WO2019210097A1 - Aptamers with stability, potency or half-life for enhanced safety and efficacy - Google Patents

Abstract

This disclosure provides approaches and compositions for improving ocular therapeutics for pediatric and other applications. In general, the compositions and methods are designed to improve vitreous retention of ocular drugs, while simultaneously minimizing serum stability and associated systemic adverse events and retaining therapeutic efficacy. Also provided are aptamers with half-lives and potencies designed to maximize therapeutic efficacy and duration, while minimizing systemic side effects.

Description

APTAMERS WITH STABILITY, POTENCY OR HALF-LIFE FOR ENHANCED

SAFETY AND EFFICACY

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 62/662,710, filed April 25, 2018, which application is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted

electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 25, 2019, is named 49644-7l0_60l_SL.txt and is 3,889 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Visual impairment is a national and global health concern that has a negative impact on physical and mental health. Visual impairment and blindness can be caused by any one of a large number of eye diseases and disorders affecting people of all ages. In some cases, treatment of a vision disorder may be attempted by inhibiting a target protein, for example, with a therapeutic agent.

[0004] Metabolism of therapeutic oligonucleotides is often mediated by extracellular nucleases present in tissues and blood, which play a critical metabolic role by catabolizing endogenous RNA and DNA, released during cell turnover, into their constituent nucleosides. There are two general types of nucleases responsible for this catabolism - exonucleases which degrade RNA and DNA from the terminus in a 5' to 3' or 3' to 5' direction, and endonucleases which cleave internal inter-nucleotide linkages within RNA and DNA strands. Combined, the extracellular nucleases responsible for RNA and DNA catabolism include activities specific for single and double-stranded RNA and DNA, as well as generally nonspecific nucleases capable of degrading any type of nucleic acid independent of its structure. Regardless of the type or specificity of the nuclease, the fundamental mechanism of action is the same, with strand scission catalyzed by hydrolysis of the individual phosphodiester bonds in the RNA or DNA backbone by nucleophilic attack of the phosphate center. DNA specific endonucleases employ water as the nucleophile, whereas the 2' OH of the ribose ring serves as the nucleophile for RNA specific endonucleases. Water also generally serves as the nucleophile for exonucleases and non-specific nucleases. SUMMARY OF THE INVENTION

[0005] Extracellular nucleases may present a tremendous hurdle to the development of oligonucleotide therapeutics, as oligonucleotides composed of unmodified RNA or DNA may be rapidly degraded in the body, thereby limiting their therapeutic utility. Medicinal chemistry efforts in the field have often focused on substitutions that reduce the rate of hydrolysis of the phosphodiester backbone, either by reducing the reactivity of the phosphate center to

nucleophilic attack or by altering the 2' position of the ribose ring to make it a less reactive nucleophile. Additional efforts have focused on creating chemistries to“cap” the termini of oligonucleotides to reduce binding to and the subsequent rate of degradation of the

oligonucleotides by exonucleases. However, most of these efforts have focused on non-ocular diseases, elucidating oligonucleotide modifications that reduce metabolism in serum, plasma, or tissue extracts, with minimal understanding of the impact of these modifications on the metabolism of oligonucleotides in the ocular compartments. Although the underlying nucleolytic mechanism of metabolism of oligonucleotides is thought to be common and well understood, the content and type of nucleases may differ between tissues, leading to differential metabolism in one tissue as compared to another. Therefore, understanding the nuclease content and specificity of the target tissue of an oligonucleotide therapeutic may be important to optimizing the therapeutic for use in the target tissue.

[0006] In some cases, a retinal therapy may be delivered by intravitreal (IVT) administration. However, efficacy and dosing intervals for retinal therapies may be limited by both the IVT residence time and toxicities caused by systemic exposure of the therapy. There is thus a need for therapeutics that can be dosed so as to improve efficacy without incurring substantial risks of toxicity.

[0007] In one aspect, a method is provided for treating a pediatric subject with an ocular disease or disorder, comprising delivering an aptamer to the eye of the pediatric subject with the ocular disease or disorder, wherein the pediatric subject is less than 18 years of age, wherein the aptamer is results in limited systemic exposure, and wherein the aptamer specifically binds to a therapeutic target in the eye of the subject. In some cases, the aptamer exhibits higher stability in a vitreous matrix than in a blood matrix or a non-ocular tissue matrix. In some cases, the modified aptamer is delivered via an intraocular or intravitreal administration. In some cases, the subject is less than 10 years of age. In some cases, the subject is less than 36 months of age. In some cases, the subject is an infant who was born prematurely. In some cases, the infant was bom at less than 37 weeks of gestational age. In some cases, the modified aptamer exhibits a greater than two-fold higher stability in the vitreous matrix compared with the stability in the blood matrix or a non-ocular tissue matrix. In some cases, the aptamer is associated with fewer systemic adverse reactions than an aptamer optimized for vitreous stability or blood stability. In some cases, the aptamer is associated with fewer systemic adverse reactions than an aptamer optimized for non-ocular tissue stability or blood stability. In some cases, the aptamer targets vascular endothelial growth factor (VEGF). In some cases, the aptamer targets platelet-derived growth factor (PDGF). In some cases, the aptamer targets angiopoietin-2 (Ang2). In some cases, the aptamer comprises an end cap on the 3’ end. In some cases, the aptamer comprises a linker on the 5’ end. In some cases, the aptamer comprises fewer than five 2^,-fluoropyrimdines. In some cases, the aptamer is at least about 80% identical to Compound 2. In some cases, the aptamer has at least about 80% sequence identity to SEQ ID NO: 3. In some cases, the limited systemic exposure is a systemic concentration of less than the IC_l0 concentration of the aptamer for its target. In some cases, the aptamer has a ¾ of at least about 20 nM. In some cases, the aptamer has a half-life of at least about 4 days in the vitreous, at least about 2 days in the blood or non-ocular tissue, or both. In some cases, the aptamer is administered at a dose of at least about 0.5 mg/eye. In some cases, the ocular concentration of the aptamer remains above the IC concentration at which 90% of the target is inhibited (IC₉₀) for at least about 4 weeks. In some cases, the blood, non-ocular tissue, or serum concentration of the aptamer is less than the concentration at which 10% of the target is inhibited (IC_l0). In some cases, the ocular concentration of the aptamer remains above the concentration at which 90% of the target is inhibited (IC₉₀) for at least about 4 weeks, and the blood, non-ocular tissue, or serum

concentration of the aptamer is less than the concentration at which 10% of the same target is inhibited (IC_l0). In some cases, the aptamer is administered no more than once every four weeks.

[0008] In one aspect, a method is provided for treating a pediatric ocular disease, the method comprising, delivering an aptamer comprising a nucleic acid sequence according to SEQ ID NO: 3 to the eye of a pediatric subject, wherein the aptamer does not contain any T - fluoropyrimidines, thereby treating the pediatric ocular disease. In one aspect, a method is provided for treating a pediatric ocular disease, the method comprising, delivering an aptamer to the eye of a pediatric subject, wherein the aptamer binds to VEGF, and wherein the aptamer does not contain any T -fluoropyrimidines, thereby treating the pediatric ocular disease.

[0009] In one aspect, a method is provided for selecting an aptamer with improved stability in a vitreous matrix compared to a blood matrix or a non-ocular tissue matrix, comprising obtaining a panel of aptamers comprising unique patterns of modifications, wherein the aptamers comprise a base sequence known to bind a therapeutic target; incubating the aptamers comprising the unique patterns of modifications in a vitreous matrix; measuring the stability of the aptamers in the vitreous matrix; and identifying the unique patterns of modifications that result in greater vitreous matrix stability. In some cases, the vitreous matrix is vitreous fluid or is designed to resemble vitreous fluid. In some cases, the method further comprises incubating the aptamers comprising the unique patterns of modifications in a blood matrix or a non-ocular tissue matrix and measuring the stability of the aptamers in the blood matrix or the non-ocular tissue matrix.

In some cases, the stability is determined by measuring the half-life of the aptamer in the vitreous matrix or the blood matrix or the non-ocular tissue matrix. In some cases, the stability is measured at a temperature of 37°C. In some cases, the blood matrix comprises serum extracted from whole blood. In some cases, the method further comprises selecting the modified aptamer when the vitreous-matrix-stability to blood-matrix-stability ratio is greater than 5: 1. In some cases, the method further comprises testing the modified aptamer for therapeutic efficacy when the vitreous matrix stability to blood matrix ratio is greater than 5: 1. In some cases, the method further comprises selecting the modified aptamer when the vitreous-matrix-stability to non ocular tissue-matrix-stability ratio is greater than 5: 1. In some cases, the method further comprises testing the modified aptamer for therapeutic efficacy when the vitreous matrix stability to non-ocular tissue matrix ratio is greater than 5: 1. In some cases, the pattern of modifications includes one or more modifications selected from the group consisting of: 2’F, 2OMe, 2’deoxy, phosphorthioate, phosphoramidate, methyl phosphonate, PEG linker, polyethylene glycol (PEG) linker, PEG spacer, stabilizing end cap, nucleic acid insertion, and truncated motif. In some cases, the measuring the stability comprises using an assay to detect fully intact aptamer. In some cases, the assay to detect fully intact aptamer is liquid

chromatography-mass spectroscopy (LC-MS). In some cases, a modified aptamer may be generated using the method described herein.

[0010] In one aspect, a pharmaceutical composition is provided suitable for administration to an eye, the pharmaceutical composition comprising an aptamer that specifically binds to a therapeutic target in the eye with a K_d of at least about 50 nM and an intraocular half-life of at least 4 days. In some cases, the intraocular half-life of the aptamer is at least 7 days. In some cases, the aptamer has a K_d value of at least about 100 nM. In some cases, the aptamer has a serum half-life of less than 4 days. In some cases, the aptamer has a non-ocular tissue half-life of less than 4 days. In some cases, the pharmaceutical composition is formulated to have a unit dose of about 0.75 mg. In some cases, the pharmaceutical composition is formulated to have a unit dose of about 1.5 mg. In some cases, ocular concentration of the aptamer remains above the IC₉₀ concentration for at least about 4 weeks. In some cases, a serum concentration of the aptamer is less than the IC_l0 of the aptamer. In some cases, the serum concentration of the aptamer does not exceed the IC_l0 of the aptamer at any time after administration. In some cases, the concentration of the aptamer in tissues other than the eye does not exceed the IC_l0 of the aptamer at any time after administration. In some cases, a non-ocular tissue concentration of the aptamer is less than the IC_l0 of the aptamer. In some cases, the serum concentration of the aptamer does not exceed the IC_l0 of the aptamer at any time after administration. In some cases, the concentration of the aptamer in tissues other than the eye does not exceed the IC_l0 of the aptamer at any time after administration.

[0011] In one aspect, an aptamer is provided that specifically binds to a therapeutic target in an eye, wherein the aptamer does not attain a serum and/or non-ocular tissue concentration above the aptamer’ s IC_l0 for the therapeutic target beyond 96 hours following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’ s IC90 for the therapeutic target. In some cases, the aptamer maintains an ocular concentration above the aptamer’ s IC₉₀ for the therapeutic target for at least 2 weeks following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’ s IC90 for the therapeutic target. In some cases, the aptamer does not attain a serum or non-ocular tissue concentration above the aptamer’ s IC_l0 for the therapeutic target at any time following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’ s IC₉₀ for the therapeutic target. In some cases, the aptamer maintains an ocular concentration above the aptamer’ s IC90 for the therapeutic target for at least 4 weeks following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’ s IC₉₀ for the therapeutic target. In some cases, the ocular concentration exceeding the aptamer’ s IC₉₀ for the therapeutic target is less than about lOO-fold or higher than the aptamer’ s IC90 for the therapeutic target. In one aspect, a pharmaceutical formulation is provided comprising an aptamer as described herein, the pharmaceutical formulation being suitable for administration to the eye. In one aspect, a method of treating a subject with an ocular disease or disorder is provided, the method comprising administering a pharmaceutical formulation to the eye of the subject with the ocular disease or disorder, thereby treating the ocular disease or disorder. In one aspect, a method of treating a subject with an ocular disease or disorder is provided comprising providing an aptamer that specifically binds to a therapeutic target in an eye at a potency capable of treating the ocular disease or disorder; and administering the aptamer to the eye of the subject with the ocular disease or disorder at a dose sufficient to achieve an ocular concentration above the aptamer’ s IC90 for the therapeutic target and such that the aptamer maintains a serum or non-ocular tissue concentration that does not exceed the aptamer’ s IC_l0 for the therapeutic target beyond 96 hours after the administering of the aptamer to the eye. In some cases, the ocular concentration of the aptamer is maintained above the aptamer’s IC₉₀ for the therapeutic target for at least 2 weeks following the administering of the aptamer to the eye of the subject at the dose sufficient to achieve an ocular concentration above the aptamer’s IC₉₀ for the therapeutic target_.

[0012] In one aspect, an aptamer suitable for administration to the eye is provided that has been specifically modified for increased metabolic stability in vitreous, such that the aptamer, when conjugated to a suitable carrier, has a half-life in rabbits of greater than about three to about four days following intravitreal administration. In some cases, the intraocular half-life of the aptamer is at least about 4.5 days. In some cases, the intraocular half-life of the aptamer is at least about 5 days. In some cases, the intraocular half-life of the aptamer is at least about 6 days. In some cases, the intraocular half-life of the aptamer is at least about 7 days. In some cases, the aptamer has a K_d value of at less than about 10 nM. In some cases, the pharmaceutical composition is formulated to have a unit dose of about 1.5 mg. In some cases, the pharmaceutical composition is formulated to have a unit dose of about 3 mg. In some cases, the pharmaceutical composition is formulated to have a unit dose of at least about 5 mg. In some cases, ocular concentration of the aptamer remains above the IC₉₀ concentration for at least about 12 weeks. In some cases, ocular concentration of the aptamer remains above the IC₉₀ concentration for at least about 16 weeks. In some cases, ocular concentration of the aptamer remains above the IC₉₀ concentration for at least about 20 weeks. In some cases, ocular concentration of the aptamer remains above the IC₉₀ concentration for at least about 24 weeks or more.

[0013] In one aspect, a method of treating a subject with an ocular disease or disorder is provided comprising providing an aptamer that specifically binds to a therapeutic target in an eye at a potency capable of treating the ocular disease or disorder; administering the aptamer to an eye of the subject with the ocular disease or disorder at a dose sufficient to achieve an ocular concentration above the aptamer’s IC₉₀ for the therapeutic target; and maintaining a serum or non-ocular tissue concentration of the aptamer that does not exceed the aptamer’s IC_l0 for the therapeutic target beyond 96 hours after the administering of the aptamer to the eye of the subject. In some cases, the method further comprises maintaining an ocular concentration of the aptamer above the aptamer’s IC₉₀ for the therapeutic target for at least 2 weeks following the administering of the aptamer to the eye of the subject at the dose sufficient to achieve an ocular concentration above the aptamer’s IC₉₀ for the therapeutic target. In some cases, the

administering comprises intravitreally injecting the aptamer into the eye of the subject. In some cases, the aptamer does not attain a serum or non-ocular tissue concentration above the aptamer’s IC10 for the therapeutic target at any time within 96 hours following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’s IC₉₀ for the therapeutic target. In some cases, the aptamer does not attain a serum or non-ocular tissue concentration above the aptamer’s IC_l0 for the therapeutic target at any time following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’s IC₉₀ for the therapeutic target. In some cases, the aptamer maintains an ocular concentration above the aptamer’s IC₉₀ for the therapeutic target for at least 4 weeks following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’s IC₉o for the therapeutic target. In some cases, the ocular concentration exceeding the aptamer’s IC₉o for the therapeutic target is less than lOO-fold higher than the aptamer’s IC₉₀ for the therapeutic target. In some cases, the aptamer is administered as part of a pharmaceutical formulation suitable for treating an eye.

[0014] In some embodiments of any of the above aspects, the aptamer has a half-life of at least about 4 days in vitreous, at least about 5 days in vitreous, at least about 6 days in vitreous, or at least about 7 days in vitreous. In some embodiments of any of the above aspects, the aptamer has a half-life of less than about 6 days in blood or non-ocular tissue, less than about 5 days in blood or non-ocular tissue, less than about 4 days in blood or non-ocular tissue, less than about 3 days in blood or non-ocular tissue, or less than about 2 days in blood or non-ocular tissue. In some embodiments, the aptamer has a half-life of at least about 4 days in vitreous and less than about 4 days in blood or non-ocular tissue. In some embodiments, the aptamer is administered via a syringe. In some embodiments, the aptamer is administered to a subject less than 18 years of age, less than 12 years of age, less than 10 years of age, or less than 8 years of age, and the method further comprises detecting the serum blood concentration of the aptamer in the subject. In some embodiments, the serum blood concentration of the aptamer in the subject is less than about 10 nM.

INCORPORATION BY REFERENCE

[0015] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0017] FIG. 1 depicts a non-limiting example of a method workflow according to an

embodiment of the disclosure. [0018] FIG. 2 depicts Compound 1 (SEQ ID NO: 1) and Compound 2 (SEQ ID NO: 2).

[0019] FIG. 3 depicts Compound 3 (SEQ ID NO: 3) and Compound 4 (SEQ ID NO: 3).

[0020] FIG. 4 depicts expected secondary structures of Compound 1 (SEQ ID NO: 1) and Compound 2 (SEQ ID NO: 2).

[0021] FIG. 5 depicts expected secondary structures of Compound 3 (SEQ ID NO: 3) and Compound 4 (SEQ ID NO: 3).

[0022] FIG. 6 illustrates the stability of Compound 1 in different biologic matrices.

[0023] FIG. 7 illustrates the stability of Compound 2 in different biologic matrices.

[0024] FIG. 8 illustrates the stability of Compound 3 in different biologic matrices.

[0025] FIG. 9 illustrates the stability of Compound 4 in different biologic matrices.

[0026] FIG. 10 depicts further examples of modified anti-PDGF aptamers (SEQ ID NOs: 1, 1,

8, 1, and 9, respectively, in order of appearance).

[0027] FIG. 11 depicts expected secondary structures of some modified anti-PDGF aptamers (SEQ ID NOs: 1, 1, 8, 1, and 9, respectively, in order of appearance).

[0028] FIG. 12 depicts further examples of modified anti-VEGF aptamers (SEQ ID NOs: 10 and 11, respectively, in order of appearance).

[0029] FIG. 13 depicts expected secondary structures of some modified anti-VEGF aptamers (SEQ ID NOs: 10 and 11, respectively, in order of appearance).

[0030] FIG. 14 illustrates the intravitreal aptamer concentration over time for three different aptamer half-lives, given a dose of 0.75 mg/eye.

[0031] FIG. 15 illustrates the intravitreal aptamer concentration over time for three different aptamer half-lives, given a dose of 1.5 mg/eye.

[0032] FIG. 16 depicts the sequence and secondary structures of Aptamers 15 (SEQ ID NO: 12) and 74 (SEQ ID NO: 12). L represents a hexylamino linker, X represents an inverted deoxythymidine, and guanosine residues highlighted in gray represent 2’-0-methyl residues.

[0033] FIG. 17 depicts the relationship between intravitreal half-life in rabbits and

hydrodynamic radius (R_h) of various macromolecules.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The disclosure herein provides methods of generating aptamer therapies, and

compositions thereof, with improved safety profiles, particularly for children. In some cases, adverse reactions caused by systemic exposure to a therapy can limit the safely-tolerated dose, and thus may limit the efficacy of the therapy. The disclosure herein provides methods for generating aptamer therapeutics, and compositions thereof, for use in the treatment of an ocular disease or disorder. In some cases, the aptamers exhibit sufficient stability in the eye for effective treatment of an ocular disease or disorder, while at the same time exhibiting lower stability in the blood and/or other non-ocular tissues, thereby causing reduced or limited systemic exposure. In some cases, the methods provided herein may be suitable for generating aptamer therapies that have longer half-lives in the eye ( e.g ., in the vitreous humor). Additionally, the methods provided herein may be suitable for generating aptamer therapies that have shorter half- lives in blood or a non-ocular tissue.

[0035] The disclosure herein provides aptamers for ocular use (e.g., for the treatment of an ocular disease or disorder), and methods of generating such aptamers for ocular use. Generally, the aptamers provided herein may exhibit sufficient stability in the eye for effective treatment of an ocular disease or disorder, while at the same time exhibiting low stability in the blood and/or non-ocular tissues, thereby causing reduced systemic exposure. This disclosure further provides methods to improve the safety and dosing interval of aptamers for the eye by altering their metabolic stability in the vitreous to vary their rate of clearance and therefore half-life in the eye. In some cases, the aptamers provided herein may possess varying degrees of potency. In some cases, the balance between potency and half-life can be adjusted in order to promote a reduction of systemic side effects while maintaining therapeutic efficacy.

[0036] The disclosure herein demonstrates that nuclease-mediated oligonucleotide metabolism in the eye may be sufficiently different from that in blood and/or non-ocular tissues such that oligonucleotide therapeutics may be differentially stabilized to substantially decrease their metabolism in the eye as compared to blood and non-ocular tissues, and thereby greatly increase the ratio of their ocular half-life as compared to their systemic half-life.

[0037] In some aspects, methods are provided for generating aptamers that have clinically relevant differential stability in two or more body fluids. In one example, the aptamers may have differential stability in the eye (e.g, vitreous) versus blood and/or non-ocular tissues. In some cases, a series of aptamers, generally each with the same or similar base sequence but with different modifications, may be screened for stability in two or more body fluids. In some examples, the aptamers may be screened by incubating each aptamer for different durations of time in either vitreous fluid, blood serum, or non-ocular tissue. By analyzing the amount of full- length aptamer remaining after each incubation, aptamers which are more stable in vitreous fluid than in blood serum or non-ocular tissue can be identified. An outline showing one non-limiting example of a screen for generating an aptamer with differential stability is shown in FIG. 1. As depicted in FIG. 1, a series of modified aptamers (e.g, each aptamer having the same or similar primary nucleotide sequence, but with each aptamer having different modifications) may be obtained or generated (100). Each aptamer may be screened for stability by incubating each aptamer in vitreous fluid (101) or blood (102), and then measuring stability of each aptamer after different incubation times (103, 104). By comparing the stability of each aptamer in vitreous fluid and blood (105), those aptamers with increased ratios of vitreous fluid stability to blood stability are identified (106).

[0038] The term“aptamer” as used herein refers to an oligonucleotide and/or nucleic acid analogues that can bind to a specific target molecule. Aptamers can include RNA, DNA, modified RNA, modified DNA, any nucleic acid analogue, and/or combinations thereof.

Aptamers can be single-stranded oligonucleotides. In some cases, aptamers may comprise more than one nucleic acid strand ( e.g ., two or more nucleic acid strands). Aptamers may bind to a target (e.g., a protein) with high affinity and specificity through non-Watson-Crick base pairing interactions. Generally, the aptamers described herein are non-naturally occurring

oligonucleotides (e.g, synthetically produced) that are isolated and used for the treatment of a disorder or a disease. Aptamers can bind to essentially any target molecule including, without limitation, proteins, oligonucleotides, carbohydrates, lipids, small molecules, and even bacterial cells. Aptamers may be monomeric (composed of a single unit) or multimeric (composed of multiple units). Multimeric aptamers can be homomeric (composed of multiple identical units) or heteromeric (composed of multiple non-identical units). Aptamers herein may be described by their primary structures, meaning the linear nucleotide sequence of the aptamer. Aptamer sequences herein are generally described from the 5’ end to the 3’ end, unless otherwise stated. Additionally or alternatively, aptamers herein may be described by their secondary structures which may refer to the combination of single-stranded regions and base-pairing interactions within the aptamer. Whereas many naturally occurring oligonucleotides, such as mRNA, encode information in their linear base sequences, aptamers generally do not encode information in their linear base sequences. Further, aptamers can be distinguished from naturally occurring oligonucleotides in that binding of aptamers to target molecules is dependent upon secondary and tertiary structures of the aptamer. Aptamers may be suitable as therapeutic agents and may be preferable to other therapeutic agents because: 1) aptamers may be fast and economical to produce because aptamers can be developed entirely by in vitro processes; 2) aptamers may have low toxicity and may lack an immunogenic response; 3) aptamers may have high specificity and affinity for their targets; 4) aptamers may have good solubility; 5) aptamers may have tunable pharmacokinetic properties; 6) aptamers may be amenable to site-specific conjugation of PEG and other carriers; and 7) aptamers may be stable at ambient temperatures.

[0039] In general,“sequence identity” refers to an exact nucleotide-to-nucleotide or amino acid- to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their“percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol., 215:403-410 (1990); Karlin And Altschul,

Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993); and Altschul et al, Nucleic Acids Res., 25:3389-3402 (1997). The program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desired degrees of sequence identity are approximately 50% to 100% and integer values therebetween. In general, this disclosure encompasses sequences with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any sequence provided herein.

[0040] In general,“modification identity” refers to two polynucleotides with identical patterns of modifications on a nucleotide-to-nucleotide level. Techniques for determining modification identity may include determining the modifications of a polynucleotide and comparing these modifications to modifications of a second polynucleotide. The percent modification identity of two sequences is the number of exact modification matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. Ranges of desired degrees of modification identity are generally approximately 50% to 100%. In general, this disclosure encompasses sequences with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% modification identity with any sequence provided herein.

[0041] The term“about,” as used herein, generally refers to a range that is 15% greater than or less than a stated numerical value within the context of the particular usage. For example,“about 10” would include a range from 8.5 to 11.5. [0042] As used herein, the term“or” is used nonexclusively to encompass“or” and“and.” For example,“A or B” includes“A but not B,”“B but not A,” and“A and B” unless otherwise indicated.

[0043] As used herein, the term“IC90”, when used in relation to an aptamer, refers to the concentration at which the aptamer inhibits 90% of a given target of the aptamer in a specified tissue or biological matrix. For example, the IC₉₀ of an aptamer intended to inhibit vascular endothelial growth factor (VEGF) in the eye, is the concentration of aptamer in the eye at which 90% of VEGF activity in the eye is inhibited. The IC90 concentration may be different in each tissue. Similarly, the term“IC_l0”, when used in relation to an aptamer, refers to the

concentration at which the aptamer inhibits 10% of a given target of the aptamer in a specified tissue or biological matrix; and the term“IC₅₀”, when used in relation to an aptamer, refers to the concentration at which the aptamer inhibits 50% of a given target of the aptamer in a specified tissue or biological matrix.

Aptamers

[0044] In some cases, the disclosure provides aptamers with unique combinations of potency and half-life, or other unique properties. This disclosure also provides novel ways of using known aptamers.

[0045] The aptamers provided herein may include DNA aptamers, RNA aptamers, or a combination thereof. In some cases, the aptamers may contain both DNA and RNA residues. In some cases, the DNA aptamers may be modified DNA aptamers. In some cases, the RNA aptamers may be modified RNA aptamers. The aptamers provided herein can include any number of modifications; often, such modifications may protect the aptamer from nuclease degradation or may enhance the stability of the aptamer under physiological conditions.

Aptamers of the disclosure may include any modifications as described herein. In some instances, DNA residues of a DNA aptamer may be substituted with RNA residues to produce a modified aptamer. For example, when a DNA residue comprising a thymine is substituted with an RNA residue, the RNA residue may comprise a thymine or a uracil. Likewise, RNA residues of an aptamer may be substituted with DNA residues to produce a modified aptamer.

[0046] The size of an aptamer provided herein may be about 15 to about 800 nucleotides. For example, the size of the aptamer may be about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, or greater than about 800 nucleotides. In some cases, the aptamers disclosed herein may be about 15 to about 45 nucleotides, about 25 to about 40 nucleotides, about 30 to about 40 nucleotides, about 30 to about 35 nucleotides, or about 35 to about 40 nucleotides. The length of the aptamer can be variable. In some cases, the length of the aptamer may be less than about 100

nucleotides. In some cases, the length of the aptamer may be greater than about 10 nucleotides.

In some cases, the length of the aptamer may be between about 10 and about 90 nucleotides.

[0047] In some cases, the aptamer can be a bi-specific aptamer. A bi-specific aptamer may include two or more aptamers conjugated to a linker molecule ( e.g ., a PEG polymer). In some cases, the two or more aptamers may be conjugated to each end of the linker molecule. In some cases, two or more aptamers may include the same aptamer bound multiple times to a linker molecule. In other cases, the two or more aptamers may include different aptamers bound to the same PEG molecule. In some examples, the two or more aptamers may bind to two or more different binding sites on a target. A bi-specific aptamer provided herein generally may recognize at least two different target molecules, e.g., a therapeutic target and a vitreous binding target.

[0048] Aptamers as described herein may include any number of modifications than can affect the function or affinity of the aptamer. For example, aptamers may be unmodified or they may contain modified nucleotides to improve stability, nuclease resistance, and/or delivery characteristics. In some preferred cases, the modifications may impact the stability or half-life of the aptamer. In some cases, the aptamers described herein may contain modified nucleotides to improve the affinity and/or specificity of the aptamers for a specific epitope. Examples of modified nucleotides include those modified with guanidine, indole, amine, phenol,

hydroxymethyl, or boronic acid (e.g, by incorporating such compounds into the aptamers).

[0049] Modifications of the aptamers contemplated in this disclosure include, without limitation, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid aptamer bases or to the nucleic acid aptamer as a whole. Modifications to generate

oligonucleotide populations that are resistant to nucleases, susceptible to nucleases, increase or decrease stability, and/or increase or decrease affinity can also include one or more substitute intemucleotide linkages, altered sugars, altered bases, or combinations thereof. Such

modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, and unusual base-pairing combinations such as the isobases, isocytidine and isoguanosine. [0050] Modifications can also include 3' and 5' modifications such as capping, e.g, addition of a 3'-3'-dT cap to increase exonuclease resistance. Examples of caps that can be added to aptamers of the disclosure include: 3'-3'-dT caps, abasic caps, C3 caps, strings of 3 dT phosphorothioate, hexylamine, hexylthiol, polyethylene glycol, or any other nucleotide caps known in the art. An end cap used in the methods of this disclosure may be an inverted deoxy thymidine (idT) cap. Caps can be added to the 3’ end of the aptamer to protect from 3’ to 5’ exonucleases. Caps can be added to the 5’ end of the aptamer to protect from 5’ to 3’ exonucleases. Caps can be added to both the 3’ and 5’ ends of the aptamer to protect from both 3’to 5’ and 5’ to 3’ exonucleases. When caps are added to both the 3’ and 5’ ends of the aptamer, the caps can be either the same or different. Some caps may provide greater protection against certain exonucleases than against other exonucleases. The protective capabilities provided by certain caps against specific exonucleases may be used to select the most appropriate cap for a given application.

[0051] In preferred cases provided herein, the modifications of the aptamers may include alterations of a sugar group, a base, and/or the phosphate background. Sugar groups that may be modified include, but are not limited to, ribose, deoxyribose, and dideoxyribose. The

modification may be at one or more positions of a sugar group. Examples of positions of a sugar that can be modified include the T position (e.g, T position of ribose), the 3’ position, the 4’ position, and the 5’ position.

[0052] In some cases, the nucleobases (or bases) of the aptamer may be modified. Bases in the aptamers that may be modified include any base, e.g, pyrimidines, or purines. Specific examples of bases that may be modified include: guanine, uridine, adenine, thymine and cytosine. The bases may be altered at any position, e.g, the 2, 3, 4, 5, or 6 position of pyrimidines; or the 2, 3, 4, 5, 6, 7, 8, or 9 position of purines. Examples of base modifications may include various modified pyrimidines and modifications with -amino (NH₂), fluoro (F), methyl (Me), and/or O-methyl (O-Me) modifications. In some cases, the aptamers described herein may comprise a -O-Me modification of the base. Examples of modified bases include 1- methyladenosine, 2-methyladenosine, N6-methyladenosine, -O-methyladenosine, 2-methylthio- N6-methyladenosine, N6-isopentenyladenosine, 2-methylthio- N6 -isopentenyladenosine, N6- (cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N 6- glycinylcarbamoyladenosine, N 6-threonylcarbamoyladenosine, 2-methylthio-N -threonyl carbamoyladenosine, N 6-methyl-N -threonylcarbamoyladenosine, N 6- hydroxynorvalylcarbamoyladenosine, 2-methylthio-N -hydroxynorvalyl carbamoyladenosine, 2'-0-ribosyladenosine (phosphate), inosine, l-methylinosine, l,2'-0-dimethylinosine, 3- methylcytidine, 5-methylcytidine, 2-O-methylcytidine, 2-thiocytidine, N 4-acetylcytidine, 5- formyl cyti dine, 5, 2'-0-di ethyl cyti dine, N 4-acetyl -2'-0-methylcyti dine, lysidine, 1- methylguanosine, N2-methylguanosine, 7-methyl guanosine, 2 -O-methylguanosine, N2,N2- dimethylguanosine, N2,2'-0-dimethylguanosine, N2,N2,2'-0-trimethylguanosine, 2'-0- ribosylguanosine (phosphate), wybutosine, peroxywybutosine, hydroxywybutosine,

undermodified hydroxywybutosine, wyosine, methylwyosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deazaguanosine, 7-aminomethyl-7- deazaguanosine, archaeosine, pseudouridine, dihydrouridine, 5-methyluridine, 2 -O- methyluridine, 5,2'-0-dimethyluridine, l-methylpseudouridine, 2'-0-methylpseudouridine, 2- thiouridine, 4-thiouridine, 5-methyl-2-thiouridine, 2-thio-2'-0-methyluridine, 3-(3-amino-3- carboxypropyl)uridine, 5-hydroxyuridine, 5-methoxyuridine, uridine 5-oxyacetic acid, uridine 5- oxyacetic acid methyl ester, 5-(carboxyhydroxymethyl)uridine, 5- (carboxyhydroxymethyl)uridine methyl ester, 5-methoxycarbonylmethyluridine, 5- methoxycarbonylmethyl-2'-0-methyluridine, 5-methoxycarbonylmethyl-2-thiouridine, 5- aminomethyl-2-thiouridine, 5 -methyl aminomethyluridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-carbamoylmethyluridine, 5-carbamoylmethyl-2'-0- methyluridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2 -O- methyluridine, 5-carboxymethylaminomethyl-2-thiouridine, N6,N6-dimethyladenosine, 2'-0- methylinosine, N4-m ethyl cyti di ne, N4,2'-0-di methyl cyti dine, 5-hydroxymethylcytidine, 3- methyluridine, 1 -methyl-3 -(3 -amino-3 -carboxypropyl) pseudouridine, 5-carboxymethyluridine, N6,2'-0-dimethyladenosine, N6,N6,2'-0-trimethyladenosine, N2, 7-dimethyl guanosine,

N2,N2,7-trimethylguanosine, 3,2'-0-dimethyluridine, 5-methyldihydrouridine, 3- methylpseudouridine, 5-formyl-2'-0-methylcytidine, l,2'-0-dimethyl guanosine, l,2'-0- dimethyladenosine, 5 -taurinom ethyl uridine, 5-taurinomethyl-2-thiouridine, 4-demethylwyosine, isowyosine, N6-acetyladenosine, 5-(isopentenylaminomethyl)uridine, 5- (isopentenylaminomethyl)-2-thiouridine, 5-(isopentenylaminomethyl)-2'-0-methyluridine,

N2, 7, 2 '-O-trimethyl guanosine, N4,N4,2 -O-trimethyl cyti dine, and 8-methyladenosine. Specific examples of modified bases include modified purine bases such as hypoxanthine, xanthine, and 7-methylguanine. Specific examples of modified bases also include modified pyrimidine bases such as 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine. Bases may also be modified by the addition of amino acid side chains and/or hydrophobic substituents, for example thymidine 5'-0-a-phenylphosphonyl-P, g-diphosphate, 3'-fluoro-3'-deoxythymidine 5'-0-a- phenylphosphonyl-b, g-diphosphate and thymidine 5'-0-a-decylphosphonyl-P, g-diphosphate. Nucleotide triphosphate analogs modified at the 5-position (R) of uridine (dUTP) may also be used, some examples of such triphosphate analogs include, but are not limited to, 5- benzylamino- carbonyl-dU (BndU); 5-naphthylmethylaminocarbonyl-dU (NapdU); 5- tryptaminocarbonyl-dU (TrpdU); and 5-isobutylaminocarbonyl-dU (iBudU).

[0053] The phosphate backbone of aptamers provided herein may have various modifications.

In some cases, the aptamers described herein can comprise one or more phosphate chemical substitutions such as phosphorthioate DNA or methyl phosphonate DNA nucleotides. The backbone of the aptamer may be modified for increased stability, for example, by substituting the T hydroxyl group on ribose.

[0054] The aptamers of the present disclosure may comprise modified nucleotides and/or nucleotide analogs. Non-limiting examples of nucleotide analogs include: 2'-0-methyl- substituted RNA, locked nucleic acid (LNA) or bridged nucleic acid (BNA), morpholino, and peptide nucleic acid (PNA). In other cases, nucleotide triphosphate analogs or CE- phosphoramidites may be modified at the 5 position to generate, for example, 5- benzyl ami nocarbonyl -2’ -deoxyuri dine (BndU); 5-[N-(phenyl-3-propyl)carboxamide]-2'- deoxyuridine (PPdU); 5-(N-thiophenylmethylcarboxyamide)-2'-deoxyuridine (ThdU); 5-(N-4- fluorobenzylcarboxyamide)-2'-deoxyuridine (FBndU); 5-(N-(l-naphthylmethyl)carboxamide)- 2'-deoxyuridine (NapdU); 5 -(N-2-naphthylmethylcarboxyamide)-2'-deoxyuridine (2NapdU); 5- (N-l-naphthylethylcarboxyamide)-2'-deoxyuridine (NEdU); 5-(N-2- naphthylethylcarboxyamide)-2'-deoxyuridine (2NEdU); 5-(N-tryptaminocarboxyamide)-2'- deoxyuridine (TrpdU); 5-isobutylaminocarbonyl-2’-deoxyuridine (IbdU); 5-(N- tyrosylcarboxyamide)-2'-deoxyuridine (TyrdU); 5-(N-isobutylaminocarbonyl-2’-deoxyuridine (iBudU); 5-(N-benzylcarboxyamide)-2'-0-methyluridine, 5-(N-benzylcarboxyamide)-2'- fluorouridine, 5-(N-phenethylcarboxyamide)-2'-deoxyuridine (PEdU), 5-(N-3,4- ethylenedioxybenzylcarboxyamide)-2'-deoxyuridine (MBndU), 5-(N- i i dizolyl ethyl carboxyamide)-2'-deoxyuridine (ImdU), 5-(N-isobutylcarboxyamide)-2'-0- methyluridine, 5-(N-isobutylcarboxyamide)-2'-fluorouridine, 5-(N— R-threoninylcarboxyamide)- 2'-deoxyuridine (ThrdU), 5-(N-tryptaminocarboxyamide)-2'-0-methyluridine, 5-(N- tryptaminocarboxyamide)-2'-fluorouridine, 5-(N-[l-(3- trimethylamonium)propyl]carboxyamide)-2'-deoxyuridine chloride, 5-(N- naphthylmethylcarboxyamide)-2'-0-methyluridine, 5-(N-naphthylmethylcarboxyamide)-2'- fluorouridine, 5-(N-[l-(2,3-dihydroxypropyl)]carboxyamide)-2'-deoxyuridine), 5-(N-2- naphthylmethylcarboxyamide)-2'-0-methyluridine, 5-(N-2-naphthylmethylcarboxyamide)-2'- fluorouridine, 5-(N-l-naphthylethylcarboxyamide)-2'-0-methyluridine, 5-(N-l- naphthylethylcarboxyamide)-2'-fluorouridine, 5-(N-2-naphthylethylcarboxyamide)-2'-0- methyluridine, 5-(N-2-naphthylethylcarboxyamide)-2'-fluorouridine, 5-(N-3- benzofuranylethylcarboxyamide)-2'-deoxyuridine (BPDGFU), 5-(N-3- benzofuranylethylcarboxyamide)-2'-0-methyluridine, 5-(N-3-benzofuranylethylcarboxyamide)-

2'-fluorouridine, 5-(N-3-benzothiophenyl ethyl carboxyamide)-2'-deoxyuri dine (BTdU)_, 5-(N-3- benzothiophenylethylcarboxyamide)-2'-0-methyluridine, 5-(N-3- benzothiophenylethylcarboxyamide)-2'-fluorouridine; 5-[N-(l-morpholino-2- ethyl)carboxamide]-2'-deoxyuridine (MOEdu); R-tetrahydrofuranylmethyl-2’ -deoxyuridine (RTMdU); 3-methoxybenzyl-2’-deoxyuridine (3MBndU); 4-methoxybenzyl-2’ -deoxyuridine (4MBndU); 3,4-dimethoxybenzyl-2’-deoxyuridine (3,4DMBndU); S-tetrahydrofuranylmethyl- 2’-deoxyuridine (STMdU); 3,4-methylenedioxyphenyl-2-ethyl-2’-deoxyuridine (MPEdU); 4- pyridinylmethyl -2’ -deoxyuridine (PyrdU); or 1 -benzi mi dazol -2-ethyl -2’ -deoxyuridine (BidEl).

In some cases, the nucleotide bonds are modified to include one or more phosphorothioate bonds.

[0055] In some cases, the aptamers described herein may be unmodified nucleic acid. In some cases, the aptamers may comprise a limited number of modifications. In some cases, the aptamers may not contain specific modifications. For example, an aptamer may be designed such that it does not contain any 2^,-fluoropyrimidines or 2’-fluoropurines. In some cases, an aptamer may be designed such that it contains no more than one, two, three, four, five, six, seven, eight, nine, or ten 2^,-fluoropyrimidines or 2’-fluoropurines. In some cases, an aptamer may be designed such that it does not contain two or more contiguous 2^,-fluoropyrimidines or 2’-fluoropurines. In some cases, an aptamer may be designed such that it does not contain two or more contiguous 2^,-fluoropyrimidines or 2’-fluoropurines in a stem or a double-stranded portion of the aptamer. In another example, an aptamer may be designed such that it does not contain a spacer or linker. In some examples, an aptamer may be designed such that it contains no more than one, two, three, or four spacers. In some cases, the aptamers may comprise modifications to either or both of the 3’ and 5’ terminal residues, but may not contain any modifications on internal residues.

[0056] In some cases, the aptamers described herein may be modified on sugars of specific nucleosides. For example, an aptamer may be designed such that one or more nucleosides comprising adenine, cytosine, or uracil may contain a ribose sugar, may contain a deoxyribose sugar, or may contain a deoxyribose modified with a 2’-0-methyl group. In some instances, one or more nucleosides comprising guanines may contain a ribose sugar, may contain a

deoxyribose sugar, or may contain a deoxyribose modified with 2’-0-methyl groups, or a deoxyribose sugar modified with 2’-fluoro groups. In some instances, one or more nucleosides may be deoxy thymine. In some cases, an aptamer of the disclosure may contain no more than one, two, three, four, five, six, seven, eight, nine, or ten guanine nucleosides with 2’-fluoro groups. In some cases, an aptamer of the disclosure may contain no more than two contiguous guanine nucleosides with 2’-fluoro groups. In some cases, an aptamer of the disclosure may contain no more than two contiguous guanine nucleosides with 2’-fluoro groups in a stem or a double-stranded region of the aptamer.

[0057] In some cases, the aptamers described herein may be bound or conjugated to one or more molecules. Any number of molecules can be bound or conjugated to aptamers, non-limiting examples including antibodies, peptides, proteins, small molecules, gold nanoparticles, radiolabels, fluorescent labels, dyes, haptens ( e.g ., biotin), other aptamers, or nucleic acids (e.g, siRNA). In some cases, aptamers may be conjugated to molecules that increase the stability, the solubility, and/or the bioavailability of the aptamer. Non-limiting examples include polyethylene glycol (PEG) polymers, carbohydrates, and fatty acids

[0058] In some cases, the aptamers described herein may include one or more vitreous binding moieties. A vitreous binding moiety as used herein may refer to a substance that binds to a structural component of the vitreous humor. In some cases, the structural component of the vitreous humor may be hyaluronic acid. In other cases, the structural component of the vitreous humor may be collagen or collagen fibers. The one or more vitreous binding moieties can be, for example, an antibody or fragment thereof, an aptamer, a peptide, a peptidomimetic, a small molecule, and the like.

[0059] In some instances, a polyethylene glycol (PEG) polymer chain may be covalently bound to the aptamer, referred to herein as PEGylation. PEGylation may increase the half-life and stability of the aptamer in physiological conditions. In some cases, the PEG polymer may be covalently bound to the 5 end of the aptamer. The PEG polymer can have a molecular weight of, for example, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, or greater. In some cases, the aptamers described herein may be PEGylated. The PEG polymer can be branched, linear, or any combination thereof, wherein the total molecular weight is as described above.

[0060] In some cases, the aptamer may contain one or more modifications in the sequence that may impact a secondary structure of the aptamer. Such structural modifications of the aptamers provided herein may be generated by modifying the internal aptamer sequence or by providing additional sequence on either the 3 or 5 end of the aptamer. Structural elements that may be added may include truncated active motifs, 3 -end, 5 -end exonuclease or endonuclease nucleolytically active motifs or other metabolic degradation active motifs. Structural elements that may be added may include loops, stems, or sequences of about 3-15 nucleotides that may be susceptible to nucleolytic cleavage, degradation, or other metabolism pathways. Sequences of unknown function can also be added and tested to determine their effect on aptamer stability.

[0061] In some cases, modifications which are known to preserve the biological activity of aptamers may be preferred. In some cases, modifications may be selected based on the likelihood of not affecting the biological function of the aptamer. In some cases, the aptamers may be designed or obtained such that modifications which are known to preserve the biological function of the aptamer are preferred.

[0062] Modifications to the aptamers provided herein can also include the presence of a linker or spacer at either end of the aptamer or within the aptamer. The linker may comprise a carbon chain ( e.g ., from about 3 to about 50 carbons), nucleic acid molecules, poly-ethylene glycol (PEG), or a combination thereof. Examples of carbon chains as linkers include, but are not limited to, an alkyl, an alkene, or an aldehyde. The carbon chain may be one or more of substituted, un- substituted, unbranched, or branched. Linkers comprising nucleic acid molecules may comprise one or more of DNA, RNA, single- stranded, double-stranded, nucleic acid bases found in nature (“natural nucleic acid bases”), or synthetic or modified nucleic acid bases (including, but not limited to, those not found naturally occurring). In some cases, the linker may comprise one or more PEG linkers (e.g., one, two, three, four, or more than four PEG linkers). In some cases, the aptamer may contain a spacer or spacers within the aptamer sequence. A non-limiting example of a spacer which may be used is hexaethylene glycol. In some cases, the aptamer may not contain a spacer, or may contain no more than one, two, three, or four spacers.

[0063] An aptamer of the disclosure may comprise a linker of any length. In some cases, the linkers may have a length of from about 3 to about 20 nm, and more preferably, from about 5 to about 10 nm. In some cases, the linkers may comprise one carbon molecule, while in other cases the linkers may comprise multiple carbon molecules (e.g, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, or more carbons). In more specific embodiments, the carbon chains may have a length of from about 10 carbons to about 20 carbons, from about 2 to about 15 carbons, or from about 11 to about 30 carbons. The nucleic acid linkers may comprise one nucleic acid molecule or multiple nucleic acid molecules (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, or more nucleic acid molecules). In some cases, the nucleic acid linkers may comprise between about 10 to about 40 nucleic acid bases (single-stranded), base pairs (double-stranded), or a combination thereof.

[0064] In some aspects, the aptamer may selectively bind to a target molecule. Target molecules of the aptamers provided herein can be, but are not limited to, proteins, peptides, nucleic acids e.g ., DNA or RNA), lipids, or even biological cells (e.g. , bacterial cells). Often, the target molecule is known or suspected of playing a biological function in the pathology of a disease or disorder such that modulating the biological activity of the target molecule may alleviate, treat, or cure the disease or disorder, or symptoms associated therewith.

[0065] In some cases, the target molecule may be used in the methods provided herein. For example, the target molecule may be used during the screening process to confirm that a modified aptamer retains the ability to bind to the target molecule. In some cases, the target molecule may be isolated (e.g., separated from its natural environment). In a non-limiting example, the target molecule may be isolated or extracted from a biological cell, a tissue, a bodily fluid, or a biological matrix. In another non-limiting example, the target molecule may be a recombinant protein (e.g, one that is produced using recombinant DNA techniques). In some cases, the target molecules may be immobilized to a solid support. In some cases, the target molecule may not be isolated. For example, the target molecule may be present in a functional assay. The functional assay may involve living cells, cell lysates, or a mixture of isolated cellular components.

Methods of the Disclosure

[0066] In some aspects, the aptamer selection methods herein may involve screening an aptamer to determine differential stability in different biological matrices. In some cases, the aptamer selection methods herein may involve creating a panel of aptamers comprising a plurality of aptamers, each of the plurality of aptamers having the same or similar base sequence, but each having different patterns of nucleic acid modifications. In some cases, each of the plurality of aptamers may have the same or similar base sequence as a known, clinically relevant aptamer. Generally, each aptamer of the aptamer panel may be individually assessed for desired properties such as differential stability, activity, and/or potency. The aptamer selection methods herein may involve performing one or more selection steps on a panel of aptamers to arrive at a final set of aptamer(s) of interest. Selection steps may include differential stability screening, as well as potency and activity screens. After the selection steps, aptamers with desired clinical properties can be identified.

[0067] The aptamer panels provided herein may generally serve as a panel of aptamers that is narrowed down after successive selection steps. A starting point for a panel provided herein may be an aptamer with a known sequence and/or known therapeutic efficacy. The panel may contain multiple variants of such aptamer, such that multiple modifications of such aptamer are represented in the panel. Subsequent selection steps may be performed in order to identify modifications with one or more desired properties, such as the ability to confer partial or complete resistance to endonuclease or exonuclease degradation. In some cases, the aptamers of the panel may be screened individually, with a single aptamer species in each screening reaction.

[0068] In some cases, several different aptamers of a panel may be designed based on an aptamer of known sequence and/or therapeutic efficacy. The designed aptamers may contain one or more modifications that may impart a desired property such as increased vitreous retention, increased vitreous stability, and/or decreased systemic stability. The aptamers may then be assessed for aptamers that exhibit desired properties. For example, aptamers with modifications designed to decrease serum half-life or non-ocular tissue half-life may be screened for additional modifications that increase ocular retention time, or vice versa. Alternatively, aptamers with modifications designed to increase metabolic stability in vitreous may be screened for additional modifications that further increase metabolic stability in vitreous to increase ocular retention time.

[0069] The aptamers provided herein may be generated from a known aptamer, with known properties, which has been modified in a range of different ways. Examples of modifications may include the use of modified nucleotides, or conjugation of different chemical entities.

[0070] In some cases, aptamers may be selected or generated to be similar to a starting aptamer. The aptamers may differ from the starting aptamer in the nucleic acid modifications. The aptamers may include aptamer modification patterns which are about 50%, greater than about 50%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, greater than about 99%, or 100% similar to each other. Several non-limiting examples of aptamers which have been designed to be similar to a starting aptamer may be seen in FIG. 10 and FIG. 11 (aptamers similar to a starting anti-PDGF aptamer), and FIG. 12 and FIG. 13 (aptamers similar to a starting anti-VEGF aptamer). In some cases, aptamers may be designed by selecting a sequence of a known therapeutic aptamer and removing all 2’-fluoro or 2’-0-Me modified bases and/or spacers.

[0071] The aptamers may differ from the starting aptamer by the presence, absence, or location of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more than 50 modifications. The starting aptamer may be a known aptamer with known properties. The starting aptamer may be a known clinical aptamer which binds a therapeutic target and has known therapeutic efficacy. For example, the known clinical aptamer may have an IC₅₀ of less than approximately 100 nM for the therapeutic target.

[0072] In some cases, the aptamers described herein can comprise at least one nucleotide modification. In some cases, at least about 0% to about 50%, or at least about 50% to about 100% of the nucleotides of the aptamer may comprise a modification. In some cases, at least about 2% to about 99%, or at least about 10% to about 90% of the nucleotides of the aptamer may comprise a modification. In some cases, at least about 1%, at least about 10%, at least about 50%, at least about 90%, or 100% of the nucleotides of the aptamer may comprise a

modification.

Differential stability screening

[0073] This disclosure further includes methods for screening aptamers to identify aptamers with particular half-lives or stabilities in a certain environment. The screen may also be conducted to identify aptamers with differential half-lives or stabilities in different environments. The methods of the screen may involve incubating modified aptamers from aptamer libraries or aptamer panels in different biological matrices. The biological matrices may comprise a component derived from a body fluid or a non-ocular tissue ( e.g ., blood, serum, vitreous, non ocular tissue homogenate). In some cases, the biological matrices may comprise a component resembling a particular component of a body fluid. After incubation in the different biological matrices, the aptamer may be assessed for stability by any suitable analytical method, or for activity through a functional assay. Generally, aptamers from the aptamer panel may be screened individually either in parallel or in series.

Biological matrices

[0074] In some cases, the biological matrices used in a screen described herein may contain biological fluids, or components designed to mimic a biological fluid in some respect. Such biological fluids include, but are not limited to: blood, serum, vitreous, lymph fluid, tissue homogenates and/or lysates, and cell lysates.

[0075] A biological matrix used in the methods of the present disclosure may be a blood matrix. Whole blood may comprise blood cells and blood plasma. Blood plasma is essentially an aqueous solution generally containing water, blood plasma proteins, nucleic acids, inorganic salts, and trace amounts of other materials. The term serum generally refers to plasma from which the clotting proteins have been removed. The blood matrix may comprise whole blood, blood plasma, blood serum, blood plasma-like solutions or blood serum-like solutions. Blood serum-like solutions may be solutions with some or all of the properties of blood serum. Blood plasma-like solutions may be solutions with some or all of the properties of blood plasma. Blood plasma may also be reconstituted plasma produced by adding water to dehydrated blood plasma. Blood serum may also be reconstituted serum produced by adding water to dehydrated blood serum. In some cases, blood matrices used in the methods provided herein may contain one or more of the following: enzymes, heparan sulfate proteoglycans, globulins, coagulation proteins, complement factors, nucleases, endonucleases, exonucleases, reversible anticoagulants ( e.g ., citrate), and metabolic enzymes.

[0076] In some particular situations, blood matrices may comprise serum. The serum may be from any biological source including human, bovine, rodent, rabbit, mouse, belted rabbit, calf, rat, horse, sheep, camel, goat, or other source of serum. The serum may be obtained directly from the animal, or purchased separately.

[0077] A biological matrix used in the methods of the present disclosure may be a vitreous matrix. The vitreous humor is mostly composed of water (-98-99% of its volume) and the remainder comprises inorganic salts, lipids, collagen fibers, hyaluronic acid, hyalocytes (the cells that supply hyaluronic acid and collagen to the vitreous) and a wide variety of proteins.

Additional components of the vitreous humor include collagen, opticin, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, globulins, coagulation proteins, complement factors, and low-molecular-weight proteins. A biological matrix provided herein may contain vitreous-like fluids with some or all of the properties of in vivo vitreous fluid. Vitreous-like fluids may contain physiological concentrations of inorganic salts, lipids, collagen fibers, hyaluronic acid, and proteins. Proteins included in vitreous-like fluids may include collagen, opticin, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, globulins, coagulation proteins, complement factors, nucleases, endonucleases, exonucleases, and metabolic enzymes. Vitreous fluid may also be reconstituted vitreous fluid produced by adding water to dehydrated vitreous fluid.

[0078] A biological matrix provided herein may contain lymph fluid. Lymph fluid is the fluid that circulates throughout the lymphatic system. The lymph fluid is formed when the interstitial fluid is collected through lymph capillaries. It is then transported through lymph vessels to be mixed back into the blood. Lymph has a composition similar to that of blood plasma, and contains white blood cells.

[0079] A biological matrix provided herein may contain a tissue homogenate and/or tissue lysate. In some cases, the tissue homogenate or tissue lysate is homogenate or lysate of a non ocular tissue. Tissue homogenates may be prepared by homogenizing a tissue sample.

Examples of tissues which may be homogenized include: liver, kidney, gut, lungs, heart, brain, central nervous system, skin, connective tissue, and muscle. In some cases, a tissue homogenate may consist of any non-ocular tissue, or of several non-ocular tissues. In some cases, the tissue or tissues comprising a non-ocular tissue homogenate or lysate may be chosen to represent tissues which are associated with side effects of a given therapy or therapeutic target. In some cases, the tissues comprising a non-ocular tissue homogenate or lysate may be chosen to represent tissues which express the therapeutic target. Homogenization may be accomplished using physical, chemical, or thermal methods. Examples of homogenization methods include mechanical disruption, such as with rotating blades, liquid homogenization wherein tissue is forced through a small space, sonication, and manual grinding. Cells may also be lysed by treatment with chemicals, high temperatures, or freeze thaw cycles. In some cases, the homogenate may be filtered or centrifuged to clear large particles from the solution.

[0080] Body fluids may be obtained from humans, rabbits, mice, rats, or any other vertebrates. Body fluids may be obtained from live animals or volunteers, or may be derived from sacrificed animals or cadavers. Body fluids may be used immediately after being collected or may be used within 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours of being collected. Body fluids may be frozen after collection for use at a later date. Anticoagulants may be added to body fluids either during or after collection.

[0081] Fluids designed to mimic biological fluids include cell culture mediums, and conditioned mediums which have been incubated in contact with cultured cells known, or suspected, to secrete factors. Fluids designed to mimic biological fluids can also include solutions of nucleases ( e.g ., exonucleases, endonucleases, RNases and/or DNases) known to be present in a given biological fluid. Examples of vertebrate nucleases, include Harbinger Transposase- Derived Nuclease (HARBI1), Apoptosis Enhancing Nuclease (AEN), FANCI-Associated Nuclease 1 (FAN1), Endonuclease G-Like (EXOG), Staphylococcal Nuclease And Tudor Domain Containing 1 (SND1), DNA Replication Helicase/Nuclease 2 (DNA2),

Apurimc/Apyrimidinic Endodeoxyribonuclease 2 (APEX2), Apurmic/Apyrimidimc

Endodeoxyribonuclease 1 (APEX1), Poly(A)-Speeific Ribonuelease (PARN), 5 -3'

Exoribonuclease 2 (XRN2), Tyrosyl-DNA Phosphodiesterase 2 (TDP2), MRE11 Homolog A, Double Strand Break Repair Nuclease (MRE11A), DEAD/H-Box Helicase 1 (DDX1), DNA Fragmentation Factor Subunit Beta (DFFB), KNA Exonuclease 2 (REX02), TatD DNase Domain Containing 3 (TATDN3), and Nei like DNA Glycosylase 1 (NEILl). A fluid designed to mimic a biological fluid may comprise a nuclease at a concentration of between about 0.001% weight/volume (w/v) and about 10% w/v, between about 0.001% w/v and about 1% w/v, between about 0.01% w/v and about 1% w/v, between about 0.05% w/v and about 1% w/v, between about 0.1% w/v and about 1% w/v, between about 0.05% w/v and about 0.5% w/v, between about 0.1% w/v and about 0.5% w/v, or between 0.5% w/v and about 1% w/v. A fluid designed to mimic a biological fluid may further comprise additional nucleases such that the total concentration of nucleases is between about 0.001% w/v and about 10% w/v, between about 0.001% w/v and about 1% w/v, between about 0.01% w/v and about 1% w/v, between about 0.05% w/v and about 1% w/v, between about 0.1% w/v and about 1% w/v, between about 0.05% w/v and about 0.5% w/v, between about 0.1% w/v and about 0.5% w/v, or between about 0.5% w/v and about 1% w/v.

[0082] Biological matrices used herein may contain a target protein or other target molecule of the aptamers to be tested. The target protein may be extracted from tissue, or may be

recombinant protein. The target protein may be present at a concentration of between about 0.0001 pg/mL and about 100 pg/mL, about 0.0002 pg/mL and about 75 pg/mL, about 0.0005 pg/mL and about 50 pg/mL, about 0.001 pg/mL and about 25 pg/mL, about 0.0025 pg/mL and about 20 pg/mL, about 0.005 pg/mL and about 15 pg/mL, about 0.0075 pg/mL and about 10 pg/mL, about 0.01 pg/mL and about 9 pg/mL, about 0.02 pg/mL and about 8 pg/mL, about 0.05 pg/mL and about 7 pg/mL, about 0.075 pg/mL and about 6 pg/mL, about 0.1 pg/mL and about 5 pg/mL, about 0.0001 pg/mL and about 0.001 pg/mL, about 0.001 pg/mL and about 0.01 pg/mL, about 0.001 pg/mL and about 0.1 pg/mL, about 1 pg/mL and about 20 pg/mL, or about 1 pg/mL and about 10 pg/mL. In some cases, the target protein or other molecule is attached to a solid substrate such as a dish, a bead, a well, or other solid substrate.

Incubation conditions

[0083] The aptamers may be incubated in the biological matrices under several different conditions. Generally, the aptamers may be incubated under in vitro conditions designed to mimic or recapitulate one or more in vivo conditions. In other cases, the aptamers may be incubated in vivo (e.g, an animal model). In some cases, the aptamers may be incubated under in vitro conditions that do not necessarily mimic an in vivo condition. The aptamers may be incubated for any length of time or at any temperature. The aptamers may generally be incubated individually, with each incubation reaction containing a single species of aptamer.

[0084] In some cases, aptamers can be screened by incubating under conditions approximating physiological vitreous, non-ocular tissue, or blood conditions. Vitreous, non-ocular tissue, and blood conditions can be approximated by use of vitreous-like fluids, non-ocular tissue-like fluids and blood-like fluids. Blood conditions may be approximated by incubating aptamers in blood matrices which may comprise extracted blood, extracted blood plasma, extracted blood serum, reconstituted serum, reconstituted plasma, or an artificial blood solution. Vitreous conditions can be approximated by incubating aptamers in vitreous matrices which may comprise extracted vitreous, reconstituted vitreous, or artificial vitreous solution. Aptamers can also be screened by incubating under conditions approximating physiological lymph conditions. Lymph conditions can be approximated by incubating aptamers in lymph matrices which may comprise extracted lymph fluid, reconstituted lymph fluid, or artificial lymph fluid. Non-ocular tissue conditions may be approximated by incubating aptamers in non-ocular tissue matrices which may comprise non-ocular tissue homogenates, non-ocular tissue lysates, or cultured cells.

[0085] Aptamers may also be screened in vivo by injecting samples into a subject, particularly, an animal subject, such as a rodent. Aptamers may be injected directly into the blood, into tissue, or into the vitreous humor. Aptamers may be injected either intravenously, intraocularly, or intravitreally. Samples of blood, tissue, and/or vitreous which contain aptamers may be taken at one or more different time points after administration.

[0086] The aptamers may be incubated at various temperatures and over various time periods in order to assess stability of the aptamers or to test other properties of the aptamers, such as activity. Aptamers can be screened by incubating at a temperature of about 37°C. Aptamers can also be screened at temperatures above 37°C. For example aptamers can be screened at a temperature of about 40°C, about 42°C, about 44°C, about 46°C, about 48°C, about 50°C, about 52°C, about 54°C, about 56°C, about 58°C, about 60°C, about 62°C, about 64°C, about 66°C, about 68°C, about 70°C, about 72°C, or higher than about 72°C. Aptamers can also be screened at temperatures below 37°C. For example aptamers can be screened at a temperature of about 36°C, about 35°C, about 34°C, about 33°C, about 32°C, about 3 l°C, about 30°C, about 29°C, about 28°C, about 27°C, about 26°C, about 25°C, about 24°C, about 23°C, about 22°C, about

2l°C, about 20°C, about l9°C, about l8°C, about l7°C, about l6°C, about l5°C, about l4°C, about l3°C, about l2°C, about l l°C, about l0°C, about 9°C, about 8°C, about 7°C, about 6°C, about 5°C, about 4°C, or less than about 4°C. The aptamers may be incubated in the biological matrices at a first temperature, and then a selection of the aptamers may be incubated in the biological matrices at a second temperature.

[0087] Aptamers can be incubated for a range of incubation times. For example, aptamers may be incubated in the biological matrices of the present disclosure for greater than 200 days, about 200 days, about 190 days, about 180 days, about 170 days, about 160 days, about 150 days, about 140 days, about 130 days, about 120 days, about 110 days, about 100 days, about 90 days, about 80 days, about 70 days, about 60 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 25 days, about 20 days, about 18 days, about 16 days, about 14 days, about 12 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1.5 days, about 1.4 days, about 1.3 days, about 1.2 days, about 1.1 days, about 1 day, about 0.9 days, about 0.8 days, about 0.7 days, about 0.6 days, about 0.5 days, about 0.4 days, about 0.3 days, about 0.2 days, about 0.1 days, or less than about 0.1 days. [0088] Aptamers can be incubated in the biological matrices at a range of concentrations. For example, the modified aptamer may be added to the biologic matrix to achieve a concentration of between about 0.001 mM and about 50 mM, about 0.002 pM and about 25 pM, about 0.005 pM and about 10 pM, about 0.01 pM and about 5 pM, about 0.025 pM and about 2 pM, about 0.05 pM and about 1 pM, about 0.075 pM and about 0.5 pM, about 0.1 pM and about 0.25 pM, about 0.1 pM and about 10 pM, about 0.1 pM and about 20 pM, or about 0.1 pM and about 15 pM.

[0089] In some cases, the incubation may be performed with or without agitation of the biological matrices. For example, the incubation step may be performed on a rocking platform or on a shaker. The incubation vessels may contain stir bars, magnetic stir bars, or beads to agitate the solution. The incubation vessels may be periodically shaken. The incubation vessels may be periodically inverted.

Aptamer activity

[0090] Aptamer activity may be measured while the aptamers are incubated in the biological matrices, or after removing the aptamers from the biological matrices. Measurements of aptamer activity while the aptamers are incubated in the biological matrices may reflect the aptamer activity in those solutions. Due to the different compositions of the biological matrices, the aptamers may have different activities in different biological matrices.

[0091] Aptamer activity against the target may be measured by functional inhibition assays to determine the IC50 value of the modified aptamers for the target in different biological matrices. Functional inhibition assays may be designed for each or any target used in the screen. Using such methods, IC₅₀ values for each or any target can be determined for each of the modified aptamers. The IC₅₀ values may be determined by assessing incubated biological matrices which have different initial concentrations of aptamer against the target. The IC50 values may also be calculated by diluting an original incubated aptamer solution. In such cases, preferably, the diluent used would match the original solution used.

[0092] Activity screening of aptamers that have been removed from the incubation solution may be useful to separate the effects of the biological matrices on the activity and the effects of the incubation on the‘retained activity’. The retained activity may be a measure of the stability of the aptamer in the solution. A retained activity IC₅₀ against the target may be calculated by creating a dilution series from a single incubated solution with aptamer. Different incubation times may result in different retained activity IC₅₀ values for a single modified aptamer and target. Aptamer stability

[0093] The stability of the aptamers in the different biological matrices may be determined after various durations of incubation with different conditions, such as temperature and agitation. Often, aptamer stability may be measured after a range of different durations of incubation at 37°C with agitation. For measuring the stability, aliquots of the aptamer may be taken at multiple different time points and total concentrations of fully intact aptamer may be assayed. Concentrations of fully intact aptamer can be measured by liquid chromatography-mass spectroscopy (LC-MS), denaturing gel electrophoresis, or by other bioanalysis methods. These data may be used to calculate the stability or half-life of the modified aptamer under the given conditions. The half-life of the aptamer in a given solution is the time taken for the incubated aptamer to degrade such that the amount of fully intact aptamer remaining in the solution is half that of the amount of fully intact aptamer initially added. The half-life of the modified aptamer in the vitreous matrix can be expressed as the vitreous matrix stability value (VMSV). The half- life of the modified aptamer in the blood matrix can be expressed as the blood matrix stability value (BMSV). The half-life of the modified aptamer in the non-ocular tissue matrix can be expressed as the non-ocular tissue matrix stability value (NOTMSV).

[0094] These methods may also be used to detect and quantify different cleavage products of the aptamers used. Detecting and quantifying the cleavage products produced as an aptamer is degraded may demonstrate the mechanism by which the aptamer was degraded and which residues are most susceptible to degradation. Knowledge of the residues most susceptible to degradation may allow for generation of additional aptamers in which susceptible residues are changed for other, more resistant residues.

[0095] The steps of the stability screen can be performed in series or in parallel. Aptamers may be screened in several different biological matrices, alternatively the aptamers may be screened in a first biological matrix and then a subset of the aptamers may be identified for further screening.

Analysis

[0096] After performing a differential stability screen, the activities and stabilities of the different aptamers in the different biological matrices can be compared to select those aptamer which have desired properties. Data from a single aptamer under different conditions can be compared, or for each different set of conditions individual aptamers can be compared against a ‘panel average’. In one example, the stability of each aptamer in a blood biological matrix or non-ocular tissue biological matrix can be compared to its stability in vitreous matrix and the aptamers with the greatest difference can be selected. In another example, all aptamers screened in vitreous matrix can be compared and those with the highest stability noted, and the identified aptamers may then be screened in a blood matrix or a non-ocular tissue matrix.

[0097] Each modified aptamer may be evaluated by the difference in IC50 for the target between vitreous and blood conditions or non-ocular tissue conditions. Aptamers showing lower IC50 in vitreous conditions than in blood conditions or non-ocular tissue conditions may be selected as particularly useful. To compare the differential activity, a ratio of activity in vitreous matrices to blood or non-ocular tissue matrices can be calculated for each modified aptamer. Modifications which result in higher activity in vitreous than in blood or non-ocular tissue may be identified. Preferred modifications may be those that result in a ratio of IC₅₀ for the target of the modified aptamer in the vitreous matrices compared to the blood or non-ocular tissue matrices that is more than: 10,000:1, 5,000:1, 4,000:1, 3,000:1, 2,000:1, 1,000:1, 900:1, 800:1, 700:1, 600:1, 500:1, 400:1, 300:1, 200:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1. These modifications can then be used to obtain further modified aptamers against different targets to test whether the modifications have similar effects on the activity of multiple different aptamers.

[0098] Each modified aptamer may be evaluated by the difference in half-life between vitreous and blood or non-ocular tissue conditions. Aptamers demonstrating longer half-lives in vitreous conditions than in blood or non-ocular tissue conditions may be selected as particularly useful.

To compare the differential stability, a ratio of stability in vitreous solution to blood or non ocular tissue solution can be calculated for each modified aptamer, e.g ., the VMSV:BMSV ratio, or VMSV:NOTMSV ratio. Modifications which result in longer half-lives in vitreous than in blood or non-ocular tissue may be identified. Preferred modifications may be those that result in a ratio of stability of the modified aptamer in the vitreous matrices compared to the blood matrices (VMSV:BMSV), or non-ocular tissue matrices (VMSV:NOTMSV) that is greater than: 10,000:1, 5,000:1, 4,000:1, 3,000:1, 2,000:1, 1,000:1, 900:1, 800:1, 700:1, 600:1, 500:1, 400:1, 300:1, 200:1, 100:1, 90:1, 80:1.70:1, 60:1, 50:1, 40:1, 30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. These modifications can then be used to obtain further modified aptamers against different targets to test whether the modifications have similar effects on stability of multiple different aptamers.

[0099] Modified aptamers may also be evaluated by the difference in half-life between vitreous and lymph conditions. Modified aptamers with increased stability in vitreous compared to lymph may be preferred. To compare the differential stability, a ratio of stability in vitreous matrices to lymph can be calculated for each modified aptamer. Modifications which result in longer half-lives in vitreous than in lymph may be identified. Preferred modifications may be those that result in a ratio of stability of the modified aptamer in the vitreous matrices compared to the lymph that is greater than: 1,000: 1, 750: 1, 500: 1, 250: 1, 100: 1, 75: 1, 70: 1, 50: 1, 40: 1,

30: 1, 25: 1, 20: 1, 15: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1.5: 1, or 1 : 1.

[00100] In some cases, a therapeutic aptamer may have a short serum exposure but may extravasate and accumulate in tissue if the therapeutic target is present, leading to adverse effects in the tissue. Accordingly, modified aptamers may also be evaluated by the difference in half- life between vitreous and tissue conditions. Modified aptamers with increased stability in vitreous compared to tissue may be preferred. To compare the differential stability, a ratio of stability in vitreous matrices to tissue can be calculated for each modified aptamer. In some cases, vitreous stability may be compared to stability in a single tissue or in several tissues. Vitreous stability may be compared to individual stability values in each of several tissues, or to an average stability value of the tissues. Examples of tissues which may be assessed for tissue stability include, but are not limited to, liver, kidney, gut, lungs, heart, brain, central nervous system, skin, connective tissue, and muscle. Modifications which result in longer half-lives in vitreous than in tissue may be identified. Preferred modifications may be those that result in a ratio of stability of the modified aptamer in the vitreous matrices compared to the tissue that is greater than: 1,000: 1, 750: 1, 500: 1, 250: 1, 100: 1, 75: 1, 70: 1, 50: 1, 40: 1, 30: 1, 25: 1, 20: 1, 15: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5:1, 4: 1, 3: 1, 2: 1, 1.5: 1, or 1 : 1.

[00101] In some cases, the preferred aptamer is an aptamer that has high stability and activity in vitreous and low stability and/or low activity in the blood or in non-ocular tissues. An aptamer with high stability and activity in vitreous and low stability and/or low activity in lymph may also be desired. In other cases, an aptamer with high stability and activity in vitreous without regard for stability or activity in non-ocular tissues may be desired. Such aptamers may be identified by comparing those aptamers with the highest vitreous stability:blood stability and/or vitreous activity:blood activity ratios and/or the highest vitreous stability. Such aptamers may also be identified by comparing those aptamers with the highest vitreous stabilitymon-ocular tissue stability and/or vitreous activitymon-ocular tissue activity ratios. A high ratio for either stability or activity may be sufficient, though high ratios for both may be preferred. A preferred aptamer selected by the methods described herein may not have the highest stability or activity in the vitreous, and it is even possible that such an aptamer may have a below average stability and/or activity in the vitreous.

[00102] In some embodiments, preferred aptamers may be aptamers without 2^,-fluoropyrimidine or 2’-fluoropurine residues. The data in Example 1 and Example 7 suggest that T - fluoropyrimidine or 2’-fluoropurine residues, while stabilizing aptamers in the serum, may destabilize the aptamers in the vitreous. The data in Example 1 and Example 7 further suggest that two or more contiguous 2’-fluoropyrimidine or 2’-fluoropurine residues, while stabilizing aptamers in the serum, may destabilize the aptamers in the vitreous. The data in Example 1 and Example 7 further suggest that two or more contiguous 2’-fluoropyrimidine or 2’-fluoropurine residues in a stem or double-stranded region of the aptamer, while stabilizing aptamers in the serum, may destabilize the aptamers in the vitreous. Indeed, in the vitreous, 2’-fluoro-modified aptamers are less stable than DNA-only aptamers. In some cases, modifications which are known to protect aptamers from endonucleases in the serum may not protect the aptamers in the vitreous. In some cases, modifications which protect aptamers from exonucleases in the serum may also protect aptamer stability in the vitreous.

Properties of aptamers

[00103] The aptamers provided herein or identified by the methods described herein may have fewer modified bases than aptamers optimized for stability in serum or non-ocular tissue. In some examples, the aptamers may contain more than about 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% unmodified nucleic acid. In further examples, the aptamers may contain less than one, two, three, four, five, six, seven, eight, nine, or ten modified bases. In particular aspects, the aptamers may contain fewer 2’-fluoropyrimidines than an aptamer optimized for stability in serum or non-ocular tissue. For example, an aptamer of the disclosure may contain fewer than one, two, three, four, five, six, seven, eight, nine, or ten 2’-fluoropyrimidine modified bases.

[00104] The aptamers described herein may have an improved half-life in a body fluid ( e.g ., blood, serum, lymph, interstitial fluid, vitreous fluid), particularly compared to the unmodified version of the molecule or to a different therapeutic, such as an antibody. In some cases, the aptamers may have an improved blood, serum, or non-ocular tissue half-life. The aptamers identified may contain modifications which improve blood, serum, or non-ocular tissue half-life by increasing or by decreasing the blood, serum, or non-ocular tissue half-life of the aptamer.

An increase in blood, serum, or non-ocular tissue half-life may be desirable in some instances, for example, to maximize in vivo exposure to the aptamer. In contrast, a decrease in blood, serum, or non-ocular tissue half-life may be desirable in some instances, for example, to minimize in vivo exposure to the aptamer and/or to minimize toxicity.

[00105] The aptamers described herein may have a decreased half-life in blood or related fluids, or non-ocular tissue, as compared to the half-life in vitreous or related fluids. The aptamers described herein may have a decreased half-life in blood or related fluids, or non-ocular tissue, as compared to other therapeutics, including antibodies. Aptamers identified by the methods described herein, may have a decreased half-life in blood, serum, or non-ocular tissue compared to a starting aptamer ( e.g ., from an aptamer panel as described herein). Aptamers identified by the methods described herein may have low systemic exposure while retaining high activity in the vitreous. In some cases, an identified aptamer as disclosed herein may have a decreased half- life in a biological fluid (e.g., blood, serum, lymph, interstitial fluid) or tissue as compared to the starting aptamer. In some cases, the aptamers described herein may have a blood, serum, or non-ocular tissue half-life of less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 3 days, less than 2 days, less than 1 day, less than 0.5 days, or less than 0.25 days in a human. In some cases, the aptamers described herein may have a blood, serum, or non-ocular tissue half-life of less than about 24 hours, less than about 18 hours, less than about 12 hours, less than about 10 hours, less than about 8 hours, less than about 6 hours, less than about 4 hours, or less than about 2 hours.

[00106] Aptamers identified by the methods described herein may be more susceptible to clearance from the blood or from a non-ocular tissue than a starting aptamer (e.g, from a panel of aptamers as described herein). For example, the selected modifications may serve to enhance clearance of the aptamer by the liver, spleen, and/or kidney. The modifications of the aptamers may enhance the stability of the aptamer against nucleases while increasing clearance of the aptamer through other mechanisms. The modifications of the aptamers may cause the aptamers to bind tightly to common blood proteins such as albumins or globulins.

[00107] In some aspects, due to their enhanced metabolic stability in the vitreous, the aptamers disclosed herein may have an improved ocular half-life as compared to other therapeutics, including antibodies. Aptamers identified by the methods described herein, may have an improved ocular half-life as compared to a starting aptamer. An improved ocular half-life generally refers to an increase in ocular half-life; but, in some cases, an improved ocular half-life may refer to a decrease in ocular half-life. In some cases, the aptamers may have an improved half-life when injected into the eye (intraocular half-life) as compared to an antibody or to the starting aptamer of the screen. In some cases, the aptamers may have an improved intravitreal half-life when injected into the eye of a human. In some cases, the aptamers described herein may have an intraocular half-life of at least 7 days in a subject (e.g, human). In some cases, the aptamers described herein may have an intraocular half-life of at least 4 days, at least 5 days, at least 6 days, least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or greater in a human.

[00108] In preferred cases, aptamers identified by the methods described herein may have an improved or increased intraocular (or intravitreal) half-life and a decreased serum or non-ocular tissue half-life, when compared to a starting aptamer of the aptamer panel. In some cases, the aptamers described herein may have an improved intraocular (or intravitreal) half-life and an increased serum or non-ocular tissue half-life. In other cases, the aptamers described herein may have an improved intraocular (or intravitreal) half-life and no change in serum or non-ocular tissue half-life.

[00109] Aptamers generally have high stability at ambient temperatures for extended periods of time. In some cases, the aptamers identified by the methods described herein may demonstrate greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 99.5%, or greater than about 99.9% activity in solution under physiological conditions at 30 days or later. In some cases, the aptamers described herein may demonstrate greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 99.5%, or greater than about 99.9% activity in solution under intravitreous physiological conditions over time. The aptamers described herein may demonstrate greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 99.5%, or greater than about 99.9% activity in solution after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more than 200 days under intravitreous physiological conditions or in vivo in the vitreous humor.

[00110] Aptamers described herein may be particularly advantageous over antibody therapies against the same target molecule as they may sustain therapeutic intravitreal concentrations of drug for longer periods of time, thus requiring less frequent administration. The aptamers described herein may have a longer intraocular half-life, and/or sustain therapeutic intravitreal concentrations of drug for longer periods of time, than antibody therapies.

[00111] In some cases, aptamers described herein may be very well tolerated when used as a therapeutic ( e.g ., for the treatment of an ocular disease or disorder). Treatment with aptamers described herein may result in fewer adverse reactions than treatment with similar therapeutics. For example, treatment with the aptamers provided herein may result in fewer types of adverse reactions compared with treatment with similar therapeutics. In some cases, treatment with the aptamers provided herein may result in an adverse reaction which is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% less severe when compared with the adverse reaction caused by treatment with similar therapeutics.

Treatment with the aptamers described herein may be better tolerated than treatment with other methods for inhibiting the same target molecule. Treatment with the aptamers described herein may allow administration of higher effective doses than treatment with similar therapeutics. An aptamer as described herein may be administered at a higher concentration than a similar therapeutic and may cause equal adverse reactions. An aptamer as described herein may be administered at a higher concentration than a similar therapeutic and cause fewer adverse reactions. An aptamer described herein may be administered at a higher concentration than a similar therapeutic ( e.g ., a different aptamer) and may cause less severe adverse reactions.

Treatment with aptamers as described herein may have no adverse reactions. Therapeutics which have similar or greater stability in blood or non-ocular tissue as compared to vitreous may have adverse effects in biological systems other than the eye. This may be of particular concern when such treatments are used in pediatric settings as the therapeutic may adversely affect normal physiology and or development.

[00112] Given that the aptamers identified by the present disclosure may have increased vitreous stability as compared to blood, serum, or non-ocular tissue stability, or may have low potency, treatment with these aptamers may be very well tolerated. Aptamers with increased vitreous stability compared to serum or non-ocular tissue stability, or with low potency, may have improved safety profiles compared to therapeutics with similar stability in both

environments. For example, an aptamer that is stable enough in the vitreous to be efficacious or to stay in the vitreous for longer residential periods but that has decreased stability in the blood, serum, or non-ocular tissue may cause decreased systemic exposure of the aptamer. In another example, an aptamer with low potency may cause decreased systemic exposure of the aptamer at doses that achieve therapeutic concentrations in the eye. Decreased systemic exposure may result in fewer adverse reactions, or less severe adverse reactions. Aptamers with increased vitreous stability compared to serum or non-ocular tissue stability may have about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10% or about 5% fewer adverse reactions than a therapeutic that lacks this property. Aptamers with increased vitreous stability compared to serum or non-ocular tissue stability, or with low potency, may cause an adverse reaction that is about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10% or about 5% less severe than the same adverse reaction caused by a therapeutic that lacks these properties. This may be of particular importance in pediatric indications where the results of inhibiting pathways involved in normal growth and development could be especially serious. In some cases, aptamers selected using the methods of this disclosure may not cause systemic adverse reactions such as cognitive and/or neuromuscular impairments.

[00113] Aptamers with increased vitreous stability compared to serum or non-ocular tissue stability, or with low potency, may also have improved safety profiles in adults. This may allow administration of therapies to patients in which they would normally be contraindicated, such as administration of anti -angiogenesis therapy in a patient with history of high blood pressure or heart attack. This may also allow administration of therapeutics to pregnant or nursing women with significantly reduced secondary exposure of the embryo, fetus or infant.

[00114] Using the results from the aptamer panel screen, it may be possible to develop a framework of rules to design aptamers that have increased stability in a first biological matrix compared to a second biological matrix. There may be several modifications that impart increased stability in a first biological matrix compared to a second biological matrix. The choice of the first modification for imparting increased stability in a first biological matrix compared to a second biological matrix may influence the choice of a second modification. For example, some modifications may act cooperatively in increasing the stability of an aptamer in a first biological matrix compared to a second biological matrix, while some modifications may negate the effect of specific other modifications. In some cases, a pattern of two or more modifications may be required. In some cases, the pattern of two or more modifications may require that the specific modifications are present in a specific order. In some cases, the pattern of two or more modifications may require that the specific modifications are present in a specific orientation.

[00115] In some aspects, an aptamer as described herein may be an anti-PDGF aptamer such as Compound 1 or Compound 2, both of which are depicted in FIG. 2; or an anti-VEGF aptamer such as Compound 3 or Compound 4, both of which are depicted in FIG. 3. In some aspects, an aptamer of this disclosure may be C6NFl₂-CAGGC-fU-A-fC-mG-[Spl8]-CGTA-mG-A-mG- CA-fU-fC-mA (SEQ ID NO: l)-[Spl8]-TGAT-fC-fC-fU-mG-idT, wherein capital A, C, T, and G are DNA; fU and fC are 2’-fluoro RNA; mA and mG are 2’-0-methyl RNA; C6NH₂ is a hexylamino linker; [Spl8] is an internal hexaethylene glycol spacer; and idT is a 3’ -3’ inverted deoxythymidine (Compound 1). In some aspects, an aptamer of this disclosure may be C6NH₂- CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-idT (SEQ ID NO: 2), wherein capital A, C, T, and G are DNA; and C6NH₂ is a hexylamino linker (Compound 2). In some aspects, an aptamer of this disclosure may be C6NH₂-fC-mG-mG-aa-fU-fC-mA-mG-fU-mG- mA-mA-fU-mG-fC-fU-fU-mA-fU-mA-fC-mA-fU-fC-fC-mG-idT (SEQ ID NO: 3), wherein capital A, C, T, and G are DNA; lower case a is RNA; fU and fC are 2’-fluoro RNA; mA and mG are 2’-0-methyl RNA; C6NH₂ is a hexylamino linker; [Sp 18] is an internal hexaethylene glycol spacer; and idT is a 3’-3’ inverted deoxythymidine (Compound 3). In some aspects, an aptamer of this disclosure may be C6NH₂-fC-ggaa-fU-fC-ag-fU-gaa-fU-g-fC-fU-fU-a-fU-a-fC- a-fU-fC-fC-g-idT (SEQ ID NO: 3), wherein capital A, C, T, and G are DNA; lower case a and g are RNA; fU and fC are 2’-fluoro RNA; mA and mG are 2’-0-methyl RNA; C6NH₂ is a hexylamino linker; [Spl8] is an internal hexaethylene glycol spacer; and idT is a 3’ -3’ inverted deoxythymidine (Compound 4). In some cases, an aptamer of this disclosure may be an aptamer comprising a nucleic acid sequence having at least about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity to a nucleic acid sequence of any one of the aptamers described above.

[00116] In some aspects, an aptamer of the disclosure may have a modification identity of greater than about 70%, about 80%, about 85%, about 90%, about 95%, or about 99% to any one of the aptamers disclosed herein. The modification identity of an aptamer may be determined by comparing each position for the presence of the same base and the same modification. A first aptamer may have greater than 80% sequence identity to a second aptamer but have less than 80% modification identity if different modifications are present on the first and second aptamers.

[00117] In some cases, an aptamer as described herein may be any aptamer from Table 1, Table 8, Table 9, or Table 10. In some aspects, an aptamer may be an aptamer which has at least about 70%, about 80%, about 90%, or about 95% modification identity to any aptamer described in Table 1, Table 8, Table 9, or Table 10.

[00118] In some cases, an aptamer of the disclosure may be an aptamer that has low stability in the serum, rapid clearance from the serum, or both, such that the maximum serum concentration of the aptamer (serum C_max) following administration to the eye is low. In some cases, when the aptamer is administered to the eye at a dose of about 0.3 mg/eye, the serum C_max may be less than about 1.5 nM, less than about 0.75 nM, or less than about 0.5 nM. In some cases, when the aptamer is administered to the eye at a dose of about 1 mg/eye, the serum C_max may be less than about 4.5 nM, less than about 2 nM, or less than about 1 nM. In some cases, when the aptamer is administered to the eye at a dose of 3 mg/eye, the serum C_max may be less than about 10 nM, less than about 8 nM, less than about 6 nM, or less than about 4 nM. In some cases, when the aptamer is administered to the eye, the serum C_max does not exceed about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, or about 0.5 nM. In some cases, the ratio of the C_{max O}f the aptamer in vitreous to serum following administration to the eye may be greater than about 100: 1, greater than about 500: 1, greater than about 1000: 1 or greater than about 10,000: 1.

Potency

[00119] In some aspects, aptamers are provided with a moderate or relatively low potency that may enable them to have an improved safety profile. A desired safety profile may result when the aptamer effectively modulates a target molecule in an ocular compartment, such as the vitreous compartment, but does not effectively modulate the target molecule in the blood and/or non-ocular tissue compartments. Methods of using such low-potency or moderate-potency aptamers to treat a subject with an ocular disease or disorder may include providing aptamers with a sufficient dose, potency, and ocular half-life to be effective in the eye, while

concomitantly minimizing the risk of systemic exposure when the aptamers exit the eye and enter systemic circulation. The methods may involve administering a low-potency or moderate- potency aptamer to the subject at a high or moderately-high dose and the aptamer may be designed to have a half-life that is long enough for the dose to remain effective over a significant period of time, such as 2-4 weeks. In some cases, the methods may involve administering a moderate-potency aptamer to the subject at a high or moderately-high dose and the moderate- potency aptamer may be designed to have a half-life that is long enough for the dose to remain effective over a significant period of time, such as 2-4 weeks.

[00120] In some particular aspects, a method may include administering a moderate-potency or low-potency aptamer such that the ocular concentration ( e.g ., vitreal concentration) of the aptamer is equal or higher than the IC₉₀ of the aptamer for the target molecule. In some cases, the method may include administering a moderate-potency aptamer such that the ocular concentration of the aptamer remains at least equal to the IC₉₀ concentration for the target molecule for 2 or 4 weeks after administration. In some cases, the method may include administering a moderate-potency aptamer such that the ocular concentration of the aptamer on the day of the administration is significantly higher than the IC₉₀ of the aptamer for the target molecule (e.g., at least 2-fold greater than the IC₉o of the aptamer for the target, 5-fold greater than the IC₉o of the aptamer for the target, at least lO-fold greater than the IC₉o of the aptamer for the target). In some further aspects, the concentration of aptamer delivered to the eye results in a systemic concentration (e.g, serum or non-ocular tissue concentration) of the aptamer that is less than the IC_l0 of the aptamer for the target molecule or less than the IC₅ of the aptamer for the target molecule. In some cases, the aptamer is not detectable in the serum or non-ocular tissue at a concentration higher than the IC_l0 of the aptamer for the target molecule within the first two, three, four, or five days after administration to the eye. In some cases, the aptamer is not detectable in the serum or non-ocular tissue at a concentration higher than the IC_l0 of the aptamer for the target molecule at any time beyond two, three, four, or five days after administration to the eye. In some cases, the aptamer is not detectable in the serum or non-ocular tissue at a concentration higher than the IC_l0 of the aptamer for the target molecule at any time after administration to the eye. In some examples, the ocular concentration of the aptamer is at least as high as ( e.g ., equal to or at least 2-fold greater than) the IC₉₀ of the aptamer for the target molecule; and the systemic concentration (e.g., serum or non-ocular tissue concentration) of the aptamer is less than the IC_l0 of the aptamer for the target molecule. In some cases, the IC90 of the aptamer for the target molecule is between about 25 nM and about 50 nM. In some cases, the IC₉₀ of the aptamer for the target molecule is greater than about 5 nM, greater than about 25 nM, greater than about 40 nM, greater than about 50 nM, or greater than about 100 nM. In some cases, the IC90 of the aptamer for the target molecule is less than about 5 nM, less than about 25 nM, less than about 30 nM, less than about 40 nM, less than about 50 nM, or less than about 100 nM. In some cases, the aptamer may have a K_d of greater than about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 100 nM, about 150 nM, about 200 nM, or about 250 nM. In some cases, the aptamer may have a K_d of less than about 10 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, or about 0.5 nM.

[00121] A low-potency or moderate-potency aptamer provided herein may have a long ocular half-life (e.g, at least 7 days, at least 10 days, at least 2 weeks, at least 4 weeks). In some cases, the low-potency or moderate-potency aptamer may have an ocular half-life that is longer than its systemic half-life, either because it has been modified to increase its ocular half-life, modified to decrease its systemic half-life, or modified to both increase its ocular half-life and decrease its systemic half-life. The potency of the aptamer may be varied relative to its half-life. For example, lower-potency aptamers may be designed to have a longer half-life. As such, in some cases, a low-potency or moderate-potency aptamer provided herein may be sufficiently stable to remain efficacious over several weeks. In some cases, a low-potency or moderate-potency aptamer provided herein may have a long ocular half-life that is sufficiently long such that the concentration of the aptamer in the eye remains above the IC90 of the aptamer for the target molecule for at least two, three, four, five, or six weeks after administration of 0.75 mg of aptamer per eye. In some cases, a low-potency or moderate-potency aptamer provided herein may have a long ocular half-life that is sufficiently long such that the concentration of the aptamer in the eye remains above the IC90 of the aptamer for the target molecule for at least two, three, four, five, or six weeks after administration of 1.5 mg of aptamer per eye. [00122] An aptamer provided herein may have a low potency and a long half-life. For example, an aptamer provided herein may have a K_d value of about 100 nM (or higher), and a half-life of 7 days, 8 days, 9 days, 10 days, or greater than 10 days. In some cases, an aptamer provided herein may have a moderate potency and a moderate or long half-life. In some cases, an aptamer provided herein may have a K_d value of about 50 nM (or higher) and a half-life of at least 5 days, 6 days, 7 days, or greater than 7 days. In some cases, an aptamer provided herein may have a low, moderate, or high potency, a long half-life in the vitreous, and a short half-life in blood and/or non-ocular tissue. For example, an aptamer provided herein may have a K_d value of about 5 nM or less; a vitreal half-life of at least about 7 days, 8 days, 9 days, 10 days, or greater than 10 days; and a serum or non-ocular tissue half-life of about 4 days, 3 days, 2 days, 1 day, or less than 1 day.

[00123] As noted herein, a desired safety profile may be one where the low-potency or moderate-potency aptamer effectively modulates a target in the vitreal compartment but does not effectively modulate the target in the blood and/or non-ocular tissue compartments. As shown in Example 5, this may be achieved in several ways. For example, an aptamer that has high potency, relatively high ocular stability, and relatively low systemic stability may be able to maintain a high stability in the vitreal compartment without achieving a high concentration in the blood and non-ocular tissue compartments. Alternatively, an aptamer with low potency and with ocular stability that is not higher than its serum or non-ocular tissue stability may be

administered at a high enough concentration to maintain an effective concentration in the vitreal compartment; however, due to the significant dilution factor between the vitreal compartment and the blood and non-ocular tissue compartments, the low-potency or moderate-potency aptamer may be prevented from reaching an effective concentration in the blood and non-ocular tissue compartments.

[00124] This disclosure provides aptamers with a potency and half-life designed to enable a significant duration ( e.g ., 2 weeks, 4 weeks, or greater) of efficacy in the eye. In some cases, this disclosure provides an aptamer that has a K_d of about 25 nM to about 50 nM (e.g., 50 nM) and a half-life of 7 days or greater. In some cases, at least 0.75 mg of such aptamer is administered to an eye. In some further cases, at least 0.75 mg of such aptamer is administered to the eye no more than once every 4 weeks. In some cases, the ocular concentration of such aptamer remains above the IC₉₀ concentration of the aptamer for the target for over 2 weeks or over 4 weeks.

[00125] In some aspects, the aptamer designed to enable a significant duration of efficacy in the eye may have a K_d from about 25 nM to about 50 nM (e.g, 50 nM) and a half-life of 4 days or greater. In some cases, at least 1.5 mg of such aptamer is administered to an eye. In some further cases, at least 1.5 mg of such aptamer is administered to an eye no more than once every 4 weeks. In some cases, the ocular concentration of such aptamer remains above the IC₉₀ concentration of the aptamer for the target for over 2 weeks or over 4 weeks.

[00126] In some aspects, the aptamer designed to enable a significant duration of efficacy in the eye may have a K_d of about 100 nM and a half-life of 7 days or greater (e.g, 8 days, 10 days). In some cases, at least 0.75 mg of such aptamer is administered to an eye. In some further cases, at least 0.75 mg of such aptamer is administered to an eye no more than once every 4 weeks. In some cases, the ocular concentration of such aptamer remains above the IC90 concentration of the aptamer for the target for over 2 weeks or over 4 weeks.

[00127] In some aspects, the aptamer designed to enable a significant duration of efficacy in the eye may have a K_d of about 100 nM and a half-life of 7 days or greater. In some cases, at least 1.5 mg of such aptamer is administered to an eye. In some further cases, at least 1.5 mg of such aptamer is administered to an eye no more than once every 4 weeks. In some cases, the ocular concentration of such aptamer remains above the IC₉₀ concentration of the aptamer for the target for over 2 weeks or over 4 weeks.

[00128] This disclosure provides for aptamers with a metabolic stability in the vitreous to enable a significant duration of efficacy in the eye (e.g, 12 weeks, 16 weeks, 20 weeks or 24 weeks and greater). In some cases, this disclosure provides aptamers that have an in vitro half-life in rabbit vitreous of about 125 to about 150 hours, and a half-life in rabbits of about 4.5 days following intravitreal administration. In some cases, at least 1.5 mg of such aptamer is administered to an eye. In some cases, at least 3 mg of such aptamer is administered to an eye. In other cases, at least 5 mg of such aptamer or more is administered to an eye. In some cases, this disclosure provides aptamers that have an in vitro half-life in rabbit vitreous of about 600 to about 700 hours and a half-life in rabbits of about 7 to about 8 days following intravitreal administration.

In some cases, at least 1.5 mg of such aptamer is administered to an eye. In some cases, at least 3 mg of such aptamer is administered to an eye. In other cases, at least 5 mg of such aptamer or more is administered to an eye.

Indications

[00129] In some aspects, the methods and compositions provided herein may be used for the treatment of ocular diseases or disorders. In some cases, the methods and compositions provided herein can be used for the prevention of an ocular disease. In other cases, the methods and compositions provided herein may be used to slow or halt the progression of an ocular disease.

In other cases, the methods and compositions provided herein may be used to cure an ocular disease. In yet other cases, the methods and compositions provided herein may be used to treat or ameliorate one or more symptoms associated with an ocular disease or disorder. In some cases, the ocular disease may be a pediatric ocular disease. In some aspects, aptamers as described herein may be used in the treatment or prevention of a disease associated with aberrant VEGF, angiopoietin-2 (Ang2), interleukin-8 (IL8), and/or platelet-derived growth factor (PDGF) signaling.

Subjects

[00130] In some aspects, the methods and compositions provided herein may be used to treat a subject in need thereof. In some cases, the subject may have, may be suspected of having, or may be at risk of developing an ocular disease or disorder. In some cases, the subject may be experiencing symptoms associated with an ocular disease or disorder. The subject can be a non human animal, for example, a non-human primate, a livestock animal, a domestic pet, or a laboratory animal. For example, a non-human animal can be an ape ( e.g ., a chimpanzee, a baboon, a gorilla, or an orangutan), an old world monkey (e.g., a rhesus monkey), a new world monkey, a dog, a cat, a mouse, a rat, a rabbit, or any other non-human animal. In some cases, the subject is a human. In some cases, the human is a patient at a hospital or a clinic.

[00131] The subject may be of any age; but, in some preferred aspects, the subject is an infant or a child. In some cases, the subject is a neonatal infant. In some cases, the subject may have an age between about 2 weeks and about 10 years. In some cases, the subject may be less than about 18 years old, less than about 17 years old, less than about 16 years old, less than about 15 years old, less than about 14 years old, less than about 13 years old, less than about 12 years old, less than about 11 years old, less than about 10 years old, less than about 9 years old, less than about 8 years old, less than about 7 years old, less than about 6 years old, less than about 5 years old, less than about 4 years old, less than about 3 years old, less than about 2 years old, less than about 1 year old, or younger. In some cases, the subject may be an infant between about 2 and about 104 weeks of age. In some cases, the subject may be about 1, about 5, about 10, about 11, about 12, about 20, about 40, about 52, about 104 or more than about 104 weeks old. In some cases, the subject may be about 2 weeks old or older. The subject may have been born at full- term; in some cases, the subject may have been born prematurely. In some cases, the subject may have been bom at about 31 gestational weeks of age or younger than about 31 gestational weeks of age. In some cases, the subject may have been born with a gestational age of about 20 to about 40 weeks, about 20 to about 30 weeks, about 25 to about 35 weeks, or about 25 to about 30 weeks. In some cases, the subject may have been bom with a gestational age of about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, or more than about 36 weeks. [00132] In some cases, the subject may be a teenager, an adult, or a senior citizen. For example, the subject may be 18 years old, 40 years old, 70 years old, or any other age.

[00133] The aptamers disclosed herein, may be administered by local ocular delivery. Non limiting examples of local ocular delivery include intravitreal (IVT), intracamarel,

subconjunctival, subtenon, retrobulbar, posterior juxtascleral, and peribulbar. In some cases, an aptamer of the disclosure may be delivered by intravitreal administration (IVT). Local ocular delivery may generally involve injection of a liquid formulation. Therefore, in some cases, the aptamers described herein may be formulated in a liquid formulation, suitable for local ocular delivery. The aptamers disclosed herein, may be administered as a single injection, or a series of single injections administered about every 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or less frequently than every 8 weeks.

EXAMPLES

[00134] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1. Aptamer stability in serum versus vitreous matrices

[00135] This example provides representative selection methods of the disclosure. In general, the selection methods involve assessing stability and/or retained activity of an aptamer in serum and vitreous conditions. The retained activity and stability is compared in order to identify aptamers (and associated modifications) with higher vitreous retained activity and stability than serum retained activity and stability. The ultimate goal of these assays is to determine a type or pattern of oligonucleotide modifications that provide differential stability in the eye as compared to systemic circulation, such that when applied to therapeutic aptamers, the aptamers possess sufficient ocular residence time to be effective when administered to the eye, and are rapidly cleared when they leak from the eye into systemic circulation. To interrogate the relative stability of specific oligonucleotide chemistries in vitreous as compared to serum matrices, the aptamers described in Table 1 were synthesized and their metabolic stability and profiles were determined. These aptamers are also depicted in FIG. 2 and FIG. 3, and predicted secondary structures are shown in FIG. 4 and FIG. 5. Table 1. Aptamer sequences

[00136] Briefly Compound 1, Compound 2, Compound 3, and Compound 4 were incubated at a 10 mM final concentration in either rabbit vitreous, rabbit serum, or human serum at 37°C.

Aliquots were removed at 0, 1, 4, 8, 24, 48, and 72 hours, and reactions were terminated by the addition of EDTA, followed by incubation at 60°C in an equal volume of Clarity^® OTX™ loading/lysis buffer (Phenomenex, CA) with proteinase K containing 2.5 mIUΊ of an internal standard oligonucleotide. Samples were vortexed and immediately frozen at -70°C.

Subsequently, all samples were thawed, extracted per the Clarity^® OTX™ 96-well protocol, dried down in a vacuum concentrator, and resuspended in 200 pL of water for analysis. High resolution accurate mass LC-MS analysis was conducted on an LTQ-Orbitrap™ mass spectrometer using approximately 1/10*¹¹ of the sample volume. Disappearance of parent compound was calculated based on the area ratios of test article/intemal standard at each time point as compared to time 0. For each test article, identity of detected oligonucleotide fragments at each time point was assigned by mass. Based on the identity and timing of each identified metabolite for each test article, metabolic profiles were assembled to identify the site of initial nuclease cleavage and subsequent metabolism of each oligonucleotide and metabolite over time. [00137] The metabolic stability of Compound 1 is shown in Table 2 and in FIG. 6. Compound 1 was an aptamer which was optimized for stability in serum by selective replacement of deoxynucleotides with 2’-fluoropyrimidines and 2’ -O-m ethyl purines (Floege J., Ostendorf T., Janssen U., Ostman A., Heldin C.H., and Janjic N. 1999. Novel Approach to Specific Growth Factor Inhibition In Vivo. Am. J. Pathol. 154: 169-179.). Additionally, it contained a 3’-inverted deoxythymidine cap and a 5’-hexylamino linker, modifications which can be used to reduce the rate of exonuclease loading and subsequent metabolism of oligonucleotides.

Table 2. Metabolic stability of Compound 1 in human serum, rabbit serum, and rabbit vitreous

[00138] In general, with respect to oligonucleotide metabolism, serum is considered a more metabolically active matrix than vitreous, and from a species perspective, rabbit serum is considered a more metabolically active matrix than human serum. Therefore, it was surprising to observe a greater percent of intact Compound 1 at 24 hours in rabbit serum than in rabbit vitreous, and to generally observe a greater metabolic stability in serum as compared to the metabolic stability in vitreous for this aptamer. Examination of the specific nature of the nuclease cleavage that initiated metabolism of Compound 1 in the respective matrices yielded unanticipated results. In human and rabbit serum, metabolism of Compound 1 was initiated by exonuclease degradation from both the 3’ and 5’ ends as expected, indicating that the T sugar modifications within the backbone of Compound 1 reduced the rate of endonuclease degradation, such that exonuclease degradation was the dominant metabolic pathway. In contrast, in rabbit vitreous, endonucleolytic cleavage at 2^,-fluoropyrimidines initiated metabolism of Compound 1. The major initial sites of endonuclease cleavage observed in rabbit vitreous were 2’- fluoropyrimidines in double-stranded regions of Compound 1 including after the 2’-fluorouridine at position 7 (numbering is from the 5’ end and with the C6NH₂ linker as position 1), and after the 2’-fluorocytidine at position 30. This finding indicated that the operative oligonucleotide metabolic pathways in vitreous were substantially different from the operative oligonucleotide metabolic pathways in serum, and that optimization of metabolic stability of oligonucleotide therapeutics for vitreous applications may be substantially different than for systemic applications. This is an unexpected result given the large body of data available for stabilization of oligonucleotide therapeutics for systemic administration.

[00139] The metabolic stability of Compound 2 is shown in Table 3 and in FIG. 7. Compound 2 was the same nucleotide sequence as Compound 1. It represented the“all DNA” version of this molecule prior to optimization of Compound 1 for serum stability (Green L.S., Jellinek D., Jenison R., Ostman A., Heldin C.H., and Janjic N. 1996. Inhibitory DNA Ligands to Platelet Derived Growth Factor B-chain. Biochemistry 35: 14413-14424). Compound 2 contained the same exonuclease blocking chemistries as Compound 1 but none of the modifications to reduce endonucleolytic metabolism.

Table 3. Metabolic stability of Compound 2 in human serum, rabbit serum, and rabbit vitreous

in human serum than in rabbit serum, and greater stability in rabbit vitreous than in serum.

However, the magnitude of the stability difference in rabbit vitreous as compared to rabbit serum was surprising, with an approximately 68-fold increase in intact aptamer in rabbit vitreous as compared to rabbit serum following 8 hours of incubation. Examination of the metabolic profiles in the respective matrices showed unanticipated differences in metabolism in the vitreous as compared to the serum. Based on studies of oligonucleotide metabolism in serum and tissues, it was anticipated that endonuclease cleavage within single-stranded loops and joining regions would be the primary sites of initial metabolism, as single stranded DNA is known to be labile in serum and tissues. However, in the vitreous matrix, metabolism of Compound 2 was an exonucleolytic process, with both the 5’ linker and 3’ inverted

deoxythymidine caps removed after >8 hours followed by rapid metabolism, with no evidence of metabolism initiated by endonucleolytic cleavage of Compound 2. In contrast, in both rabbit and human serum, metabolism of Compound 2 was initiated by endonucleolytic cleavage, with cleavage after the deoxyadenosine at position 25, which was a joining region proximal to a loop formed by C26, C27, and A28, as the major initial endonuclease cleavage site. [00141] The metabolic stability of Compound 3 is shown in Table 4, and in FIG. 8. Compound 3 was an aptamer which was optimized for stability in serum and tissues by use of T - fluoropyrimidines in the selection process with post-hoc selective replacement of 2’-ribopurines e.g ., natural RNA) with T -O-methyl purines, wherever tolerated (Ruckman J., Green L.S., Beeson J., Waugh S., Gillette W.L. Henninger D.D., Claesson-Welsh L., and Janjic N. 1998. 2’Fluoropyrimidine RNA-based Aptamers to the l65-Amino Acid Form of Vascular Endothelial Growth Factor (VEGF165): Inhibition of Receptor Binding and VEGF-induced Vascular Permeability Through Interactions Requiring Exon-7-encoded Domain. J. Biol. Chem. 273: 20556-20567). Additionally, it contained a 3’-inverted deoxythymidine cap and a 5’-hexylamino linker, modifications widely used to reduce the rate of exonuclease loading and subsequent metabolism of oligonucleotides.

Table 4. Metabolic stability of Compound 3 in human serum, rabbit serum, and rabbit vitreous

[00142] The metabolic stability of Compound 3 was unanticipated, with metabolism occurring substantially more rapidly in rabbit vitreous than in rabbit serum or human serum. Examination of the metabolic profiles of Compound 3 in the respective matrices showed unanticipated differences between the serum and vitreous. In the vitreous matrix, metabolism of Compound 3 was initiated by endonucleolytic cleavage following T -fluoropyrimidines in double-stranded regions of the aptamer, in particular, the 2’-fluorocytidine at position 26. Additional minor endonucleolytic cleavages occurred following the 2’-fluorouridine at position 29, which was also in a double-stranded region of the aptamer, and after the 2’-fluorouridine at position 7. This observation was consistent with the unexpected metabolic processing of Compound 1 in the vitreous, where metabolism was initiated by endonucleolytic cleavage after 2^,-fluoropyrimidine residues, largely in double-stranded regions of the aptamer. Endonucleolytic cleavage was coupled with rapid exonuclease cleavage. In contrast, in serum matrices, metabolism of Compound 3 followed the anticipated metabolic process, with metabolism initiated by exonuclease degradation from both the 3’ and 5’ ends, indicating that the T sugar modifications within the backbone of Compound 3 reduced the rate of endonuclease degradation in the serum, such that exonuclease degradation was the dominant metabolic pathway.

[00143] The metabolic stability of Compound 4 is shown in Table 5 and in FIG. 9. Compound 4 had the same nucleotide sequence as Compound 3. It represented the 2’-hydroxypurine, T - fluoropyrimidine version of this molecule prior to further post-hoc optimization of Compound 4 for serum stability (Ruckman J., Green L.S., Beeson J., Waugh S., Gillette W.L. Henninger D.D., Claesson-Welsh L., and Janjic N. 1998. 2’Fluoropyrimidine RNA-based Aptamers to the l65-Amino Acid Form of Vascular Endothelial Growth Factor (VEGF165): Inhibition of Receptor Binding and VEGF-induced Vascular Permeability Through Interactions Requiring Exon-7-encoded Domain. J. Biol. Chem. 273: 20556-20567). Compound 4 contained the same exonuclease blocking chemistries as Compound 3 but lacked modifications of 2’-hydroxypurine residues to reduce endonucleolytic metabolism.

Table 5. Metabolic stability of Compound 4 in human serum, rabbit serum, and rabbit vitreous

[00144] The metabolic stability of Compound 4 was interesting. It showed the expected trend of greater stability in human serum than rabbit serum. However, rather than following the expected trend of increased metabolic stability in rabbit vitreous as compared to rabbit serum, the metabolic stability of Compound 4 was comparable in rabbit vitreous and rabbit serum.

Ribonucleotides, especially in single-stranded regions, are highly labile in serum and tissue matrices (Beigelman L., McSwiggen J.A., Draper K.G., Gonzalez C., Jensen K., Karpeisky A.M., Modak A.S., Matulic-Adamic J., DiRenzo A.B., Haeberli P., Sweedler D., Tracz D.,

Grimm, S., Wincott F.E., Thackray V.V. and Usman N. 1995. Chemical Modification of Hammerhead Ribozymes. J. Biol. Chem. 270: 25702-25708), and it was thus anticipated that metabolism of Compound 4 would initiate and be driven by endonucleolytic cleavage following ribopurines. However, examination of the metabolic profile of Compound 4 in the respective matrices provided unanticipated results. In serum matrices, metabolism of Compound 4 was initiated as expected by endonucleolytic cleavage following ribopurines, including following adenosines in single-stranded regions of the aptamer at positions 14, 22, 24, as well as others. In contrast, in vitreous, metabolism of Compound 4 was initiated by endonucleolytic cleavage following 2^,-fluoropyrimidines, including after 2’-fluorocytidine in single-stranded regions including at positions 8 and 23, as well as 2’-fluorouridine in double-stranded regions including at position 25.

[00145] The metabolic stability of Compound 6 is shown in Table 6. Compound 6 was similar to Compound 2; however, Compound 6 contained the internal hexaethylene glycol spacers of Compound 1. Compound 6 showed higher stability in the rabbit vitreous matrix than the rabbit serum matrix, however, it was degraded in both solutions by 24 hours.

Table 6. Metabolic stability of Compound 6 in human serum, rabbit serum, and rabbit vitreous

[00146] The metabolic stability of Compound 7 is shown in

7. Compound 7 was the same nucleotide sequence as Compound 3; Compound 7, however, lacked the 3’ end cap. Compound

7 showed slightly higher stability in the rabbit vitreous matrix than in the rabbit serum matrix at early time points, however, degraded to less than 10% in both matrices by 24 hours.

Table 7. Metabolic stability of Compound 7 in human serum, rabbit serum, and rabbit vitreous

[00147] In summary, this data showed that metabolism of oligonucleotides in the vitreous occurred by a different process than observed in serum and tissues. In particular, stabilization chemistries developed to reduce endonucleolytic cleavage of oligonucleotides in serum and tissues, such as 2’-fluoropyrimidine substitutions, did not appear to confer substantial protection from metabolism to oligonucleotides in the vitreous. Surprisingly, in these sets of experiments, 2’-fluoropyrimidines were frequently sites at which metabolism was initiated by endonuclease cleavage, even in the presence of chemistries considered labile, such as unmodified DNA and RNA residues, and when these residues were within double-stranded regions. These experiments were directed at identifying oligonucleotide compositions that exhibited enhanced stability in vitreous as compared to serum matrices. In this initial set of compounds, all DNA aptamers with end-capping modifications were unexpectedly found to be promising compositions, with substantially greater stability in vitreous as compared to serum. Given that metabolism of all DNA aptamers with end-capping modifications in the serum was initiated by endonucleolytic cleavage and in the vitreous by exonucleolytic cleavage, it is anticipated that further modification of such compositions to reduce the rate of exonucleolytic metabolism may provide for compositions with greater metabolic stability in the vitreous without enhancing serum or tissue stability.

Example 2. Further stability studies

[00148] Additional constructs are designed around well-characterized aptamers and include only modifications known to maintain active secondary structure with retention of target affinity and pharmacologic activity. The constructs include: (1) anti-thrombin aptamers (see Table 8); (2) anti-PDGF aptamers (see Table 9) with the indicated modifications; and (3) anti-VEGF aptamers (see Table 10) with the indicated modifications. The anti-PDGF and anti-VEGF aptamers of Table 9 and Table 10 are also depicted in FIGS. 10-13. Stock solutions of aptamers from

Tables 8-10 are assayed for stability in different matrices as in Example 1 in order to analyze aptamer stability in serum and vitreous conditions.

Table 8. Anti-thrombin aptamer constructs

Table 9. Anti-PDGF aptamer constructs

Table 10. Anti-VEGF Aptamer Constructs

Example 3. Analysis of Aptamer Retained Activity in Serum and Vitreous Conditions

[00149] Aliquots from VEGF aptamer samples described in Example 1 and Example 2 are used in parallel in order to analyze retained activity of the aptamers under serum and vitreous conditions. A phospho-VEGFR2 capture enzyme-linked immunosorbent assay (ELISA) is performed on each sample. Briefly, various concentrations of the aptamers are mixed with human VEGF121 in DMEM 0.2% FBS before being added to serum-starved 293/VEGFR- 2/KDR cells. Cells are then washed and lysed. Phosphorylation of VEGFR-2/KDR at tyrosine residues is then quantified using a capture ELISA assay. Aptamers with high retention of activity in vitreous matrices are of particular interest. For all aptamers retained activity levels are found to match levels expected based on stability.

Example 4: In vivo stability of aptamers

[00150] The aptamers identified in Example 1 and Example 2 are further studied in vivo. The aptamers are injected into Sprague Dawley rats by either intravenous injection or intravitreal injection. Blood and vitreous fluid samples are taken at 5 time points (0, 2, 4, 8, and 24 hours). Blood samples are collected in EDTA-coated tubes containing nuclease inhibitors. Blood samples are briefly centrifuged at low speed to clear blood cells from serum. Serum and vitreous samples are analyzed as in Example 1 and Example 3 for aptamer stability and retained activity.

Example 5: Considerations for Anti-VEGF Therapy for retinopathy of prematurity (ROP)

[00151] To achieve a suitable anti-VEGF therapy for ROP, which maintains efficacy for 4 weeks following a single IVT injection without exceeding the IC_l0 in serum, there is a fairly large design space (formulation, half-life, potency) that could provide a suitable compound.

Compounds with an IC₉₀ of about 50 nM may provide the simplest path to a suitable compound, as they would meet efficacy and serum IC_l0 criteria while remaining within formulation and half- life specifications that are readily deliverable without substantial experimentation or new technology solutions.

[00152] An in silico model was developed to determine the effects of different anti-VEGF aptamer concentrations, potencies, and half-lives on the efficacy of VEGF inhibition in both vitreous and serum. Conditions which will maintain a vitreous concentration higher than the IC₉₀ for at least 4 weeks, without ever exceeding a serum concentration of IC_l0, may be preferred.

[00153] VEGF protein was assayed in both vitreous and serum of subjects with retinopathy of prematurity, and concentrations used in the model were as follows: vitreous concentrations of 360-8,882 pg/ml which equated to 9-200 pM; and serum concentrations of 11-135 pg/ml which equated to 0.3-3 pM. For the purpose of the model, it was assumed that the fraction of VEGF bound by the anti-VEGF aptamer was the percentage VEGF inhibition. It was further assumed that the maximum efficacy in the vitreous required the concentration of aptamer to be at or higher than the IC₉₀, and that avoiding systemic effects required a serum concentration below the IC_10.

[00154] The aptamer was assumed to be formulated at 30 mg/ml or 60 mg/ml oligo weight. The injection volume was assumed to be 25 pL, which resulted in a dose of 0.75 mg/eye or 1.5 mg/eye, depending on the concentration. The molecular weight of the anti-VEGF aptamer was assumed to be 12 kDa; therefore, the maximum dose at 0.75 mg/eye is 62.5 nmoles and at 1.5 mg/eye is 125 nmoles.

[00155] To calculate the vitreal concentration, the volume of the vitreous was assumed to be 4 ml. Given that assumption, the maximum concentration (Cmax) at a dose of 0.75 mg/eye was -15 mM and the Cmax at a dose of 1.5 mg/eye was -30 mM. The model was run using half-lives of 4, 7, and 10 days and assuming the aptamer was effective between concentrations of Cmax to the IC₉₀ concentration.

Table 11. VEGF binding in vitreous vs. aptamer affinity

[00156] Given the low VEGF concentrations in the vitreous, target occupancy is driven by the concentration of aptamer, as shown in Table 11. The fraction of VEGF bound is insensitive to VEGF concentration in the range of VEGF concentrations found in babies with ROP.

[00157] The duration of efficacy vs. aptamer affinity was modeled using a one-phase exponential decay model. Dashed lines in FIG. 14 and FIG. 15 represent the IC₉₀ concentration when the aptamer I is 5 nM (50 nM), 50 nM (500 nM) and 100 nM (1 mM). The data is also shown in Table 12 and Table 13

Table 12. Aptamer concentration with time (0.75 mg/eye)

Table 13. Aptamer concentration with time (1.5 mg/eye)

[00158] As shown in FIG. 14 and Table 12, if the aptamer potency is - 5 nM, about a four week duration of efficacy can be achieved with a half-life of 4 days, or greater, at a dose of 0.75 mg/eye. If the aptamer potency is about 50 nM, then about four weeks duration of efficacy can be achieved with a half-life of 7 days or greater at a dose of 0.75 mg/eye. Near 4 weeks duration of efficacy can be achieved with a half-life of 4 days at a dose of 1.5 mg/eye (see FIG. 15 and Table 13) while with a potency of 100 nM, near 4 weeks duration of efficacy can be achieved with a half-life of 7 days at 0.75 mg/eye and is well covered if the half-life is 10 days. Increasing the dose to 1.5 mg/eye provides clear potential for a 4-week duration of efficacy with a half-life of 7 days or greater.

Table 14. VEGF binding in serum vs. aptamer affinity

[00159] Similar to the vitreous, serum target occupancy is driven by the concentration of aptamer. Aptamer to VEGF binding was only modeled for a VEGF concentration of 3 pM as the fraction of VEGF bound is insensitive to the concentration of VEGF over the range of 0.3-3 pM. In the Avery study, the serum C_max for an anti-VEGF aptamer was 0.52 nM after delivery of 0.3 mg/eye in adults (Avery RL, Castellarin AA, Steinle NC, et al. Br J Ophthalmol Published online first: doi: 10.1 l36/bjophthalmol-20l4-305252). In a rabbit PK study, where rabbit systemic volume of distribution is likely comparable to an infant (based on similar body weight), serum C_max ranged from 1 to 6 nM in rabbits administered 1.5 mg of aptamer to an eye. In summary, a potency (K_d) of 5 nM provides for 4 weeks of efficacy over a range of anticipated IVT half-lives at 0.75 mg/eye, but raises potential concerns regarding the risk of exceeding the serum ICi₀ of 0.5 nM. A potency this high may require engineering of the aptamer for enhanced serum clearance to limit C_max achieved, for example, by introducing modifications to enhance metabolism of the aptamer in the serum.

[00160] A potency (K_d) of the anti-VEGF aptamer of 50 nM has the potential to provide for 4 weeks of efficacy if the half-life is 7 days or greater following IVT administration at 0.75 mg/eye. A potency in this range appears to provide a good safety option with minimal engineering given that the ICio in serum would be 5 nM. Given that the aptamer will sustain considerable dilution in the serum versus its concentration in the eye, it should be relatively easy to maintain the serum aptamer concentration at a level less than 5 nM.

[00161] A potency of 100 nM of the anti-VEGF aptamer has the potential to provide 4 weeks of efficacy only if the half-life following IVT injection is at least 10 days or if the dose can be increased to 1.5 mg/eye. The safety profile of a molecule in this potency range would be expected to be very good and readily achievable.

[00162] A potency centered around an IC₉₀ of 50 nM (25 to 50 nM) appears to provide a path to achieving efficacy for 4 weeks following a single IVT injection while also providing the desired safety profile. Molecules of higher potency (IC₉₀ of 5 nM or less) may meet suitable efficacy parameters over a large range of IVT half-life, but would require further engineering to minimize serum exposure to keep the serum concentration below the IC_10. Alternatively, with an IC₉₀ of 5 nM, there could be substantial room to lower the dose, which may reduce serum exposure while still maintaining efficacy for at least 4 weeks. Lower potency molecules in the 100 nM IC₉₀ range could be suitable, but would require formulation at high concentration and maintenance of a half-life in the 7 to 10 day range.

Example 6: Treatment of Retinopathy of Prematurity

[00163] In this example, three neonates are diagnosed with Retinopathy of Prematurity (ROP). The first neonate is treated with 0.75 mg/eye of an anti-VEGF aptamer by intravitreal administration. The K_d of the anti-VEGF aptamer is 50 nM, and the ocular half-life is 7 days or greater. The second neonate is treated with 1.5 mg/eye of an anti-VEGF aptamer by intravitreal administration. The K_d of the anti-VEGF aptamer is 50 nM, and the ocular half-life is 4 days or greater. The third neonate is treated with 1.5 mg/eye of an anti-VEGF aptamer by intravitreal administration. The K of the anti-VEGF aptamer is 100 nM, and the ocular half-life is 7 days or greater. The serum concentration of the anti-VEGF aptamer in the first two neonates remains below 5 nM, which is the ICio of the first and second compounds in serum. The serum concentration of the anti-VEGF aptamer in the third neonate remains below 10 nM, which is the IC io of the third compound in serum. The patients are treated once every 4 weeks. After six months of treatment, one year of treatment, and every six months thereafter, the patients are assessed for stabilization of ROP and for adverse side effects. Examples of side effects which may be assessed include mental and psychomotor impairment deficits as defined in Lien et al. (Lien R, Yu M-H, Hsu K-H, Liao P-J, Chen Y-P, Lai C-C, et al. 2016. Neurodevelopmental Outcomes in Infants with ROP and Bevacizumab Treatment PLoS ONE 11(1): e0l480l9.). As a result of the treatment, all three patients experience few of the side effects normally associated with anti-VEGF ocular therapy.

Example 7. Improvement of metabolic stability in vitreous

[00164] The methods provided in Example 1 were used to improve the metabolic stability of a specific aptamer in the vitreous to provide an improved half-life following intravitreal administration. Aptamer 15 (FIG. 16) was an aptamer against complement factor D (fD). It was composed of 2’-0-methyl A, C, and U, and 2’-fluoroguanosine residues, and included a 5’- hexylamino linker and a 3’-inverted deoxythymidine as exonuclease caps (represented by L and X, respectively, in FIG. 16). Aptamer 15 was subjected to metabolic profiling as described in Example 1. Surprisingly, and consistent with data presented in Example 1 and Example 2, the primary site of metabolism in rabbit vitreous was an endonuclease cleavage between the T - fluoroguanosine residues at positions 36 and 37 in the terminal stem of Aptamer 15. Notably, the primary site of metabolism was associated with the presence of at least two contiguous T - fluoro residues present in a stem, similar to the primary metabolism observed for Compound 3 in vitreous in Example 1. To potentially improve the metabolic stability of Aptamer 15 in the vitreous, 2’-0-methylguanosines were substituted for the 2’-fluoroguanosines in the terminal stem of Aptamer 15 to yield Aptamer 74 (FIG. 16). The substitution of 2’-0-methylguanosine for 2’-fluoroguanosine at these positions was well tolerated with a minimal impact on the potency of Aptamer 74 as compared to Aptamer 15 (see Table 17).

Table 15. Aptamer sequences

[00165] To compare the metabolic stability of Aptamers 15 and 74, in vitro metabolic stability experiments were conducted as described in Example 1 with minor modifications, as follows. First, Aptamers 15 and 74 were site specifically labeled on the hexylamino linker with the fluorophore AlexaFluor^® 647 to enable analysis and quantification of intact aptamer using polyacrylamide gel electrophoresis. Second, samples were incubated in rabbit vitreous at a 0.5 mM final aptamer concentration at 37°C, and following incubation for 0, 24, 48, or 72 hours, reactions were terminated by the addition of a lOx volume of a solution consisting of 95% formamide, 1% SDS and were immediately frozen. Finally, separation of full-length aptamer from metabolites was achieved by 15% polyacrylamide-urea denaturing gel electrophoresis, and quantification of full-length aptamer over time was performed using a Li-COR gel imager and manufacturers’ software. To estimate the metabolic stability for each aptamer, the fraction of full-length aptamer remaining at 24, 48, and 72 hours was plotted as compared to the starting material (time 0), and the points fit using a single-phase exponential decay to determine the in vitro half-life in rabbit vitreous.

[00166] The results of this experiment are provided in Table 16. Substitution of 2’-0- methylguanosine for 2’-fluoroguanosine at sites of primary metabolism in the terminal stem of Aptamer 15 led to a substantial increase in metabolic stability as demonstrated by the greater half-life of Aptamer 74 as compared to Aptamer 15 in rabbit vitreous. These results provided direct data that an increase in 2’-0-methyl content and reduction in 2’-fluoro content improved the metabolic stability of aptamers in the vitreous. It was also notable that Aptamer 15, which contained a reduced level of 2’-fluoro residues as compared to Compound 3, exhibited greater stability than Compound 3 in rabbit vitreous (see Table 4). Further, these data demonstrated the utility of metabolic profiling in the vitreous to identify primary sites of metabolism to enable site-specific substitution to reduce the rate of aptamer metabolism in the target tissue.

Table 16. Metabolic stability of Aptamer 15 vs. Aptamer 74 in rabbit vitreous

Example 8: Improvement of metabolic stability in vitreous provides for a greater half-life following intravitreal administration in rabbits

[00167] Prior to evaluation in vivo , Aptamers 15 and 74 were conjugated to a 40 kDa branched PEG to reduce their rate of distribution from the vitreous, so as to better evaluate the impact of metabolic stabilization on the rate of clearance following intravitreal administration. Briefly, a concentrated feed solution consisting of aptamer in DMSO, 16 to 25 mM borate and water was combined with a solution consisting of several equivalents 2,3-Bis(methylpolyoxyethylene-oxy)- l-{3-[(l,5-dioxo-5-succinimidyloxy, pentyl)amino]propyloxy} propane (for example,

SUNBRIGHT® GL2-400GS2) in acetonitrile, and incubated at approximately 35°C for approximately 1 hour with mixing to effect conjugation of the PEG to the amine moiety of the hexyl amine linker present on the 5' terminus of the aptamer. Following the pegylation reaction, each PEG-aptamer was purified by anion exchange chromatography to collect the pegylated aptamer and remove unreacted PEG and unreacted aptamer. Anion exchange purified PEG- aptamers were desalted by ultrafiltration into water prior to functional characterization. The pegylated versions of Aptamers 15 and 74 were termed aptamers P01 and P04, respectively.

[00168] The potency of the PEG-aptamers P01 and P04 compared to their non-pegylated counterparts was determined in an alternative complement-dependent hemolysis assay. As shown in Table 17, these aptamers tolerated pegylation well, with each PEG-aptamer exhibiting a modest increase in potency compared to its non-pegylated counterpart as determined by their respective IC50 values in this assay.

Table 17. IC₅₀ values for select PEG and non-PEG aptamers

[00169] The impact of increased metabolic stability on clearance following intravitreal administration was determined by measuring the half-life of Aptamers P01 and P04 following intravitreal administration to rabbits. For evaluation of Aptamer P01, sixteen New Zealand White rabbits, two rabbits per timepoint (four eyes), were treated with 1.5 mg/eye of aptamer P01 administered by intravitreal injection. Vitreous and plasma samples were taken at 1, 8, 24, 48, 96, 168, 240, and 336 hours post-Aptamer P01 administration with individual samples being obtained from the left and right eye of each animal at each timepoint. Vitreous and plasma samples were also obtained from two placebo treated animals to serve as controls for sample analysis. The concentration of Aptamer P01 was measured in the vitreous and plasma over time following administration using a dual hybridization ELISA assay. For evaluation of Aptamer P04, sixteen New Zealand White rabbits, one to two rabbits per timepoint (two to four eyes), were treated with 3.0 mg/eye of Aptamer P04 administered by intravitreal injection. Vitreous and plasma samples were taken at 1, 8, 24, 96, 168, 336, 672, 1008, and 1440 hours post- Aptamer P04 administration with individual samples being obtained from the left and right eye of each animal at each timepoint. The study duration for Aptamer P04 was increased due to the expected longer half-life of P04 as compared to P01. Vitreous and plasma samples were also obtained from two placebo treated animals to serve as controls for sample analysis. The concentration of Aptamer P04 was measured in the vitreous and plasma over time following administration using an anion exchange HPLC assay.

[00170] Vitreous Aptamer P01 and P04 were distributed following a single IVT injection and their concentrations declined over time. The maximum concentration observed post

administration for Aptamer P01 was approximately 1,018 pg/mL, or approximately 76 mM based on aptamer molecular weight, within 1 hour of dosing (first sampling time point). At day 14, the vitreous Aptamer P01 concentration was approximately 62 pg/mL, or approximately 4.6 pM based on aptamer molecular weight. The maximum concentration observed post administration for Aptamer P04 was approximately 1,678 pg/mL, or approximately 124.7 pM based on aptamer molecular weight, within 24 hours of dosing. At day 60, the vitreous Aptamer P01 concentration was approximately 0.4 pg/mL, or approximately 26 nM based on aptamer molecular weight. Vitreous PK parameters for Aptamers P01 and P04 as determined by non-compartmental analysis are provided in Table 18. The estimated vitreous half-life of Aptamer P01 was approximately 117 hours, or 4.7 days. By comparison, the estimated vitreous half-life of Aptamer P04 was substantially longer at 177 hours, or 7.4 days. The improvement in intravitreal half-life observed for Aptamer P04 compared to Aptamer P01 demonstrated that increasing the metabolic stability of an aptamer by judicious choice of T sugar modification chemistry lead to reduced clearance from the vitreous as evidenced by the increased intravitreal half-life of Aptamer P04.

Table 18. Estimated vitreous PK parameters following IVT administration for Aptamers P01 and P04

[00171] By comparison, the pegylated aptamer Macugen^®, which has been well-studied following IVT administration in animals and humans, has a vitreous half-life in rabbits of approximately 80 hours, or 3.3 days, and a vitreous half-life in humans of approximately 10 days (“MACUGEN^®, Drugs at FDA;

https://www.accessdata.fda.gov/drugsatfda_docs/label/20l l/02l756s0l8lbl.pdf ).

[00172] Based on the comparison of the respective vitreous half-life in rabbits of Aptamers P01 or P04 versus Macugen^®, in combination with the vitreous half-life of Macugen^® in humans, the estimated vitreous half-life of Aptamer P01 or P04 in humans following IVT administration would be anticipated to be greater than 10 to about 15 days. In retinal disease states, the target concentrations are typically less than 1 nM and often less than 250 pM. Given the high potency of aptamers such as P01 or P04, a vitreous aptamer concentration of approximately 0.4 nM to 4 nM would be sufficient to provide complete to near complete (approximately 90%) target occupancy or inhibition in the vitreous or retina in a retinal disease state. With a vitreous half- life of 10 to about 15 days, IVT administration of 1 mg (based on aptamer weight) of Aptamer P01 or P04, would provide near complete or complete suppression of target activity for approximately 20 to 25 weeks, or 4-6 months. With these same assumptions, IVT administration of 5 mg (based on aptamer weight) of Aptamer P01 or P04, would provide near complete or complete suppression of IL8 activity for approximately 26 to 38 weeks, or 6-10 months.

Example 9. Relationship between metabolic stability and half-life following intravitreal administration in rabbits

[00173] Prior research has demonstrated a linear relationship between hydrodynamic radius and half-life for intravitreal administration of macromolecules (see, for example, Schatz et al. Mol. Pharmaceutics 13 : 2996-3003, 2016 and US 2017/0096479 Al). One of the findings of note in Schatz et al. was that the pegylated aptamer pegaptanib (MACUGEN^®) demonstrated an unexpectedly short half-life in rabbits, approximately 3.3 days, based on its hydrodynamic radius as compared to biologies such as ranibizumab, albumin, bevacizumab, pegylated Fab fragments or large molecular weight hyaluronic acid. The underlying reason for the unexpectedly short intravitreal half-life of pegaptanib was unclear at the time of the Schatz et al. publication.

Compound 3 in Example 1 is the aptamer portion of pegaptanib, and it is notable that the in vitro metabolic stability of Compound 3 was substantially less than that of Aptamer 15 or Aptamer 74 (half-life in vitro in rabbit vitreous of less than 24 hours compared to approximately 143 and 660 hours, respectively, for Aptamers 15 and 74). Based on the data presented in Example 8, it was hypothesized that the explanation for the unexpectedly short half-life of pegaptanib following intravitreal administration in rabbits was due to its metabolic stability in vitreous, such that its clearance was limited by metabolism in the vitreous as opposed to distribution from the vitreous, which is governed by molecular size. In FIG. 17, the relationship between half-life and hydrodynamic radius from Schatz et al. and US 2017/0096479 Al is reproduced, with the data from Aptamers P01 and P04 overlaid on the graph. As Aptamers P01 and P04 are conjugated to a similar branched 40 kDa PEG as pegaptanib, and the hydrodynamic radius is largely determined by the PEG moiety, the hydrodynamic radius of Aptamers P01 and P04 was assumed to be the same as pegaptanib for this analysis. In FIG. 17, as the metabolic stability of the aptamer in vitreous increased from that of the aptamer moiety of pegaptanib to the aptamer moiety of P01 to the aptamer moiety of P04, the half-life increased and approached, or exceeded the predicted half-life based on the relationship between half-life and molecular size established for biologies. This analysis indicated that improvement of aptamer metabolic stability in vitreous shifted the rate-limiting step in clearance from metabolism to distribution from the vitreous, demonstrating the utility of designing aptamers with improved metabolic stability using the methods and teachings described herein.

[00174] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a pediatric subject with an ocular disease or disorder comprising: delivering an aptamer to the eye of the pediatric subject with the ocular disease or disorder, wherein the pediatric subject is less than 18 years of age, wherein the aptamer exhibits limited systemic exposure and wherein the aptamer specifically binds to a therapeutic target in the eye of the subject.

2. The method of claim 1, wherein the aptamer exhibits higher stability in a vitreous matrix than in a blood matrix.

3. The method of claim 1 or 2, wherein the aptamer exhibits higher stability in a vitreous matrix than in a non-ocular tissue matrix.

4. The method of any one of claims 1-3, wherein the modified aptamer is delivered via an intraocular or intravitreal administration.

5. The method of any one of claims 1-4, wherein the subject is less than 10 years of age.

6. The method of any one of claims 1-5, wherein the subject is less than 36 months of age.

7. The method of any one of claims 1-6, wherein the subject is an infant who was born

prematurely.

8. The method of claim 7, wherein the infant was born at less than 37 weeks of gestational age.

9. The method of any one of claims 1-8, wherein the modified aptamer exhibits a greater than two-fold higher stability in the vitreous matrix compared with the stability in the blood matrix.

10. The method of any one of claims 1-9, wherein the modified aptamer exhibits a greater than two-fold higher stability in the vitreous matrix compared with the stability in the non ocular tissue matrix.

11. The method of any one of claims 1-10, wherein the aptamer is associated with fewer systemic adverse reactions than an aptamer optimized for vitreous stability or blood stability.

12. The method of any one of claims 1-11, wherein the aptamer is associated with fewer systemic adverse reactions than an aptamer optimized for non-ocular tissue stability.

13. The method of any one of claims 1-12, wherein the aptamer targets vascular endothelial growth factor (VEGF).

14. The method of any one of claims 1-13, wherein the aptamer targets platelet-derived

growth factor (PDGF).

15. The method of any one of claims 1-14, wherein the aptamer comprises an end cap on the 3’ end.

16. The method of any one of claims 1-15, wherein the aptamer comprises a linker on the 5’ end.

17. The method of any one of claims 1-16, wherein the aptamer comprises fewer than five 2’- fluoropyrimidines.

18. The method of any one of claims 1-17, wherein the aptamer is at least about 80%

identical to Compound 2.

19. The method of any one of claims 1-18, wherein the aptamer has at least about 80%

sequence identity to SEQ ID NO: 3.

20 The method of any one of claims 1-19, wherein the limited systemic exposure is a

systemic concentration of less than the IC_l0 concentration of the aptamer for the therapeutic target.

21 The method of any one of claims 1-20, wherein the aptamer has a K_d of at least 20 nM.

22 The method of any one of claims 1-21, wherein the aptamer has a half-life of at least about 4 days in vitreous, at least about 2 days in blood or non-ocular tissue, or both.

23. The method of any one of claims 1-22, wherein the aptamer is administered at a dose of at least about 0.5 mg/eye.

24. The method of claim 23, wherein an ocular concentration of the aptamer remains above the IC90 concentration of the aptamer for the target for at least about 4 weeks.

25. The method of any one of claims 1-24, wherein a tissue concentration of the aptamer is less than the IC_l0 of the aptamer for the therapeutic target.

26. The method of claim 25, wherein the tissue concentration of the aptamer is measured in a tissue selected from the group consisting of: liver, kidney, gut, lungs, heart, brain, central nervous system, skin, connective tissue, and muscle.

27. The method of any one of claims 1-24, wherein serum concentration of the aptamer is less than the IC_l0 of the aptamer for the therapeutic target.

28. The method of any one of claims 1-27, wherein the aptamer is administered no more than once every four weeks.

29. A method of treating a pediatric ocular disease, the method comprising, delivering an

aptamer comprising a nucleic acid sequence according to SEQ ID NO: 3 to the eye of a pediatric subject, wherein the aptamer does not contain any 2^,-fluoropyrimidines, thereby treating the pediatric ocular disease.

30 A method of treating a pediatric ocular disease, the method comprising, delivering an

aptamer to the eye of a pediatric subject, wherein the aptamer binds to vascular endothelial growth factor (VEGF), and wherein the aptamer does not contain any 2’- fluoropyrimidines, thereby treating the pediatric ocular disease.

31. A method for selecting an aptamer with improved stability in a vitreous matrix compared to a blood or non-ocular tissue matrix, comprising:

a. obtaining a panel of aptamers comprising unique patterns of modifications,

wherein the aptamers comprise a base sequence known to bind a therapeutic target; b. incubating the aptamers comprising the unique patterns of modifications in a

vitreous matrix;

c. measuring the stability of the aptamers in the vitreous matrix; and

d. identifying the unique patterns of modifications that result in greater vitreous

matrix stability.

32. The method of claim 31, wherein the vitreous matrix is vitreous fluid or is designed to resemble vitreous fluid.

33. The method of claim 31 or 32, further comprising incubating the aptamers comprising the unique patterns of modifications in a blood matrix or non-ocular tissue matrix and measuring the stability of the aptamers in the blood matrix or non-ocular tissue matrix.

34. The method of any one of claims 31-33, wherein the stability is determined by measuring the half-life of the aptamer in the vitreous matrix, the blood matrix, or the non-ocular tissue matrix.

35. The method of any one of claims 31-34, wherein the stability is measured at a

temperature of 37°C.

36. The method of claim 33, wherein the blood matrix comprises serum extracted from whole blood.

37. The method of claim 33, further comprising selecting the modified aptamer when the vitreous-matrix-stability to blood-matrix-stability ratio is greater than 5: 1.

38. The method of claim 33, further comprising testing the modified aptamer for therapeutic efficacy when the vitreous matrix stability to blood matrix ratio is greater than 5: 1.

39. The method of claim 33, further comprising selecting the modified aptamer when the

vitreous-matrix-stability to non-ocular tissue matrix stability ratio is greater than 5: 1.

40. The method of claim 33, further comprising testing the modified aptamer for therapeutic efficacy when the vitreous matrix stability to non-ocular tissue matrix ratio is greater than 5: 1.

41. The method of any one of claims 31-40, wherein the pattern of modifications includes one or more modifications selected from the group consisting of: 2’F, 2OMe, 2’deoxy, phosphorthioate, phosphoramidate, methyl phosphonate, PEG linker, polyethylene glycol (PEG) linker, PEG spacer, stabilizing end cap, nucleic acid insertion, and truncated motif.

42. The method of any one of claims 31-41, wherein the measuring the stability comprises using an assay to detect fully intact aptamer.

43. The method of any one of claims 31-42, wherein the assay to detect fully intact aptamer is liquid chromatography-mass spectroscopy (LC-MS).

44. A modified aptamer generated using the method of any one of claims 31-43.

45. A pharmaceutical composition suitable for administration to an eye, the pharmaceutical composition comprising an aptamer that specifically binds to a therapeutic target in the eye with a K_d of at least about 50 nM and an intraocular half-life of at least 4 days.

46. The pharmaceutical composition of claim 45, wherein the intraocular half-life of the

aptamer is at least 7 days.

47. The pharmaceutical composition of claim 45 or 46, wherein the aptamer has a K_d value of at least about 100 nM.

48. The pharmaceutical composition of any one of claims 45-47, wherein the aptamer has a serum half-life of less than 4 days.

49. The pharmaceutical composition of any one of claims 45-47, wherein the aptamer has a non-ocular tissue half-life of less than 4 days.

50. The pharmaceutical composition of claim 49, wherein the non-ocular tissue half-life of the aptamer is measured in a tissue selected from the group consisting of: liver, kidney, gut, lungs, heart, brain, central nervous system, skin, connective tissue, and muscle.

51. The pharmaceutical composition of any one of claims 45-50, wherein the pharmaceutical composition is formulated to have a unit dose of about 0.75 mg.

52. The pharmaceutical composition of any one of claims 45-50, wherein the pharmaceutical composition is formulated to have a unit dose of about 1.5 mg.

53. The pharmaceutical composition of any one of claims 45-52, wherein ocular

concentration of the aptamer remains above the IC₉₀ concentration of the aptamer for the therapeutic target for at least about 4 weeks.

54. The pharmaceutical composition of any one of claims 45-53, wherein the serum

concentration of the aptamer is less than the ICio of the aptamer for the therapeutic target.

55. An aptamer that specifically binds to a therapeutic target in an eye, wherein the aptamer does not attain a serum concentration above the aptamer’ s ICio for the therapeutic target beyond 96 hours following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’ s IC₉₀ for the therapeutic target_.

56. The aptamer of claim 55, wherein the aptamer does not attain a non-ocular tissue concentration above the aptamer’ s IC_l0 for the therapeutic target at any time following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’ s IC₉₀ for the therapeutic target.

57. The aptamer of claim 56, wherein the non-ocular tissue concentration of the aptamer is measured in a tissue selected from the group consisting of: liver, kidney, gut, lungs, heart, brain, central nervous system, skin, connective tissue, and muscle.

58. The aptamer of either one of claims 55 or 56, wherein the aptamer maintains an ocular concentration above the aptamer’ s IC₉₀ for the therapeutic target for at least 2 weeks following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’ s IC₉₀ for the therapeutic target_.

59. The aptamer of either one of claims 55 or 58, wherein the aptamer does not attain a serum or non-ocular tissue concentration above the aptamer’ s IC_l0 for the therapeutic target at any time following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’ s IC₉₀ for the therapeutic target.

60. The aptamer of any one of claims 55, 58, or 59, wherein the aptamer maintains an ocular concentration above the aptamer’ s IC90 for the therapeutic target for at least 4 weeks following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’ s IC₉₀ for the therapeutic target_.

61. The aptamer of any one of claims 55-60, wherein the ocular concentration exceeding the aptamer’ s IC₉₀ for the therapeutic target is less than lOO-fold higher than the aptamer’ s IC₉₀ for the therapeutic target.

62. A pharmaceutical formulation comprising the aptamer of any one of claims 55-61, the pharmaceutical formulation being suitable for administration to the eye.

63. A method of treating a subject with an ocular disease or disorder, the method comprising administering the pharmaceutical formulation of claim 62 to the eye of the subject with the ocular disease or disorder, thereby treating the ocular disease or disorder.

64. A method of treating a subject with an ocular disease or disorder comprising;

a. providing an aptamer that specifically binds to a therapeutic target in an eye at a potency capable of treating the ocular disease or disorder; and

b. administering the aptamer to the eye of the subject with the ocular disease or disorder at a dose sufficient to achieve an ocular concentration above the aptamer’ s IC₉₀ for the therapeutic target and such that the aptamer maintains a serum or non-ocular tissue concentration that does not exceed the aptamer’ s IC_l0 for the therapeutic target beyond 96 hours after the administering of the aptamer to the eye.

65. The method of claim 64, wherein the aptamer maintains a non-ocular tissue concentration that does not exceed the aptamer’ s IC_l0 for the therapeutic target beyond 96 hours after the administering of the aptamer to the eye.

66. The method of claim 64, wherein the non-ocular tissue concentration of the aptamer is measured in a tissue selected from the group consisting of: liver, kidney, gut, lungs, heart, brain, central nervous system, skin, connective tissue, and muscle.

67. The method of claim 64, wherein the ocular concentration of the aptamer is maintained above the aptamer’ s IC₉₀ for the therapeutic target for at least 2 weeks following the administering of the aptamer to the eye of the subject at the dose sufficient to achieve an ocular concentration above the aptamer’ s IC90 for the therapeutic target_.

68. A method of treating a subject with an ocular disease or disorder comprising:

a. providing an aptamer that specifically binds to a therapeutic target in an eye at a potency capable of treating the ocular disease or disorder;

b. administering the aptamer to an eye of the subject with the ocular disease or

disorder at a dose sufficient to achieve an ocular concentration above the aptamer’ s IC₉₀ for the therapeutic target; and

c. maintaining a serum or non-ocular tissue concentration of the aptamer that does not exceed the aptamer’ s IC_l0 for the therapeutic target beyond 96 hours after the administering of the aptamer to the eye of the subject.

69. The method of claim 68, further comprising maintaining an ocular concentration of the aptamer above the aptamer’ s IC₉₀ for the therapeutic target for at least 2 weeks following the administering of the aptamer to the eye of the subject at the dose sufficient to achieve an ocular concentration above the aptamer’ s IC₉o for the therapeutic target

70. The method of any one of claims 63-69, wherein the administering comprises

intravitreally injecting the aptamer into the eye of the subject.

71. The method of any one of claims 63-70, wherein the aptamer does not attain a serum or non-ocular tissue concentration above the aptamer’ s IC_l0 for the therapeutic target at any time within 96 hours following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’ s IC₉₀ for the therapeutic target.

72. The method of any one of claims 63-71, wherein the aptamer does not attain a serum or non-ocular tissue concentration above the aptamer’ s IC_l0 for the therapeutic target at any time following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’ s IC₉₀ for the therapeutic target.

73. The method of any one of claims 63-71, wherein the aptamer maintains an ocular concentration above the aptamer’ s IC₉₀ for the therapeutic target for at least 4 weeks following administration of the aptamer to the eye at an ocular concentration that exceeds the aptamer’ s IC90 for the therapeutic target_.

74. The method of any one of claims 63-73, wherein the ocular concentration exceeding the aptamer’ s IC₉₀ for the therapeutic target is less than lOO-fold higher than the aptamer’ s IC₉₀ for the therapeutic target.

75. The method of any one of claims 63-74, wherein the aptamer is administered as part of a pharmaceutical formulation suitable for treating an eye.

76. The method of any one of claims 1-28, wherein the aptamer has a half-life of at least about 4 days in vitreous, at least about 5 days in vitreous, at least about 6 days in vitreous, or at least about 7 days in vitreous.

77. The method of any one of claims 1-28, wherein the aptamer has a half-life of less than about 6 days in blood or non-ocular tissue, less than about 5 days in blood or non-ocular tissue, less than about 4 days in blood or non-ocular tissue, less than about 3 days in blood or non-ocular tissue, or less than about 2 days in blood or non-ocular tissue.

78. The method of any one of claims 1-28, wherein the aptamer has a half-life of at least about 4 days in vitreous and less than about 4 days in blood or non-ocular tissue.

79. The method of any one of claims 1-28, or 76-78, wherein the aptamer is administered via a syringe.

80. The method of any one of claims 1-28, or 76-79, wherein the aptamer is administered to a subject less than 18 years of age, less than 12 years of age, less than 10 years of age, or less than 8 years of age, and further comprising detecting the serum blood concentration of the aptamer in the subject.

81. The method of claim 80, wherein the serum blood concentration of the aptamer in the subject is less than about 10 nM.