, for example, be selected to target either the methionine initiation codon or splice sites (to block splicing). ASOs typically include modifications to enhance stability, e.g., are phosphorothioated, which does not enhance RNase H activity or influence solubility. In some embodiments, ASO may include ribose modifications, e.g., substitution of the hydrogen at the 2-position by an O-alkyl group and locked nucleic acid technology (LNA)] that reduce conformational plasticity.
Small inhibitory RNAs are transcribed endogenously with RNA Polymerase II (Pol III). Thus, in some embodiments, the promoter is a U6 promoter, H1 promoter, or 7SK promoter. In some embodiments the promoter is a variant of a naturally occurring human U6, H1, or 75K promoter, e.g., as described by Gao et al, Transcription 8:275-287, 2017. An exemplary U6 promoter is shown at SEQ ID NO:15.
In some embodiments, a multigene cargo as described herein comprises an RNA polymerase II promoter, i.e., a promoter sequence to initiate transcription from RNA polymerase II, such as a promoter for transcription of a transgene encoding a polypeptide such as CFHT; and a U6 or H1 promoter to drive expression of an inhibitory RNA molecule. In some embodiments, the second promoter is a U6 or H1 promoter.
In some embodiments, a single polymerase III promoter, e.g., a human H1 promoter, can be used to drive expression of both an inhibitory RNA and a protein-encoding transgene. (see, for example, Gao et al, Molecular Therapy: Nucleic Acids 14:32-40, 2019).
As discussed above, agents that bind and complement proteins and disease modifier proteins are delivered. These binding agents are encoded in multigene cargo and delivered to and expressed in cells. Binding agents are particularly useful for diminution of proteins produced in liver or other non-occular tissues. Exemplary binding agents are aflibercept (see Section 3.23 above) and single chain antibodies, nanobodies, bivalent nanobodies and the like.
Aflibercept is a recombinant fusion protein that binds VEGF-A that is used in treatment of AMD. VEGF expression in the retinal pigment epithelium (RPE) is associated with neovascular AMD.
Aflibercept is a recombinant inhibitory receptor (or “trap”) that binds VEGF and is used as a treatment for AMD. Exemplary DNA sequences encoding aflibercept are provided at SEQ ID NOs:6-8. Aflibercept-based gene therapy has been described (see, e.g., Kiss et al., 2020, “Analysis of Aflibercept Expression in NHPs following Intravitreal Administration of ADVM-022, a Potential Gene Therapy for nAMD” Mol Ther Methods Clin Dev. 18:345-353; Holash et al., 2002, “VEGF-Trap: a VEGF blocker with potent antitumor effects” Proc Natl Acad Sci USA 99:11393-8.). According to the present invention, a transgene encoding aflibercept, or a variant thereof, is encoded in multigene cargo and administered to the eye.
In one approach an antibody, e.g., an anti-complement protein antibody, is encoded in the multigene cargo and administered to a subject. Preferred antibodies are small, such as single chain antibodies (ScFv), single domain antibodies (nanobodies), dimerized nanobodies, bispecific nanobodies or bivalent nanobodies. In one approach, one or more nanobodies are encoded in multigene cargo. Nanobodies are single domain antibodies derived from heavy chain only antibodies found in the camelid family such as llamas. See, e.g., Jovlevska, 2020, The “Therapeutic Potential of Nanobodies” BioDrugs. 34:11-26; Zarantonello et el., 2021, “Nanobodies Provide Insight into the Molecular Mechanisms of the Complement Cascade and Offer New Therapeutic Strategies” Biomolecules 11:298; Pardon et al., 2014, “A general protocol for the generation of Nanobodies for structural biology”. Nat. Protoc. 9:674-693. Their relatively small size relative to human IgG make nanobodies suitable for DNA based delivery. In some embodiments humanized nanobodies are used. See Kazemi-Lomedasht et al., 2018, Design of a humanized anti vascular endothelial growth factor nanobody and evaluation of its in vitro function, Iran J Basic Med Sci. 21(3):260-266.
In some cases a bivalent nanobody is encoded in multigene cargo. Bivalent nanobodies are known therapeutic agents. For example, a single chain VHH bivalent nanobody targeting von Willebrand factor (vWF) has recently been approved for thrombotic thrombocytopenic purpura (aTTP) and several other antibodies are in clinical development (Morrison C., Nat Rev Drug Dis 18: 485-487, 2019). Examples include bispecific nanobodies targeting CFP and EGFR to kill tumor cells (Pedersen D. V., et al., Mol Immunol 124:200-210, 2020) or bispecific nanobodies targeting SARS-CoV-2 spike protein and albumin to block viral entry and to increase nanobody stability (Tijink, et al., Mol Cancer Ther 8:2288-2297, 2008).
In one approach, nanobodies or scFv antibodies are selected to be cross-reactive for (and thereby target) two or more related proteins. Of particular interest are nanobodies or scFv antibodies that bind two or more CFH related proteins (e.g., CFHR1/2, CFHR1/2/3/4/5, CFHR1/4). Methods for selection of cross-reactive antibodies are known. CFH related proteins are highly homologous and cross-reactive antibodies can be selected by raising or selecting antibodies against a highly conserved sequences or epitopes. The same strategy can be used to select anti-CFHR antibodies specific for specific CFHR(s) or that do not bind CFH or CFHT. In one approach two VHH nanobodies are linked via a peptide linker, to generate bispecific inhibitors with improved properties to uniquely inhibit multiple FHR family members. In some embodiments, nanobodies will be screened for cross-reactivity to homologous family members (e.g. FHR-1 nanobodies with affinity for FHR-2 or FHR-4 nanobodies with affinity for FHR-3), but such antibodies lack any appreciable affinity for CFH or CFHT proteins. In some embodiments, nanobodies will be constructed as a monotherapy to decrease the amount of endogenous complement pathway competition induced by FHR-1, FHR-2, FHR-3 and FHR-4 proteins or combined in a multigene therapy vector with CFHT or CFH and/or CFI and hpRNAs targeting C7 to provide therapeutic benefit in AMD patients. Nanobodies developed to target FHR-1 and/or FHR-4 can be used to block complement activation as single VHH domains, as bivalent VHH domains, or as bispecific VHH domains fused by a flexible linker region.
Nanobodies that specifically bind FHR-1 were produced as described in EXAMPLE 1 (See Section 10, below). Anti-FHR-1 nanobodies with cross-reactivity to CFH (FHR-1-CFH), CFHT (FHR-1-CFHT), and FHR-2 (FHR-1-FHR-2) were identified. Anti-FHR-1 nanobodies with cross-reactivity to FHR-2, FHR-3, FHR-4 and/or FHR-5, without cross-reactivity to CFH and/or CFHT are preferred. In one approach an anti-FHR-1 nanobody with cross-reactivity to FHR-2 is encoded in multigene cargo for delivery to cells. In one approach an anti-FHR-1 nanobody with cross-reactivity to FHR-3 is encoded in multigene cargo for delivery to cells. In one approach an anti-FHR-1 nanobody with cross-reactivity to FHR-2 is encoded in multigene cargo for delivery to cells. In one approach an anti-FHR-1 nanobody with cross-reactivity to FHR-5 is encoded in multigene cargo for delivery to cells. In one approach an anti-FHR-1 nanobody with cross-reactivity to two or more FHRs (FHR-2, FHR-3, FHR-4 and/or FHR-5) is encoded in multigene cargo for delivery to cells.
In certain aspects monotherapies are also contemplated by the present invention. As used herein, “monotherapy” refers to gene therapy in which a viral or nonviral vector delivers a single activity to a cell (i.e., carries cargo encoding a single hpRNA, nanobody or optimized CFHT.
An exemplary vector for hpRNA monotherapy comprises a cargo expressing an hpRNA described in TABLE 7. An exemplary vector for administering a nanobody can deliver a nanobody described in Example 1. An exemplary vector for monotherapy encodes an optimized CFHT as described in Section 3.4 above. In one approach an oCFHT is expressed from a cargo that does not include other activities.
In one approach, a cargo encoding an anti-FHR-1 nanobody with cross-reactivity to FHR-2 is encoded in cargo for delivery to cells. In one approach an anti-FHR-1 nanobody without cross-reactivity to CFH or CFHT is encoded in cargo for delivery to cells. In one approach a single bispecific anti-FHR-1/anti-FHR-4 nanobody with cross-reactivity to FHR-2 and FHR-3 is encoded in cargo for delivery to cells.
This section describes methods for coordinated expression of multiple gene products encoded in a single cargo delivered to the target ocular tissue.
Viral vectors are reviewed generally in Bulcha et al., 2021, “Viral vector platforms within the gene therapy landscape” Sig Transduct Target Ther 6, 53.
In some embodiments, a gene therapy vector is an adeno-associate virus vector (AAV). AAV is a primarily non-integrative virus with desirable safety characterises. However, AAV's limited packaging capacity means there are notable challenges to using this vector to deliver multiple (e.g., 2 or 3) activities. The total packaging capacity of AAV is about 4.7 Kb, including ITRs, and the vector can accommodate about 4.4 Kb of additional sequence including protein and/or inhibitory RNA encoding sequences and regulatory elements.
Multiple AAV serotypes are suitable for use in vectors of the present invention, including AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV113, AAVrh.74, AAV2.7m8, AAV.ANC80, Anc80L65m, AAVDJ, pseudotyped AAV, and derivatives thereof. In one approach the serotype is selected from AAV5, AAV4, AAV2, AAV8, AAV9, and AAVrh8r. In some embodiments, the vector is a derivative of an AAV2 serotype, e.g., AAV2.7m8. In one approach AAV2 is used.
Most of the AAV wildtype genome is deleted from AAV vectors, but the vector retains functional flanking ITR sequences. The Right ITR is the identical reverse complement of the Left ITR (so that a single 5′-3′ nucleotide sequence can define both ITRs). A certain degree of mismatch between the left and right ITRs is tolerated. AAV can comprise ITRs that are of a heterologous serotype in comparison with the capsid serotype (e.g., AAV2 ITRs with AAV5, AAV6, or AAV8 capsids). Various ITRs are known and are suitable for use with AAV2-type vector. In some embodiments, the AAV2 vector comprises a 128-bp ITR (SEQ ID NO:48). AAV vectors are well described in the scientific literature. See, e.g., Wang et al., “Adeno-associated virus vector as a platform for gene therapy delivery.” Nat Rev Drug Discov 18: 358-378 (2019); Li, et al., “Engineering adeno-associated virus vectors for gene therapy.” Nat Rev Genet 21: 255-272 (2020); Zolotukin et al., 2002, Production And Purification Of Serotype 1, 2, And 5 Recombinant Adeno-Associated Viral Vectors” Methods 28:158-167; Aponte-Ubillus et al., 2018, “Molecular design for recombinant adeno-associated virus (rAAV) vector production” Applied microbiology and biotechnology 102.3:1045-1054; Naso et al., 2017, “Adeno-Associated Virus (AAV) as a Vector for Gene Therapy” BioDrugs 31:317; and Penaud-Budloo et al., 2018., “Pharmacology of Recombinant Adeno-associated Virus Production” Molecular Therapy: Methods & Clinical Development 8:166-180.
In some embodiments, the vector for introducing a construct encoding multiple activities as described herein is a lentiviral vector. The basic structure of a lentiviral vector for gene therapy includes long terminal repeats (LTRs) that flank a nucleic acid sequence to be expressed by a cell. The LTRs can be divided into three elements: U3, R and U5. In one example, the viral construct comprises an inactivated or self-inactivating 3′ LTR from a lentivirus. The 3′ LTR may be made self-inactivating by any method known in the art. For example, the U3 element of the 3′ LTR can contain a deletion of its enhancer sequence, e.g., the TATA box, Sp1 and NF-kappa B sites. As a result of the self-inactivating 3′ LTR, the provirus that is integrated into the host genome will comprise an inactivated 5′ LTR. In some embodiments, the 5′ LTR of the lentiviral vector is truncated, relative to the native LTR. In some embodiments, the 5′ LTR comprises SEQ ID NO:49. In some embodiments, the 3′ LTR of the lentiviral vector is a Delta-U3 that comprises the polynucleotide sequence SEQ ID NO:50. Lentiviral vectors are well described in the medical literature. See, e.g., Milone, M. C., O'Doherty, U. Clinical use of lentiviral vectors. Leukemia 3:1529-1541 (2018); Esmaeili et al., 2020, Concise review on optimized methods in production and transduction of lentiviral vectors in order to facilitate immunotherapy and gene therapy, Biomedicine & Pharmacotherapy, 17:59-68.
Other viral vectors (including retroviruse, recombinant adenovirupox virus, alphavirus, retrovirus, arenavirus, measles, rabies, and herpes viruse based vectors may be used to deliver the cargo to the target tissue. Each vector system is well-understood by those of skill in the art. For general reviews see, e.g., Keeler et al., 2017, “Gene Therapy 2017: Progress And Future Directions” Clin Transl Sci (2017) 10, 242-248, incorporated by reference; Moore et al., 2017, “Gene Therapy For Age-Related Macular Degeneration” Expert Opinion on Biological Therapy 17:10: 1235-1244; Ochakovski et al., 2017, “Retinal Gene Therapy: Surgical Vector Delivery In The Translation To Clinical Trials” Frontiers in Neuroscience 11; Schon et al., 2015, “Retinal Gene Delivery By Adeno-Associated Virus (Aav) Vectors: Strategies And Applications” European Journal of Pharmaceutics and Biopharmaceutics 95:343-352; Naso et al., 2017, “Adeno-Associated Virus (AAV) As A Vector For Gene Therapy” BioDrugs 31:317; Dunbar et al., 2018, “Gene therapy comes of age,” Science 359: 6372, all incorporated herein by reference.
In some embodiments, delivery vehicles such as liposomes, nanocapsules, nanoparticles, microspheres, and the like, may be used to facilitate administration of the vectors and/or to deliver other nucleic acids, including shRNa, miRNA, or siRNA to target cells. See, e.g., Itaka and Kataoka, 2009, “Recent development of nonviral gene delivery systems with virus-like structures and mechanisms,” Eur J Pharma and Biopharma 71:475-483; Thomas et al., 2020, “Biodegradable Polymers for Gene Delivery” Molecules 24(20): 3744doi: 10.3390/molecules24203744; Wooff et al., 2020, Small-Medium Extracellular Vesicles and Their miRNA Cargo in Retinal Health and Degeneration: Mediators of Homeostasis, and Vehicles for Targeted Gene Therapy, Frontiers in Cellular Neuroscience 14:1662-5102; Hong et al., “Functional Nanostructures for Effective Delivery of Small Interfering RNA Therapeutics.” Theranostics 4.12 (2014): 1211-1232. PMC. Web. 13 Sep. 2018, each of which is hereby incorporated by reference in its entirety for all purposes. Expression strategies
Various molecular strategies can be used to express two or more gene products from a single gene therapy vector, including use of multiple promoters, use of bidirectional promoters, use of IRES elements, use of ribosome skipping and cleavage factors. In some embodiments, a Pol 11 promoter drives expression of a sequence encoding an inhibitory RNA and an RNA polymerase II promoter to drive expression of a polypeptide gene product.
In some embodiments, two or more gene products are expression in a single cistron. (see, e.g., Shaimardanova et al., “Production and Application of Multicistronic Constructs for Various Human Disease Therapies” Pharmaceutics 11:580, 2019). Thus, for example, in some embodiments, the multigene construct can comprise one promoter that drives expression of one or more gene products. Accordingly, such constructs can comprise various elements including IRES sequences and/or sequences encoding peptides that are cleaved.
Incorporation of an internal ribosome entry site (IRES) can be employed to efficiently co-express multiple gene products from the same promoter. In some embodiments, a construct to express two or more different genes from a gene therapy vector can comprise an internal ribosome entry site (IRES). IRES elements can bypass the ribosome scanning model of 5′-methylated Cap dependent translation and begin translation at internal sites. IRES elements from members of the picornavirus family (e.g., polio and encephalomyocarditis) have been described, as well an IRES from a mammalian mRNA. In some embodiments, multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. In some embodiments, multiple genes are expressed using a single promoter/enhancer to transcribe a single message.
In some embodiments, the gene products encoded by the vector are expressed bicistronically or multicistronically (i.e., two or more polypeptides, or inhibitory RNAs, are expressed from a single mRNA) by including one or more ribosomal skipping elements that encode a ribosome skipping polypeptide (also referred to herein as a “self-cleaving” peptide”) between a nucleic acid encoding a first polypeptide and a nucleic acid encoding a second polypeptide or inhibitory RNA. Similarly, three polypeptides can be expressed from one mRNA by including two ribosomal skipping elements. See, for example, Chang et al., 2015, “Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells,” MAbs 7(2): 403-412.
A “ribosomal skipping element” refers to a nucleotide sequence that encodes a short peptide sequence that generates two peptide chains from translation of one mRNA molecule. In some embodiments, the ribosomal skipping element encodes a peptide comprising a consensus motif of DX1EX2NPG wherein X1 and X2 are independently selected from any amino acid. In some embodiments, the ribosomal skipping element encodes a peptide comprising a consensus motif of DX1EX2NPG wherein X1 is V or I and X2 is any amino acid. In some embodiments, the ribosomal skipping element encodes a Thosea asigna virus 2A peptide (T2A), a porcine teschovirus-12A peptide (P2A), a foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), a cytoplasmic polyhedrosis virus 2A peptide (BmCPV 2A), or a flacherie virus of B. mori 2A peptide (BmIFV 2A). Such peptides are typically from 18-22 amino acids in length.
While not wishing to be bound by theory, it is hypothesized that ribosomal skipping elements function by terminating translation of the first peptide chain and re-initiating translation of the second peptide chain; or by cleavage of a peptide bond in the peptide sequence encoded by the ribosomal skipping element by an intrinsic protease activity of the encoded peptide, or by another protease in the environment (e.g., cytosol). Exemplary self-cleaving peptide sequences are shown as SEQ ID Nos: 31, 33, 35 and 37, and examples of nucleic acid sequences that may be used to introduce the sequences is shown as SEQ ID Nos: 30, 32, 24, and 36 respectively.
In some approaches a glycine- and/or serine-containing linker sequence is introduced upstream from the self-cleaving peptide sequence to enhance cleavage. In some embodiments, a Gly-Ser spacer, e.g., a sequence Gly-Ser-Gly or Ser-Gly-Ser-Gly, is incorporated at the N terminus of the 2A sequence. Exemplary linker sequences include GlyGlyGlySer (SEQ ID NO:56), which can be repeated, e.g., the sequence can be present “n” times, e.g., where n=1-10 or 1-20; and GlyGlyGlyGlySer (SEQ ID NO:57), which can be present in the linker “n” times, e.g., where n=1-10 or 1-20. A furin cleavage site may be added upstream of the self-cleaving peptide sequence to eliminate or minimize amino acids appended to the amino terminus. Added amino acids at the amino terminus of the protein in position 2 can be removed with the removal of the signal peptide, for example, to generate a mature protein.
In some embodiments, a nucleic acid sequence encoding a furin cleavage site may be incorporated into a multigene construct. In some embodiments, a furin cleavage site is upstream of the ribosomal skipping peptide, either with or without a Gly-Ser spacer. In some embodiments, a minimal furin cleavage site is Arg-X-X Arg (e.g., SEQ ID NO:39). In some embodiments, the cleavage site is Arg-X-(Lys/Arg)-Arg. In some embodiments, a furin cleavage site comprises GGRGRR (SEQ ID NO:39). In some embodiments, a furin cleavage site comprises GGRGRRGG (SEQ ID NO:40). In some embodiments, the furin cleavage site replaces an IRES and/or a ribosomal skipping peptide.
The relative levels and locations of polypeptide expression can be controlled by various promoters, including constitutive and cell-specific (e.g. RPE and choroidal endothelial cells, melanocytes, fibroblasts) promoters, e.g., promoters such as cytomegalovirus enhancer-chicken beta actin, CBA or CAG; small cytomegalovirus enhancer-chicken beta actin (smCBA); vitelliform macular dystrophy 2, VMD2; retinal pigment epithelium 65, RPE65; fms-related tyrosine kinase 1, FLT; von Willebrand factor, VWF and Endoglin, ENG) promoters; or promoters such as synthetic cytomegalovirus enhancer-vitelliform macular dystrophy 2, BEST1-v3-EP454 promoter enhancer; synthetic cytomegalovirus enhancer-retinal pigment epithelium 65, RPE65-F2/R20-EP415; and synthetic cytomegalovirus enhancer-retinal pigment epithelium 65, RPE65-F6/R26-EP419) promoters. In some embodiments the promotor is a large CMV enhancer and chicken beta actin promoter (CBA) promoter, BEST1-EP-454 promoter enhancer or spleen focus forming virus (SFFV) promoter (Hoffmann, D., et al., Gene Ther 24:298-307, 2017). Exemplary promoter sequences are provided at SEQ ID NOS:10-14, 16, 51, 185-191, 238, and 239.
Additional non-limiting examples of suitable eukaryotic promoters (i.e., promoters functional in eukaryotic cells) include a herpes simplex virus thymidine kinase gene promoter, early and late SV40 promoters, long terminal repeats from retrovirus, a human elongation factor-1 promoter, a bovine growth hormone promoter, a murine stem cell virus promoter, a phosphoglycerate kinase-1 locus promoter, and a ubiquitin gene promoter.
It will be understood by those of skill in the art that regulatory (promoter/enhancer) sequences can tolerate a certain degree of variation retaining the regulatory transcription regulatory function. In certain embodiments described herein in which a promoter/enhancer is designated, a substantially identical sequence (e.g., a sequence with at least about 90% identity, preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% nucleotide identity over the entire promoter/enhancer sequence) is contemplated as a suitable substitute for the exemplified sequence. As is well known in the art, variation is tolerated in the relationship (e.g., distance and orientation) between enhancers and promoters. Promoter variants and alternative promoters can be generated using well known techniques including truncation analysis, identification of transcription factor binding sites, motif analysis, and the like (see, e.g., Boeva, Frontiers in Genetics Vol. 7, Article 24, 2016); see Hageman and Richards WO2020019002A1, describing promoter analysis) to identify active promoters. Identification of active promoters can also comprise analysis of promoter activity as measured by art known means.
In some embodiments, a CBA (chicken beta-actin) promoter is used to drive expression of a gene product encoded by a cargo. In one embodiment, the CBA promoter includes a CMV enhancer sequence, the beta actin promoter, a spacer, a chicken b-actin intron, an intron acceptor b-globin, and a beta globin exon 3. An exemplary CBA promoter has a sequence of SEQ NO:12, or is a variant thereof with at least about 90% or 95% sequence identity to SEQ ID NO:12.
In some embodiments, a smCBA (small modified chicken beta-actin) promoter is used to drive expression of a protein encoded by a transgene. See U.S. Pat. No. 8,298,818. In one embodiment, the smCBA promoter includes a CMV enhancer sequence, the beta actin promoter, a spacer, a chicken b-actin intron, an intron acceptor b-globin, and a beta globin exon 3. An exemplary sequence is provided at SEQ ID NOs: 10 and 11. It will be recognized that the sequence can be modified without loss of biological activity, and variants of the sequence with at least about 90% or about 95% sequence identity can be used.
In some embodiments, a sctmCBA promoter is used to drive transcription of a gene product encoded by a cargo. In one embodiment, the sctmCBA promoter includes a CMV enhancer sequence, the beta actin promoter, a spacer, and a truncated chicken b-actin intron. An exemplary smtmCBA promoter is set forth as SEQ ID NO.:18. It will be recognized that the sequence can be modified without loss of biological activity, and variants of the sequence with at least about 90% or about 95% sequence identity can be used.
In some embodiments, a BEST1-EP-454 promoter is used. An exemplary BEST1-EP-454 enhancer promoter is provided at SEQ ID NO:238. An exemplary BEST1-V3 promoter is provided at SEQ ID NO:239. It will be recognized that this sequence can be modified without loss of biological activity, and variants of the sequence with at least about 90% or about 95% sequence identity can be used.
In one embodiment, a form of VMD2 promoter is used (see, e.g., SEQ NO:13). VMD2 has 680 bases from BEST1-743 and a 97 base 3′ enhancer sequence from SV40 intron. An illustrative 624 base promoter sequence is provided at SEQ ID NO:51. It will be recognized that this sequence can be modified without loss of biological activity, and variants of the sequence with at least about 90% or about 95% sequence identity can be used.
In one embodiment, a truncated RPE65 promoter is used. An exemplary sequence is provided at SEQ ID NO:14. It will be recognized that this sequence can be modified without loss of biological activity, and variants of the sequence with at least about 90% or about 95% sequence identity can be used.
Other useful promoters include (i) TYR Promoter (SEQ ID NO:185); (ii) TYRP1(SEQ ID NO:186); Choroid-enriched highly active promoters FLT1 (SEQ ID NO:187); VWF (SEQ ID NO:188); MGP (SEQ ID NO:189); RGS5 (SEQ ID NO:190); and SFFV (SEQ ID NO:191).
Bidirectional promoters drive the expression of two adjacent genes coded on opposite DNA strands. Bidirectional promoters include naturally occurring and synthetic promoters.
In some embodiments, a bidirectional promoter comprises phosphoglycerate kinase 1(PGK1) and elongation factor 1 alpha EF1a in a back-to-back configuration (Goding & Mann, Gene Terh. 2011, August:18(8):817-26). In some embodiments, a bidirectional promoter comprises a chicken beta-actin promoter duplication in opposing directions with a CMV enhancer in the middle.
In some embodiments, the promoter is a bidirectional mouse CMV promoter, which comprises the mouse CMV immediate early 1 promoter in one direction and the mouse CMV immediate early 2 promoter in the opposite directions. Such a promoter may further comprises enhancers, such as the natural mouse CMV enhancer, that comprises a major immediate early 1 enhancer and a major immediate early 2 enhancer. Such bidirectional promoters are described, e.g., in European Pat. No. EP1601776 and U.S. Pat. No. 10,570,417.
In some embodiments, the bidirectional promoter comprises a promoter that drives expression of protein in one direction and an RNA in the other direction. For example, in some embodiments, such a promoter comprises a Pol 11 promoter that contains 3 external control elements: a distal sequence element (DSE), a proximal sequence element (PSE), and a TATA box; and (2) a second basic Pol III promoter that includes a PSE and a TATA box fused to the 5′ terminus of the DSE in reverse orientation. For example, in the H1 promoter, the DSE is adjacent to the PSE and the TATA box, and the promoter can be rendered bidirectional by creating a hybrid promoter in which transcription in the reverse direction is controlled by appending a PSE and TATA box derived from the U6 promoter. See, e.g., U.S. Pat. No. 9,907,863 (describing use of the H1 promoter to express CRISPR gRNA with the H1 promoter sequence as a bidirectional promoter to express Cas9 nuclease).
In some embodiments, the viral vector further comprises a posttranscriptional regulatory element, such as a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) or shortened versions of WPRE) to enhance expression of transgenes delivered using the viral vector (e.g., SEQ ID NO:25 and 26).
Constructs for expression complement regulatory sequences as described herein include polyadenylation sequences. Exemplary polyadenylation sequences include sequences derived from the bovine Growth Hormone bGH polyadenylation signal (e.g., SEQ ID NO:29); sequences derived from the HSV Thymidine Kinase polyadenylation signal (e.g., SEQ ID NO:28); and sequences derived from the SV40 polyadenylation signal (e.g., SEQ ID NO:27).
Sequences encoding regulatory elements, protein or RNA encoding elements or other elements may be contiguous or may be separated by spacer sequences. For example and not limitation spacer sequences are generally 10-100 bp in length. For example and not limitation exemplary spacers are provided as SEQ ID Nos: 41 and 43.
TABLES 8-10 describe exemplary constructs.
The administration and dose of the gene therapy vectors of the invention will depend on factors such as the complement dysregulation disease being treated. For example, the gene therapy vector may be administered by systemic administration (e.g., intravenous injection or infusion), local injection or infusion (e.g., subretinal, suprachoroidal, intravitreal, transscleral, trans corneal or other ocular), by use of an osmotic pump, by electroporation, by application (e.g., eye drops) and by other means. It is contemplated that transgenes of the invention may be introduced into, and expressed in, a variety of cell types including neural retinal cell types (such as rod photoreceptor cells, cone photoreceptor cells, ganglion cells), RPE, ciliary epithelial, scleral, iris, choroidal (such as choroidal endothelial cells, melanocytes, fibroblasts) and other ocular cells. See Xue et al., “Technique Of Retinal Gene Therapy: Delivery Of Viral Vector Into The Subretinal Space” Eye 31:1308-1316, 2017; Moore et al., 2017, “Gene Therapy For Age-Related Macular Degeneration” Expert Opinion on Biological Therapy 17:10: 1235-1244; Ochakovski et al., 2017, “Retinal Gene Therapy: Surgical Vector Delivery In The Translation To Clinical Trials” Frontiers in Neuroscience 11; Schon et al., 2015, “Retinal Gene Delivery By Adeno-Associated Virus (Aav) Vectors: Strategies And Applications” European Journal of Pharmaceutics and Biopharmaceutics 95:343-352.
In one approach, the vector is administered via a suprachoroidal injection, thereby allowing the agent access to choroidal, scleral and the RPE cells. See Ding et al., “AAV8-vectored suprachoroidal gene transfer produces widespread ocular transgene expression”, J Clin Invest 129(11):4901-4911, 2019. Also see Emami- and Yiu, Medical and Surgical Applications for the Suprachoroidal Space, Int Ophthalmol Clin 59(1):195-207, 2019. In one approach, the agent is administered via subretinal injection. See Xue et al., “Technique Of Retinal Gene Therapy: Delivery Of Viral Vector Into The Subretinal Space” Eye 31:1308-1316, 2017. In another approach, the agent can be injected into the vitreous. Kansar et al., “Suprachoroidal Delivery of Viral and Nonviral Gene Therapy for Retinal Disease”, J Ocular Pharmacol Ther 36:610.1. The amount of agent administered will be an “effective amount” or a “therapeutically effective amount,” i.e., an amount that is effective, at dosages and for periods of time necessary, to achieve a desired result. A desired result would include an improvement in a symptom associated with AMD progression. In another approach, the vector (viral or nonviral) is delivered to the eye by electroporation (Lebreton et al US patent application 20210128911; Behar-Cohen U.S. patent application Ser. No. 17/144,341; Bejjani et al, 2007, Survey Ophthalmol 52:196-208; Bloquel et al, 2006, Adv Drug Delivery Rev 58:1224-42).
As noted previously, complement dysregulation is an underlying element in many conditions and it is contemplated that these conditions can be treated using the methods described herein above. Treatable conditions include anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Alzheimer's disease, atypical hemolytic uremic syndrome, acute kidney injury, age-related macular degeneration, antibody-mediated rejection, antiphospholipid syndrome, acute respiratory distress syndrome, Berger's disease, bullous pemphigoid, C3 glomerulopathy, C3 glomerulonephritis, cold agglutinin disease, chronic obstructive pulmonary disease, cardiopulmonary bypass, central serous chorioretinopathy, dense deposit disease, delayed graft function, early onset macular drusen, glaucoma, Guillain-Barré syndrome, generalized myasthenia gravis, granulomatosis with polyangiitis, graft versus host disease, hereditary angioedema, hidradenitis suppurativa, hematopoietic stem cell transplant-related thrombotic microangiopathy, IgA nephropathy, ischemia/reperfusion, immune complex-mediated membranoproliferative glomerulonephritis, immune-mediated necrotizing myopathy, idiopathic polypoidal choroidal vasculopathy, kidney transplant, lupus nephritis, membranous nephropathy, microscopic polyangiitis, multiple sclerosis, neuromyelitis optica (spectrum disorder), pyoderma Gangrenosum, paroxysmal nocturnal hemoglobinuria, proliferative diabetic retinopathyrheumatoid arthritis/osteoarthritis, systemic inflammatory response syndrome, systemic lupus erythematosus, Stargardt Disease, severe sepsis, septic shock, thrombotic microangiopathy, warm-type autoimmune hemolytic anemia and many others (Ricklin et al. 2016, 2018; Zelek et al 2019; Harris et al. 2018; Morgan & Harris 2015; Thurman and Holers 2006; Barlow 2017; Gavriilaki and Brodsky 2020; Zipfel et al. 2006; Schmidt et al. 2016; Mullins et al. 2017; Mohlin et al. 2017; Jha et al. 2007; Perkins et al. 2020; Taylor et al. 2019; Clark and Bishop 2018; Mandava et al 2020; de Jong et al. 2021; Bosco et al. 2018; Kaye 2020; Silverman and Wong 2019; Sohn et al. 2007; Xu and Chen 2016; Veszeli et al. 2014).
Nanobodies were produced at Yurogen Biosystems (Worcester, MA) by camelid immunization with FHR-1A protein (Yurogen). PBMC were isolated from selected camelids and a VHH-contained cDNA library was prepared. Antigen-specific VHH were identified using a plate-based method and a VHH-specific detection antibody.
A total of 1536 individual B-cells were isolated 70 days after FHR-1 protein immunization and supernatant screened for FHR-1 direct binding. VHH positive clones were detected using a rabbit anti-camelid VHH antibody. A binding signal cut-off of 0.5 RLU (relative light units) in the direct FHR-1 ELISA identified 246 clones and 0.9 RLU signal cut-off resulted in 45 clones (2.93% of total). See TABLE 11.
The top 45 FHR-1 positive VHH nanobody clones were screened against recombinant his-tagged CFH, CFHT, FHR-2 and FHR-4 proteins (0.5 mg/mL identical to the original FHR-1 binding assay). Approximately 71% of the FHR-1 nanobody clones exhibit cross-reactivity to CFH, 2.2% to CFHT, 38% to FHR-2 and 0% to FHR-4 protein (RLU cut-off of ≥0.1). See TABLE 12.
As noted above, where an exogenous protein, promotor, or regulatory element is described it is contemplated that variants could be used in place of the exemplified sequences, including naturally occurring variants of proteins and nucleic acids, and nonnaturally occurring sequences that are substantially identical to a reference sequence that is, or that encodes a protein that is, functionally similar to the reference sequence. Functional similarity can be determined using art-known assays for protein or nucleic acid function. Typically a variant that has a level of activity that is 90% or greater of that measured for the reference protein is functionally similar to the reference protein. This section describes certain assays for activity and/or quantity of various polynucleotides or encoded proteins. However, it will be understood that assay conditions can be adjusted. Further, it is understood that the scientific literature is repeat with assays for complement protein activity, promoter activity and the like and that the skilled PR actioner can easily identify alternative or additional assays. It is further understood that assay conditions can be adjusted.
The C3b binding assay uses C3b protein (Complement Technology, Cat. #A114) diluted with 50 mM carbonate coating buffer (pH 9.6) at a final concentration of 50 μM. A total of 100 μl of C3b/carbonate solution was added to each well of a black MaxiSorp 96-wellmicroplate, covered and incubated overnight at 4° C. Blank control wells were incubated with carbonate coating buffer containing no C3b protein. Wells were washed 3 times with 300 μl PBST then blocked for 1 hour with reagent dilution buffer (1% BSA in PBS). Wells were washed again 3 times with 300 μl PBST. Dilutions of recombinant protective CFH, CFHT, oCFHT or supernatant from transiently transfected or transduced Cos-7 cells were prepared in reagent dilution buffer, added to wells in triplicate and incubated for 1 hour at room temperature. The plate was washed as above then incubated for 1 hour with anti-CFH/CFHT biotinylated aCTM87b antibody (OX-24, AbCam, Cat. #ab112197) at 1:1000 dilution. The plates were washed as above, then incubated for 1 hour with high sensitivity Streptavidin-HRP (Pierce Cat. #21130) at 1:10,000, washed as above then incubated for 5 minutes with SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Scientific, Cat. #37069) before detection using the BioTek Synergy4 plate reader.
To measure binding of CFH, CFHT, and oCFHT proteins to CRP (R&D Systems, Cat. #1707-CR/CF) or MDA-LDL ligands (Cell Biolabs, Cat. #STA-212), microtiter plates(Maxisorp, Nunc) were coated overnight at 4° C. at a concentration of 2.5 μg/ml in PBS. Wells treated with only PBS or non-modified LDL (Cell Biolabs, Cat. #STA-241) served as negative ligand binding controls. After three washes in PBST, plates were blocked with SuperBlock T20 (PBS) Blocking Buffer (Thermo Scientific, Cat. #37516) for 1 hour at room temperature. Various dilutions of recombinant protective CFH, CFHT, or oCFHT or supernatant from transiently transfected or transduced Cos-7 cells were added in blocking buffer and incubated at room temperature for 1 hour. The relative binding of CFH, CFHT, or oCFHT proteins was assessed using biotinylated aCTM87b followed by high sensitivity Streptavidin-HRP and signal was detected as described above.
LPS-dependent Alternative Pathway (AP) Assay to Monitor CFH, CFHT, oCFHT and/or CFI Protein Activity AP activation was determined using an established ELISA-based assay (Harder M J et al. (2015) J. Immunol. 196:866-876) with minor modifications. In brief, 50 μl LPS solution (50 g/ml) from Salmonella typhimurium (Sigma-Aldrich, Cat. #L7261) was coated onto 96-well plates (Maxisorp; Nunc) in PBS overnight at 4 C, followed by washing three times with PBS+Tween 20. Plates were then blocked with 1% BSA/PBS for 1 hour at room temperature. Various dilutions of recombinant protective CFH, CFHT, oCFHT, CFI or supernatant from transiently transfected or transduced Cos-7 cells were mixed with 30 μl 25% normal human serum containing 10 mM MgEGTA. The mixture of analytes in serum was added to LPS-coated wells and incubated for 1.5 hours at 37° C. prior to washing and subsequent exposure to HRP conjugated goat anti-human C3 (MP Biomedicals, Cat. #855237) at 1:10,000 dilution in 1% BSA/PBS for 1 hour at room temperature. After washing three times with PBST, C3b deposition on the plate was indirectly detected using SuperSignal ELISA Pico Chemiluminescent Substrate and the BioTek Synergy 4 plate reader. PBS and EDTA (final concentration 5 mM) were used as positive and negative controls, respectively, and cell culture supernatant provided tissue media control. All responses were normalized to the activity achieved when only PBS was added in the absence of a CFH, CFHT, oCFHT and/or CFI protein regulators. All data were plotted using a nonlinear regression log (inhibitor) versus response (three parameters) model in Prism 9.0.
For fluid-phase alternative pathway activity, various dilutions of recombinant protective CFH, CFHT, CFI or supernatant from transiently transfected or transduced Cos-7 cells were mixed with 15 μl of CFH-depleted normal human serum (Complement Technology, Cat. #A337), 5 μl of Mg EGTA (0.1 M) and 25 μl of rabbit erythrocytes (Complement Technology, Cat. #B300) (5×108/ml in GVB) in a final volume of 100 μl of GVB. The reaction mixture was incubated at 37′C for 30 min and stopped by adding 1 ml of GVBE(GVB containing 10 mM EDTA). After centrifugation at 1000 g for 3 min, the absorbance of 100 μl of the supernatant from each sample was determined at 412 nm using the BioTek Synergy 4 plate reader. The percentage of lysis was normalized by setting 100% lysis to be equal to the degree of lysis occurring in the presence of normal human serum (Complement Technology, Cat. #NHS). CFI-Dependent Cofactor Activity Assay to Monitor CFH, CFHT, oCFHT, and/or CFI Protein Activity.
Co-Factor Activity Assay to Monitor CFH, CFHT, oCFHT and/or CFI Protein Activities
A fluid-phase assay was used to measure cofactor activity. Various dilutions of recombinant protective CFH, CFHT, oCFHt, CFI or supernatant from transiently transfected or transduced Cos-7 cells were incubated with C3b (Complement Technologies, Cat. #A114) and CFI (Complement Technologies, Cat. #A138) in a total volume of 20 μl PBS for 20 min at 37′C. For each reaction, 526 nM of C3b and 22.7 nM of CFI were used with varying concentrations of CFH, CFHT, or oCFHT proteins or Cos-7 supernatant. In some examples, CFI protein was eliminated from the experiment to monitor CFI activity in Cos-7 supernatant. The assay was stopped with the addition of 3 μl 10× NuPAGE sample reducing agent (Life Technologies, cat. #NP0009) and heated for 7 min at 95° C. Samples were electrophoresed on a 4-12% NuPAGE Bis-Tris gel at 200 V for 45 min to maximize separation of C3b breakdown products. Visualization of C3b and iC3b bands was accomplished by Coomassie blue staining (Life Technologies, Cat. #LC6065). The band intensity of C3b α-chain, C3b β-chain, and the 68-kDa and 43-kDa iC3b products were measured using Multi Gauge software (version 3.0) on the LAS4000 image analyzer and plotted using Prism GraphPad 9.0 software.
Aflibercept levels in Cos-7 cell culture supernatant after transfection or transduction with plasmid DNA were measured using an ELISA based assay (Kiss et al, Mol Ther Methods Clin Dev. 2020, 18: 345-353). Briefly, MaxiSorp plates were coated with 100 μl/well recombinant human VEGFA (rhVEGFA; R&D Systems Cat. #293-VE-010/CF) at a concentration of 1 μg/ml in coating buffer (R&D Systems, Cat. #DY006 Systems) and incubated overnight at 4′C. After being washed with wash buffer (KPL, VWR Cat. #95059-132), the plate was blocked with 300 μl/well Pierce protein-free blocking buffer (ThermoFisher Scientific, Cat. #37572) for 1.5 hr at room temperature. Afterward, the plate was washed then 1:25 diluted Cos-7 cell culture supernatant in blocking buffer and 100 μl added/well and incubated for 2 hr at room temperature. The plate was washed again and 100 μl/well anti-human Fcγ-specific antibody conjugated to horseradish peroxidase (HRP) (Jackson Immuno Research, Cat. #109-035-008) at 500 ng/ml in bovine serum albumin (1% in PBS) was added to the wells. After a washing step, 100 μl/well SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Cat. #37069) was added to the wells and luminescence signal was measured using the BioTek Synergy 4 plate reader. EYLEA (Regeneron Pharmaceuticals) purchased from the Moran Eye Center Pharmacy was used to generate the Aflibercept ELISA protein standard curve and determine concentrations from cell culture supernatant samples.
The VEGF inhibitory activity of Aflibercept in Cos-7 cell culture supernatant was measured using a VEGF Bioassay kit from Promega (Cat. #GA2001) following the supplied protocol. Methods are described briefly below. Control (ELYEA) and cell culture supernatant samples were 2-fold serially diluted in kit supplied assay buffer (DMEM medium with 10% FBS). The assay procedure is as follows—1. Prepare cell culture supernatant by serial dilution in assay buffer. In our assay, ELYEA was used as positive control with a starting concentration of 500 ng/ml (3×=1.5 μg/ml). 2. Prepare 3×VEGF at EC80 concentration in assay buffer. The expected EC80 of VEGF is approximately 20 ng/ml (3×=60 ng/ml). 3. Transfer 0.4 ml of KDR/NFAT-RE HEK293 cells to a 15 ml conical tube containing 4.6 ml of assay buffer. Mix well by gently inverting 1-2 times. Dispense 25 μl of the cell suspension to each well of 96-well assay plate. 4. Dispense 25 μl of the 3×ELYEA and cell culture supernatant serial dilutions to the 25 μl of pre-plated cells. 5. Dispense 25 μl of the 3×EC80 VEGF mixture prepared to wells. The final assay volume is 75 μl. 6. Cover assay plate with a lid and incubate in a 37° C., 5% C02 humidified incubator for 6 hours. 7. Remove the assay plate from the incubator and equilibrate to room temperature for 10-15 minutes. 8. Add 75 μl of Bio-Glo Reagent to the wells, taking care not to create bubbles. 9. Incubate at room temperature for 5-10 minutes. 10. Measure luminescence using BioTek Synergy 4 plate reader and plot data using Prism GraphPad 9.0 software.
The C7 capture antibody (Complement Technology #A224, 1 mg/ml) was diluted 1:32000 in 50 mM carbonate coating buffer (pH 9.6); a total of 100 μl of antibody/carbonate solution was added to each well of a black MaxiSorp 96-well microplate, covered and incubated overnight at 4° C. Plates were washed using the BIOTEK EL404 Microplate washer consisting of 3 cycles of 350 μl PBST with 5 seconds of soak time and no shaking followed by aspiration then blocked for 1.5 hours with reagent dilution buffer (1% BSA in 1×PBS). Human C7 protein standards (Complement Technology #A124, 1 mg/ml) were diluted in RDB just prior to assay. Standards and Cos-7 supernatant samples (100 μl) were added to each well in duplicate and incubated for 1.5 hours at room temperature. Normal human serum standard (Quidel Cat. No. A100) and C7-depleted serum (Complement Technology Cat. #A324, 1 mg/ml) were added to each assay plate as positive and negative controls, respectively. The plate was washed as above then incubated for 1.5 hours with sandwich antibody at 1:5000 dilution (ThermoFisher Cat. #MA5-30114, 1 mg/ml). The plate was washed as above and then incubated for 1 hour with anti-rabbit-HRP (Jackson ImmunoResearch, Cat. #111-035-045) at 1:10,000 dilution. The plate was washed as above, then incubated for 5 minutes with SuperSignal ELISA pico chemiluminescent substrate (ThermoFisher Scientific, Cat. #37069) before detection using the BioTek Synergy4 plate reader.
This example shows that C7 gene expression is upregulated in RPE/choroid in AMD, and that C7 serum levels are elevated are elevated in AMD. We further demonstrate that hpRNA can be used to decrease C7 levels in vitro.
We measured C7 mRNA levels in the RPE-choroid and retina of donors with AMD risk-associated genotypes, e.g., homozygous Chr1-Risk (G1), Chr1-I62 (G8), Chr1-Del (G10) and homozygous Chr10-Risk (G25, G26, G27, G28, G29, G30) genotypes. See
Plasma levels of C7 protein were measured in individuals with homozygous Chr. 1 risk genotypes using a Human C7 ELISA as described in Example 3, above. In this population, a 32% increase in plasma C7 protein level was observed in individuals with late stage AMD compared to individuals with no clinical AMD. See
Cos-7 cells were transiently transfected with 50 ng human C7 cDNA expression construct and 50 or 100 ng of control (5′ GCACTACCAGAGCTAACTCAGATAGTACT 3′, SEQ ID NO:241) or a C7-specific shRNA plasmid DNA (see Table 7). C7 protein in supernatant was detected using a Human C7 ELISA as described in Example 3, above. Three independent shRNA plasmid DNAs (A, B and D) targeting C7 mRNA reduced C7 protein. See
Recombinant AAV2 viral particles containing expression constructs in which a U6 promoter is employed to express shRNA_A, shRNA_B, or shRNA_D were produced to assess reduction of endogenously expressed C7 in two neuroblastoma cell lines (SK-N-AS and SK-N-SH). The hpRNA sequences were each cloned into a pCTM297 KanR vector using standard restriction enzyme techniques. The constructs for shRNA_B and shRNA_D employ the same backbone vector. Plasmid pCTM297 also contains a cDNA sequence encoding CFHT (SEQ ID NO:9). Plasmid construction is described in greater detail in Section 10.5.
The sequences (sense-hairpin-antisense) for shRNA_A, shRNA_B and shRNA_D are as follows:
SK-N-AS (ATCC, Cat. #CRL-2137) and SK-N-SH (ATCC, Cat. #HTB-11) cells were maintained in Dulbecco's Modified Eagle's Medium with 10% FBS. Cells were plated at 10,000 cells per well in a 96-well plate format one day prior to rAAV2 transduction. The following day, rAAV2 particles were added to wells with 75 μl of DMEM. Viral particles at various multiplicity of infection (10E6, 10E5, 10E4, 10E3, 10E2, 10E1 and 10E0 vg/cell) were added to wells and 24 hours post-transduction, replaced with fresh media. After an additional 72 hours, supernatant was collected and tested for C7 level using a C7 ELISA.
As shown in
Percent C7 protein reduction is shown in the lower panel of
Overall, these results indicate that C7 shRNAs are effective in down-regulating endogenous C7 protein expression in neuroblastoma cells lines, with shRNA_A and shRNA_B observed as the most effective in this experiment. Because C7 protein levels are much lower in the RPE-choroid region of human eyes, the results provide evidence that shRNA sequences will reduce C7 protein to levels that will significantly alter the amount of membrane attack complex (MAC).
To confirm that AAV2 based multigene therapy vectors are capable of expressing another transgene, we also determined protective CFHT protein levels expressed in the same rAAV2 viral vectors. Supernatant used to assess C7 levels after hpRNA knockdown were similarly tested at each MOI in both cell lines for protective CFHT protein. As shown in
Reduction of C7 protein via multigene constructs that include expression of C7 inhibitory RNA (e.g., C7 shRNA_A, shRNA_B or shRNA_D) will reduce the amount of membrane attack complex (MAC) and cell death in the RPE-choroid region. Expression of optimized CFHT proteins by multigene constructs will additionally improve negative regulation of the alternative complement pathway.
Activities of oCFHT proteins containing additional SCR7 domains at the C-terminus and/or an FHR-1 SCR1/2 coding sequence at the N-terminus were evaluated. Human codon-optimized polynucleotides encoding one, two, or three CFHT SCR7 domains were cloned in-frame with the 3′ end of a protective CFHT cDNA to generate a total of two, three, or four SCR7 domains. Human-codon-optimized FHR-1 SCR1/2 coding sequences were also cloned in frame with the 5′end of protective CFHT cDNAs with one, two, three, and four SCR7 domains. The protective CFHT and oCFHT proteins are designated as follows: CFHT.0 (unmodified CFHT), mCFHT.1 (modified CFHT having one additional SCR7 domain at the C-terminus); mCFHT.2 (modified CFHT having two additional SCR7 domains at the C-terminus); mCFHT.3 (modified CFHT having three additional SCR7 domains at the C-terminus). CFHT proteins having an SCR1/2 coding sequence at the 5′ end are designated by reference to “dimer” or inclusion of a “d” in the CFHT protein designation, e.g., dCFHT.0 refers to CFHT modified to have a dimer at the N-terminus. mCFHT.1, mCFHT.2, and dCFHT cDNA and protein sequences are provided in SEQ ID NOS:246-251.
The plasmid constructs expressing optimized and unmodified CFHT proteins were based on plasmid pCTM297. pCTM297 was constructed from plasmid pCTM261 by replacing the AmpR selectable marker with KanR. vCTM261 (the AAV2 genome carried by the plasmid corresponding to pCTM261) is described in PCT publication WO 2020/019002A1. vCTM261 contains a codon-optimized sequence encoding a truncated complement factor H (CFHT) polypeptide, a CBA promoter, a bovine Growth Factor (bGH) polyadenylation sequence. ITR sequences corresponding to SEQ ID NO:18 of WO 2020/019002A1 (and its reverse complement) were used in vCTM261.
In constructs expressing oCFHT, the CFHT-encoding sequence was modified to add a downstream (3′ end) sequence encoding one, two, or three additional C-terminal SCR7 cDNA domains and/or modified upstream (5′ end) to add an in-frame FHR-1 SCR1/2 homodimerization domain.
In some implementations, the plasmid was further modified to add a second gene sequence. For example, plasmids pCTM467-pCTM472 each include a U6 promoter-C7 shRNA hairpin sequence (see, Table 18).
In detail, the 6× His-tag cDNA was excised from Leader 6× His-TEV (GeneArt) and inserted into pCTM297 using StuI/AgeI cloning to produce pCTM447. The modified CFHT constructs containing one, two, or three additional C-terminal SCR7 cDNA domains were constructed by removal of GeneArt inserts with BamHI/SacI for subcloning into pCTM447 to generate pCTM459, pCTM460, and pCTM461. The dimerization domain of CFHR1, encoded by SCR1/2 was also synthesized by GeneArt and subcloned by StuI/AgeI digestion into pCTM459-461 to generate pCTM462-pCTM465 constructs. Table 15 provides a summary of 6×-His tagged CFHT and oCFHT expression constructs.
CFHT expression for the plasmids listed in Table 15 was evaluated by transient expression in HEK293 cells. Cell culture supernatant containing secreted protein was assessed 48 hours post-transfection for CFHT protein levels using ELISA, and relative activity assessed using plate-based ligand binding and activity assays. Expression of CFHT.0, His-CFHT.0, His-mCFHT.1, and His-mCFHT.2 was observed, with CFHT.0 exhibiting the highest expression level. Expression of His-mCFHT.3 was not detected in this experiment. When the SCR1/2 dimerization domain of human FHR-1 protein is added to the N-terminal region of CFHT, we detect robust expression of dCFHT.0, but no detectable expression of modified dimer CFHT proteins with one, two, or three extra SCR7 domains (pCTM463-pCTM465). The pCTM395 construct served as a negative plasmid DNA control for all studies and does not have any detectable CFHT protein signal as expected. Western blots were also performed and depicted the increased size of CFHT proteins for pCTM447 (His-CFHT.0), pCTM459 (His-mCFHT.1), pCTM460 (His-mCFHT.2) and a broad band for pCTM462 (His-dCFHT.0), which was possibly due to N-linked glycosylation of the FHR-1 SCR2 domain.
Activities of the modified CFHT proteins was also compared to unmodified CFHT.0 and His-CFHT.0 proteins using the exemplary C3b, CRP and MDA-LDL assay protocols at the end of this section.
For C3b assays, 25 ng/mL of CFHT protein-containing supernatant was added to plates coated with C3b. A 43-, 99- and 22-fold increase in ligand binding was observed with mCFHT.1, mCFHT.2, and dCFHT.0, respectively, relative to control (see, Table 16). CFHT.0 (pCTM297) exhibited only a 1.5-fold increase in C3b binding relative to control supernatant at 25 ng/mL. Comparable results of significantly increased binding activity for mCFHT.1, mCFHT.2 and dCFHT.0 proteins were also observed for MDA-LDL- and CRP-coated plates (also summarized in Table 16). All three of the modified proteins performed significantly better than CFHT.0 expressed in supernatant (pCTM297 and pCTM447) or purified recombinant CFHT-I62 protein.
We then evaluated supernatants in an LPS-driven AP assay, summarized in Section 10.3.2. The mCFHT.1, mCFHT.2 and dCFHT.0 proteins again exhibited much better negative control of C3b deposition than CFHT.0 and rCFHT proteins (see, Table 16). The activities of recombinant CFHT-I62, CFHT.0- and His-CFHT.0-containing supernatants were negligible in the LPS-driven assay when tested at 25 ng/mL and lower.
As noted above, a summary of all optimized CFHT and control CFHT protein activities assessed in this experiment is shown in Table 16. These studies demonstrate that modified CFHT proteins that contain extra SCR7 domains or a dimerization domain are functionally more potent than native protective CFHT protein.
We next tested the activity of purified recombinant mCFHT.1 and dCFHT.0 proteins purified from transfected HEK293 cells in comparison to unmodified wild-type CFHT.0 protein in C3b, MDA-LDL and CRP binding assays. In agreement with the activities observed above, activity of purified recombinant mCFHT.1 and dCFHT proteins were significantly improved, relative to CFHT.0, in C3b, MDA-LDL and CRP binding assays. Binding results are summarized in Table 17.
Binding of the three recombinant proteins to C3d ligand was also evaluated. A four-fold increase in mCFHT.1 binding) and two-fold increase in dCFHT.0 binding were observed (Table 17).
Overall, the ability of the modified CFHT proteins to bind to key ligands indicates that modified CFHT proteins will control multiple events in the alternative complement pathway at much lower doses than CFHT.0.
We prepared AAV2 viral particles to transduce Cos-7 cell and express mCFHT.1, mCFHT.2, dCFHT.0 and mdCFHT.1 in cell culture supernatants. Cos-7 (ATCC, Cat. #CRL-1651) cells were maintained in Dulbecco's Modified Eagle's Medium with 10% FBS. Cells were plated at 10,000 cells per well in a 96-well plate format one day prior to rAAV2 transduction. The following day, rAAV2 particles were added to wells with 75 μl of DMEM containing 0.2% FBS at 1E+0 to 1E+5 in 10-fold increments in duplicate. The next day, cultures were replaced with fresh media and 3 days post-transduction supernatants were collected. The AAV2 constructs are shown in Table 18.
Supernatants were assayed in an MDA-LDL assay using the basic protocol described in Section 10.3.1. When supernatants for each MOI were added to MDA-LDL coated plates, a robust signal was detected for both mCFHT.1 and dCFHT.0 at the two highest MOIs tested (
To confirm that mCFHT proteins have retained critical alternative complement pathway activity, we tested recombinant proteins in a decay acceleration activity (DAA) assay (described in Exemplary Assays below) that monitors the formation of the C3 convertase, C3bBb. Wells treated with increasing amounts of recombinant CFHT.0, mCFHT.1 and dCFHT.0, exhibited a potent reduction in the amount of C3 convertase in a dose-dependent manner. The unmodified CFHT recombinant protein exhibited an IC50 of 1.95 nM while mCFHT.1 is 8-fold less active (IC50=15.5 nM) and mdCFHT.1 is ˜14-fold less active (IC50=15.5 nM) when tested under these conditions (
In summary, AAV2-produced optimized CFHT proteins mCFHT.1 and dCFHT.0 from Cos-7 supernatant and purified recombinant proteins potently bind to MDA ligand, inhibit LPS-driven AP activity and influence decay acceleration of C3 convertase (C3bBb).
Cell culture media samples were diluted 1:100 with ELISA assay reagent diluent buffer (RDB, 1×PBS+1% BSA) for CFHT protein quantitation. Black MaxiSorp plates were coated with capture antibody anti-CFHT (aCTM113) at 1:600 in Maxisorp coating buffer (50 mM carbonate, pH 9.6) and placed overnight at 4° C. After washing with PBST (PBS with 0.05% Tween-20), plates were blocked with RDB for 90 minutes at room temperature. Plates were then washed again and 100 μl of diluted cell culture supernatant samples were added to each well and incubated for 90 minutes at room temperature. Plates were washed as above followed by CFHT detection with biotin conjugated aCTM87b (OX24 antibody, Thermo Fisher Scientific Cat. #MA5-17735) at 1:800, washing and incubation for 20 minutes with StreptAvidin-HRP (ThermoFisher Scientific, Cat. #21130) at 1:10,000 dilution followed by incubation with SuperSignal ELISA Pico Chemiluminescent Substrate (ThermoFisher Scientific, Cat. #37069) before detection using the BioTek Synergy 4 plate reader. A CFHT (in-house developed) protein standard curve was generated to determine concentration for each sample. CFHT protein concentration in cell culture supernatant was plotted in Prism 9 software with average and standard deviation plotted for duplicate transductions.
To measure binding of recombinant CFHT protein to malondialdehyde modified LDL (MDA-LDL, Cell Biolabs Cat. #STA-212), a black MaxiSorp 96-well microplates were coated with 100 μl of 2.5 μg/ml MDA-LDL in PBS overnight at 4′C. After 3 washes in PBST, plates were blocked with SuperBlock T20 (PBS) Blocking Buffer (ThermoFisher Scientific, Cat. #37516) for 90 minutes at room temperature. 30 μl of cell culture supernatant was combined with 70 μl of SuperBlock T20 Blocking Buffer and then added to each well and incubated for 90 minutes at room temperature. Plates were then incubated for 1 hour with biotin conjugated aCTM87b (OX24 antibody, Thermo Fisher Scientific Cat. #MA5-17735) at 1:800 to detect CFHT protein followed by washing in PBST. Plates were next incubated for 20 minutes with StreptAvidin-HRP (ThermoFisher Scientific, Cat. #21130) conjugated secondary antibody at 1:10,000 dilution. Finally, plates were incubated for 5 minutes with SuperSignal ELISA Pico Chemiluminescent Substrate (ThermoFisher Scientific, Cat. #37069) followed by detection using a BioTek Synergy 4 plate reader. MDA-LDL ligand binding for Cos-7 supernatant was plotted in Prism 9 software with average and standard deviation plotted for duplicate transductions.
Alternative pathway (AP) activation was determined using an established direct ELISA (Harder M J et al. (2015) J. Immunol. 196:866-876) with minor modifications. In brief, 50 μl of LPS solution (50 μg/ml, from Salmonella enterica serotype typhimurium, Sigma-Aldrich Cat. #L7261) was coated on black 96-well Maxisorp plate in PBS overnight at 4° C. After washing with PBST, plates were blocked with RDB for 90 minutes at room temperature. To evaluate the impact of CFHT protein on AP activation, 30 μl of cell culture supernatant sample was mixed with 30 μl of 25% pooled normal human serum (Complement Tech. Cat. #NHS) containing 10 mM MgEGTA. As controls, 30 μl of recombinant rCFHT (I62-Y402), mCFHT.1 and dCFHT.0 proteins in PBS were serially diluted (100-0.14 nM final concentration) and mixed with 30 μl of 25% NHS containing 10 mM MgEGTA. The 60 μl mixture of supernatant or recombinant CFHT and serum was then added to LPS-coated wells and incubated for 90 minutes at 37′C prior to washing and subsequent exposure to HRP conjugated goat anti-human C3 at 1:10000 (MP Biomedicals, Cat. #855237) for 1 hour at room temperature. After 3 washes with PBST, C3b deposition in each well was indirectly detected using SuperSignal ELISA Pico Chemiluminescent Substrate and the BioTek Synergy 4 plate reader. PBS and EDTA (5 mM final concentration) were used as positive and negative controls, respectively. All dose responses were normalized to activity achieved when only PBS was added to the well. The percent change in C3b deposition relative to PBS was plotted in Prism 9 software with average and standard deviation plotted for duplicate transductions.
The DAA in fluid phase was performed by an ELISA-based method (Michelfelder S et al. 2015, J Am Soc Nephrol 28: 1462-1474). Briefly, 250 ng C3b in PBS was immobilized on Maxisorp plates overnight at 4° C. In order to generate the C3 convertases (C3bBb), 400 ng Factor B (CompTech Cat. #A135) and 25 ng Factor D (CompTech Cat #. A136) were incubated with immobilized C3b in phosphate buffer containing 2 mM nickel chloride, 25 mM sodium chloride and 4% BSA for 2 h at 37° C. After washing three times in PBST (0.05% Tween-20 in PBS), increasing concentrations (0.137 nM-100 nM) of recombinant CFH_I62 (SCTM produced) or mCFHT protein (Yurogen) in buffer containing 25 mM sodium chloride and 4% BSA were added to the preformed C3 convertase complex and incubated for 40 min at 37′C. The Bb fragments that remain bound to C3b were detected by an anti-factor B polyclonal antibody (CompTech Cat. #A235) at 1:10,000 dilution in reagent diluent buffer (RDB; 1% BSA in PBS). Finally, the plates were incubated with HRP-conjugated mouse anti-goat (Jackson Immuno research Cat. #205-035-108) at 1:10,000 dilution in RDB followed by SuperSignal ELISA Pico Chemiluminescent Substrate (ThermoFisher Scientific, Cat. #37069) to indirectly detect protein. The preformed C3 convertase without regulators was included as a positive control and the C3 proconvertase (C3bB) formation (FB without adding FD) was included as a negative control. All protein variant dose responses were normalized to activity achieved when only buffer was added to the well and results were plotted in Prism 9 with average and standard deviation plotted for duplicate wells.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
All publications, patents, patent applications, and accession numbers cited herein are hereby incorporated by reference with respect to the material for which they are expressly cited.
TCGTGCAGGGCAAGAGCATCGATGTGGCCTGCCACCCTGGATACGCCCTGCCTAAGGCCCAGAC
CACCGTGACCTGCATGGAAAATGGATGGTCCCCCACCCCCCGGTGCATCAGAGTG
TCCTTCACC
This application claims benefit of U.S. provisional application No. 63/234,644, filed Aug. 18, 2021, which is herein incorporated by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/040800 | 8/18/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63234644 | Aug 2021 | US |
