Truncated tissue plasminogen activator proteins and use thereof

Abstract

Provided herein are truncated tissue plasminogen activator (tPA) variants and vectors (e.g., AAV vectors) encoding the tPA variants. Also provided are pharmaceutical compositions comprising the tPA variants and vectors, and therapeutic uses of the pharmaceutical compositions to treat intraocular pressure (IOP)-associated conditions, such as glaucoma.

Description

TRUNCATED TISSUE PLASMINOGEN ACTIVATOR PROTEINS AND USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Provisional Patent Application No. 63 / 539,251, filed on September 19, 2023, the entireties of which are hereby incorporated herein by this reference.FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to truncated tissue plasminogen activator (tPA) variant proteins and gene therapy vectors encoding such variant proteins useful in treating intraocular pressure (lOP)-associated conditions, such as glaucoma, and use thereof.BACKGROUND

[0003] Glaucoma is a group of eye diseases that cause vision loss and blindness by damaging the optic nerve. High intraocular pressure (IOP) is a major risk factor for glaucoma. Steroid- induced IOP elevation is caused by a decrease in aqueous humor outflow facility. Although the exact mechanism remains unclear, increased extracellular matrix (ECM) deposition in the trabecular meshwork has been detected in both glaucomatous human specimens and animal models of the disease.

[0004] Tissue plasminogen activator (tP A) has been found to reduce steroid-induced IOP elevation in animal models. As components of the fibrinolytic system, tPA and urokinasetype plasminogen activator (uPA, also known simply as urokinase) are two serine proteases that activate plasminogen by proteolytic cleavage. The cleavage converts plasminogen to plasmin, which has the ability to degrade fibrin and dissolve blood clots. tPA is also believed to modulate ECM remodeling and has been implicated in cell proliferation and migration, either through plasmin or independent of it.

[0005] tP A also functions as a cytokine by promoting intracellular signaling cascades and regulating gene expression, following interactions with cell surface receptors, such as low- density-lipoprotein receptor-related protein 1 (LRP-1) and N-methyl-D-aspartate receptor (NMD AR). It has been reported that an S478A substitution, which abrogates the catalytic activity of tPA. does not affect its cytokine activity.

[0006] tPA has been used in the eye for dissolving blood or fibrin clots in the treatment of vitreous hemorrhage and after glaucoma surgery'. Long-term tPA administration is typically not recommended to avoid, among other things, the risk of excessive ocular bleeding / hemorrhage. Despite the efforts that have been made to date, there is still a need for other therapeutic interventions that provide a sustained reduction of IOP without the issues associated with the long term administration of tPA.SUMMARY OF THE DISCLOSURE

[0007] Applicant has previously developed methods of treatment for an intraocular pressure (lOP)-associated condition using variant tPA, see U.S. Patent Nos.: US2021 / 022045 and US2015 / 0366953; and International Application Publication No. WO2014 / 126997, the entire contents of which are incorporated herein by reference. The present disclosure is based upon Applicant’s further development of additional truncated tissue plasminogen activator (tPA) variants useful for reducing intraocular pressure (IOP), and the unexpected effects of various of these different variant tPAs on this endpoint IOP reduction.

[0008] Specifically, as discussed below, Applicant has now developed variant tPA molecules containing alterations of the tPA protein sequence, and particularly variants containing different combinations of the different domains of the tPA protein, as described below. The Examples provided show that these different tPA variants have different and relatively unexpected effects on the model systems provided for assaying IOP.

[0009] These tPA variants that Applicant describes offer a number of advantageous properties as opposed to naturally occurring tPA and previous tPA variants described by Applicant. First, the tPA variants have high cytokine activity, which increases (i.e., upregulate) the expression of matrix metalloproteinases (MMPs) that can improve outflow facility7. Second, the tPA variants lack catalytic activity7, thereby eliminating the risk of excessive ocular bleeding or hemorrhage upon long-term administration. Third, the deletion of one or more domains of tPA, e.g.. the Kringle 1 domain and / or the catalytic domain, reduces the size of the tPA variant thereby enabling the delivery of a nucleic acid encoding the tPA variant by a viral delivery vector having a small upper limit of size, such as a self- complementary7AAV (scAAV) vector. Such delivery methods are particularly suitable for treating chronic ocular diseases.

[0010] Accordingly, in one aspect, the disclosure provides a tPA (e.g, human tPA) variant that comprises a Finger domain and a Kringle 2 domain of tPA, but not a Kringle 1 domain ora functional fragment thereof. The Finger domain can have an amino acid sequence at least 70% (e.g, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 2. The Kringle 2 domain can have an amino acid sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 5. The tPA variant can lack a Kringle 1 domain that has the amino acid sequence of SEQ ID NO: 4. Alternatively, the tPA variant can include an amino acid sequence no greater than 50% (e.g, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) identical to SEQ ID NO: 4.

[0011] A tPA variant of the present disclosure can further lack an active catalytic domain of tPA. For example, the tPA variant can have a deletion or truncation of the catalytic domain, e.g, lacking the amino acid sequence of SEQ ID NO: 6 or including an amino acid sequence no greater than 50% (e.g, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) identical to SEQ ID NO: 6. Alternatively, the tPA variant can have a mutation in an active site of the tPA protease, e.g, substitution of alanine for the serine at position 168 of SEQ ID NO: 6.

[0012] Depending upon the circumstance, a tPA variant can further include an EGF-like domain of tPA. The EGF-like domain can have an amino acid sequence at least 70% (e.g, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 3. As such, the tPA variant includes a Finger domain, a Kringle 2 domain, and an EGF-like domain of tPA. The three domains can be fused such that a C-terminus of the Finger domain is linked, directly or indirectly (e.g.. via a peptide linker, such as the amino acid sequence of Lys-Ser), to an N- terminus of the EGF-like domain, and a C-terminus of the EGF-like domain is linked, directly or indirectly (e.g, via a peptide linker 3-20 amino acids in length, such as a peptide tinker having the amino acid sequence of SEQ ID NO: 7 or 59), to an N-terminus of the Kringle 2 domain.

[0013] A tPA variant of the present disclosure can further include a tail region. For example, the tail region can have an amino acid sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%. at least 90%. at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 8. The tail region can be linked to a C-terminus of the Kringle 2 domain, e.g., by a peptide bond.

[0014] It is further contemplated that, depending upon the circumstances, a tPA variant of the present disclosure can comprise a catalytic domain fragment of tPA. For example, the catalytic domain fragment can have an amino acid sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 9 and include a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 9. The catalytic domain fragment of tPA can be linked at a C-terminus of the tPA variant, e.g., to a C-terminus of the tail region.

[0015] A number of specific tPA variants have been designed and tested that have an amino acid sequence of SEQ ID NO: 10, 11, and 12. Also contemplated are tPA variants having an amino acid sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 10. 1 1. or 12.

[0016] Certain tPA variants of the present disclosure have a shorter length than wild-type tPA. For example, a tPA variant disclosed herein can have a length in the range of 125-400, 125-350, 125-300, 125-250, 125-200, or 125-150 amino acids.

[0017] The present disclosure also provides a vector that includes a nucleic acid sequence encoding a tPA variant disclosed herein. For proper secretion of the tPA variant, the nucleic acid sequence optionally further encodes a signal peptide at an N-terminus of the tPA variant. For example, the tPA variant can have an amino acid sequence at least 70% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 13, which includes the signal peptide and a pro-domain of tPA cleaved from mature tPA. The engineered tPA variants, for example, the variants having amino acid sequences of SEQ ID NOs: 10, 11, and 12, can further include the signal peptide and pro-domain of tPA, resulting in immature protein sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively. Immature tPA variants having an amino acid sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 14. 15. or 16 are also contemplated. The vector can further include a promoter, e.g, a promoter active in trabecular mesh work cells, operably linked to the nucleic acid molecule encoding the tPA variant.

[0018] The vector disclosed herein can be a viral vector, such as an AAV vector. For example, a self-complementary AAV vector can be used for increased transgene expression efficiency. The AAV vector can be pseudotyped with capsid suitable for ocular cell transduction, such as capsid proteins VP1, VP2, and VP3 comprising amino acid sequences at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NOs: 17, 18, and 19, respectively.

[0019] The tPA variant or the vector disclosed herein can be used as a medical treatment or in the preparation of a medicament. For example, the present disclosure provides a method of increasing outflow facility in a subject in need thereof. The method comprises administering to the subject an effective amount of a tPA variant or a vector disclosed herein. Also provided is a method of treating chronic elevated intraocular pressure (e.g., open angle glaucoma or juvenile open angle glaucoma) in a subject in need thereof. The method comprises administering to the subject an effective amount of atPA variant or a vector disclosed herein. Exemplary routes of administration include intracameral injection, intravitreal injection, topical application, via an implantable device, or via implantation of cells that produce the tPA variant.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 illustrates a sequence alignment of human, mouse, and sheep tPA. The area of the sequence for each of the Finger domain. EGF-like domain, Kringle 1 domain, Kringle 2 domain, and catalytic domain are outlined.

[0021] FIG. 2A and 2B illustrate exemplary tPA variant constructs of the present disclosure. FIG. 2A illustrates exemplary ovine tPA variant constructs. FIG. 2B illustrates exemplary human tPA variant constructs. The human tPA variant having the amino acid sequence of SEQ ID NO: 11 includes a longer linker between the EGF-like domain and the Kringle 2 domain. This longer linker differs from the shorter linker in SEQ ID NO: 10 by insertion of an amino acid sequence RATSE (SEQ ID NO: 57).

[0022] FIG. 3 illustrates MMP9 gene expression in human cerebral microvascular endothelial cells transfected with Ovine tP A Variants 1, 2, and 3.

[0023] FIG. 4A and 4B are graphs showing the results of a steroid-induced mouse model of outflow facility reduction. FIG. 4A shows measured IOP in eyes treated with an AAV vector encoding a tPA variant (scAAV-PLATvar) or a negative control(scAAV-GFP). FIG. 4B shows the outflow facility in control (scAAV-GFP) and tPA variant (scAAV-PLATvar) treated eyes.

[0024] FIG. 5 is a line graph showing the measured IOP in a sheep model of steroid- induced IOP increase, where the eyes are treated with an AAV vector encoding a tPA variant (scAAV-PLATvar) or a negative control (scAAV-GFP).DETAILED DESCRIPTION

[0025] The present disclosure is based, in part, upon development of truncated tissue plasminogen activator (tPA) variants useful for reducing intraocular pressure (IOP). The tPA variants disclosed herein have various advantages over wild type tPA by having high cytokine activity, low or no catalytic activity, and / or a reduced size facilitating gene delivery. Various aspects of the disclosure are set forth in the following sections.Definitions

[0026] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below. Unless defined otherwise, all technical and scientific terms have the same meaning as is commonly understood by one of ordinary skill in the art to which the embodiments disclosed belongs.

[0027] The terms "a" and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0028] As used herein, a “mature” tPA sequence includes neither a signal peptide sequence nor a pro-peptide sequence, both of which are known to be cleaved off from naturally occurring tPA protein in extracellular space. As used herein, a “immature” tPA sequence contains a signal peptide sequence, a pro-peptide sequence, or both. Unless explicitly stated to the contrary, if an embodiment references a tPA variant or a tPA sequence, both an immature tPA sequence and a mature tPA sequence are contemplated.

[0029] As used herein, a “functional fragment” of a protein or protein domain refers to any portion of the whole protein or protein domain that substantially retains one or more activities, such as catalytic, binding, or signaling activities, of the reference protein or protein domain. For example, a “functional fragment” of a catalytic domain encompasses any fragment still capable of performing the catalytic activity of the catalytic domain. As another example, where a protein domain binds a protein partner, a “functional fragment” of theprotein domain encompasses any fragment still capable of binding the protein partner. As another example, wherein a protein domain binds a protein partner and the binding to the protein partner initiates a signaling cascade, a '‘functional fragment’’ of the protein domain encompasses any fragment still capable of binding the protein partner and initiating the signaling cascade. As another example, where a protein domain facilitates trafficking of a protein to an intracellular compartment, a “functional fragment’’ of the protein domain encompasses any fragment still capable of facilitating trafficking of the protein to the intracellular compartment.

[0030] As used herein, percent “identity” between a query sequence (e.g, a query amino acid sequence or nucleotide sequence) and a reference sequence is defined as the percentage of amino acid residues or nucleotides in the query7sequence that are identical to the amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as Basic Local Alignment Search Tool (BLAST), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. For example, BLAST analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (see Karlin et al., (1990) PROC. NATL. ACAD. Set. USA 87:2264- 2268; Altschul, (1993) J. MOL. EVOL. 36:290-300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25:3389-3402) are tailored for sequence similarity searching. For a discussion of basic issues in searching sequence databases see Altschul et al., (1994) NATURE GENETICS 6: 119- 129. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0031] As used herein, the term “operably linked” in the context of a transcription control element means that the transcription control element e.g., promoter or enhancer) is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription of the polynucleotide by RNA polymerase.

[0032] As used herein, the term '‘pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0033] As used herein, the terms ‘‘subject'’ and “patient” are used interchangeably and refer to an animal, such as a mammal including, but not limited to, non-primate (e.g, cow, pig, horse, cat, dog, rat, mouse, etc.) and primate (e.g., monkey or human), preferably a human.

[0034] As used herein, the terms “treat,” “treating,” and “treatment,” and other grammatical equivalents as used in this disclosure, refer to clinical intervention to achieve one or more therapeutic benefits. Therapeutic effects of treatment include without limitation, preventing recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. A treatment may eradicate or ameliorate the underlying disorder being treated, or eradicate or ameliorate one or more of the physiological symptoms associated with the underlying disorder.

[0035] As used herein, the terms “therapeutically effective amount” and “effective amount” are used interchangeably to refer to the amount of a compound (e.g, a tPA variant, vector, or pharmaceutical composition of the present disclosure) sufficient to result in treatment of a subject in need thereof, according to the “treatment” as defined above.

[0036] The terms “about” and “approximately” indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. In one non-limiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%. When values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another aspect. It is also understood that there are a number of values disclosed in this disclosure, and that each value is also disclosed as “about” that particular value in addition to the value itself.

[0037] Throughout the description, where compositions are described as having, including, containing, incorporating, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is intended that compositions and methods are inclusive or open-ended and do not exclude additional, unrecited components or steps. It is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited steps.tPA Variants

[0038] tPA is naturally present as a protease that functions to breakdown blood clots. tPA catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for fibrin degradation and consequent clot breakdown. The proteolytic action of tPA is dependent on the presence of serine-478 at its catalytic active site. Change of serine-478 to an alanine (S478A) result in loss of tPA enzy matic activity. It has been found, however, that tPA also functions as a cytokine that binds cell surface receptors low-density -lipoprotein receptor- related protein 1 (LRP-1) and N-methyl-D-aspartate receptor (NMD AR). The S478A mutation does not abrogate the ability of tPA to bind these receptors to stimulate downstream signaling, particularly signaling that results in the increases expression of matrix metalloproteinases (MMPs), such as MMP-9, MMP-2, and MMP-13. This signaling pathway is of particular interest since it has a positive effect on regulation of aqueous humor outflow facility by tPA.General and human tPA variants

[0039] Human tPA is encoded by the human PLAT gene. There are at least three splice variants of human tPA. The sequence of a human tPA isoform 1 is set forth in GenBank Accession No. NM_000930.5. The sequence of a human tPA isoform 3 is set forth in GenBank Accession No. NM_033011.4. The sequence of a human tPA isoform 4 is set forth in GenBank Accession No. NM_001319189.2. tPA variants of these naturally -occurring isoforms can be recombinant, modified, or synthetic and can include derivatives, analogs, and fragments of a tPA amino acid sequence.

[0040] In some embodiments, the amino acid sequence of a wild-type, immature human tPA is the amino acid sequence of UniProt ID P00750-1 :MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKTQMIYQQ HQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALYF SDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSA LAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPA CSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSM1L1GKVYTAQNPSAQAL GLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQF RIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFP PHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSS RCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP (SEQ ID NO: 1)

[0041] In some embodiments, the amino acid sequence of a mature, human tPA is set forth in SEQ ID NO: 32: SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSC SEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGT WSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPW CYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNS MILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYC DVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGI LISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFD DDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGK HEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQA NLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLD WIRDNMRP (SEQ ID NO: 32) wherein SEQ ID NO: 32 is the mature amino acid sequence of SEQ ID NO: 1 where the signal peptide and pro-peptide sequences of SEQ ID NO: 13 have been cleaved.

[0042] A wild-type, immature human tPA (e.g.. having the amino acid sequence of SEQ ID NO: 1) comprises, from N-terminus to C-terminus, a signal peptide, a pro-peptide, a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a catalytic domain. A mature human tPA (e.g., having the amino acid sequence of SEQ ID NO: 32) comprises, from N-terminus to C-terminus, a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a catalytic domain. The mature human tPA can be cleaved, e.g., by plasmin or a kallikrein, into two polypeptide chains. Chain A includes, from N-terminus to C-terminus, the Finger domain, the EGF-like domain, the Kringle 1 domain, and the Kringle 2 domain. Chain B includes the catalytic domain that has protease activity . Exemplary sequences of these domains are provided in Table 1 below. Certain adjacent domains are connected by peptide linkers, which can be readily recognized by aligning the sequences of the domains with SEQ ID NO: 1.Table 1: SEQ ID NO: 1 human tPA domains

[0043] Other investigational molecules exist with similar activity, some of them modified (2nd and 3rd generation) rtPAs, some are molecules that share similarities with tPA but come from other organisms, for example, anistreplase, duteplase, monteplase, lanoteplase, pamiteplase, amediplase, desmoteplase, staphylokinase. snake venom plasminogen activators such as TSV- PA (Trimeresurus stejnegeri venom plasminogen activator), Haly-PA (Agkistrodon halys venom plasminogen activator), LV-PA (Lachesis muta muta venom plasminogen activator), and recombinant chimeric tPAs such as GHRP-SYQ-K2S (whichincludes the tPA kringle 2 domain, K2S, and the tPA serine protease domain, glycyl-histidyl- arginyl-prolyl) and GHRP- scu-PA-32K (glycyl-histidyl-arginyl-prolyl-single-chain urokinase-type plasminogen activator). For a review, see Flemmig and Melzig (Flemmig, M„ etal., (2012) J. PHARM. PHARMACOL. 64(8): 1025-1039).

[0044] In some embodiments, atPA variant of the present disclosure comprises a Finger domain, a Kringle 2 domain, but not a Kringle 1 domain or a functional fragment thereof, of tPA (e.g., human tPA). In some embodiments, the tPA variant comprises a Finger domain, a Kringle 2 domain, but not a Kringle 1 domain of tPA (e.g, human tPA). Also contemplated are tPA variants comprising a functional fragment of a Finger domain, a functional fragment of a Kringle 2 domain, but not a Kringle 1 domain or a functional fragment thereof, of tPA (e.g, human tPA).

[0045] In some embodiments, the tPA variant comprises a Finger domain of human tPA. It is contemplated that the term "Finger domain,” as used herein, includes full-length Finger domains as well as functional fragments thereof that have at least 50%. at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of an activity of a corresponding full-length Finger domain (e.g, a human full-length Finger domain having the amino acid sequence of SEQ ID NO: 2). It is further contemplated that an activity' of the Finger domain or a functional fragment thereof can be measured in an isolated polypeptide consisting of just the full-length Finger domain or the fragment, or can be measured in a larger protein that further incorporates other domains, e.g., a Kringle 2 domain, or both a Kringle 2 domain and an EGF-like domain, or all the other domains and regions of tPA Chain A except a Kringle 1 domain. Without wishing to be bound by theory', it is hypothesized that the Finger domain interacts with LRP-1. Accordingly, in some embodiments, a functional fragment of the Finger domain is a fragment that, when contained in a tPA variant, allows the tPA variant to bind LRP-1, relative to a corresponding tPA variant that does not contain the Finger domain. In some embodiments, the tPA variant comprising a functional fragment of the Finger domain binds LRP-1 at an dissociation constant (KD) level lower than, equal to, or no higher than 10 times of the KD level at which a corresponding tPA variant that comprises a full- length, wild-type Finger domain binds LRP-1, as measured by an in vitro binding assay such as Surface Plasmon Resonance (SPR) assay.

[0046] In some embodiments, the tPA variant comprises a Finger domain of human tPA comprising a Finger domain comprising an amino acid sequence that is at least 70% (e.g, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%) identical to SEQ ID NO: 2. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 2. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 2. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 2. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 2. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 2. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 2. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 2. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 2. In some embodiments, the Finger domain comprises the amino acid sequence of SEQ ID NO: 2.

[0047] In some embodiments, the tPA variant comprises a Kringle 2 domain of human tPA. It is contemplated that the term ‘’Kringle 2 domain,” as used herein, includes full-length Kringle 2 domains as well as functional fragments thereof that have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of an activity of a corresponding full-length Kringle 2 domain (e. , a human full-length Kringle 2 domain having the amino acid sequence of SEQ ID NO: 5). It is further contemplated that an activity of the Kringle 2 domain or a functional fragment thereof can be measured in an isolated polypeptide consisting of just the full-length Kringle 2 domain or the fragment, or can be measured in a larger protein that further incorporates other domains, e.g.. a Finger domain, or both a Finger domain and an EGF-like domain, or all the other domains and regions of tPA Chain A except a Kringle 1 domain. Without wishing to be bound by theory, it is hypothesized that the Kringle 2 domain interacts with LRP1 and / or NMD AR. Accordingly, in some embodiments, a functional fragment of the Kringle 2 domain is a fragment that, when contained in a tPA variant, allows the tPA variant to bind LRP1 and / or NMD AR, relative to a corresponding tPA variant that does not contain the Kringle 2 domain. In some embodiments, the tPA variant comprising a functional fragment of the Kringle 2 domain binds LRP1 and / or NMD AR at an dissociation constant (Ko) level lower than, equal to, or nohigher than 10 times of the KD level at which a corresponding tPA variant that comprises a full-length, wild-type Kringle 2 domain binds LRP1 and / or NMD AR, as measured by an in vitro binding assay such as Surface Plasmon Resonance (SPR) assay.

[0048] In some embodiments, the tPA variant comprises a Kringle 2 domain of human tPA comprising a Kringle 2 domain comprising an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 5. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 5. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 5. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 5. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 5. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 5. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 5. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 5. In some embodiments, the Kringle 2 domain comprises the amino acid sequence of SEQ ID NO: 5.

[0049] In some embodiments, the tPA variant does not comprise a Kringle 1 domain of tPA. It is contemplated that the term ‘’Kringle 1 domain,” as used herein, includes full-length Kringle 1 domains as well as functional fragments thereof that have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of an activity of a corresponding full-length Kringle 1 domain (e.g., a human full-length Finger domain having the amino acid sequence of SEQ ID NO: 4). Such tPA variants can still contain a nonfunctional fragment of a Kringle 1 domain. Without wishing to be bound by theory, the Kringle 1 domain reduces the amount and / or cy tokine activity of tPA in the extracellular space. In some embodiments, the tPA variant comprises a non-functional fragment of the Kringle 1 domain that has no greater than 10%, 20%, 30%, 40%, or 50% of an activity of a corresponding full-length Kringle 1 domain. In some embodiments, a non-functionalfragment of the Kringle 1 domain is a fragment that, when contained in the tPA variant, does not reduce the amount of the tPA variant in the extracellular space by more than 10%, 20%, 30%, 40%, or 50%, relative to a corresponding tPA variant that does not contain the Kringle 1 domain. In some embodiments, a non-functional fragment of the Kringle 1 domain is a fragment that, when contained in the tPA variant, does not reduce the secretion of soluble tPA variant to the extracellular space by more than 10%, 20%, 30%, 40%, or 50%, relative to a corresponding tPA variant that does not contain the Kringle 1 domain. In some embodiments, a non-functional fragment of the Kringle 1 domain is a fragment that, when contained in the tPA variant, does not impair the ability of the tPA variant to decrease IOP by more than 10%, 20%, 30%, 40%, or 50%, relative to a corresponding tPA variant that does not contain the Kringle 1 domain. It is contemplated that the activity of a Kringle 1 domain or a fragment thereof can be measured in an isolated polypeptide consisting of just the Kringle 1 domain or the fragment, or can be measured in a larger protein that further incorporates other domains, e.g, a Finger domain and a Kringle 2 domain; a Finger domain, an EGF-like domain, and a Kringle 2 domain; or all the other domains and regions of tPA Chain A.

[0050] In some embodiments, the tPA variant does not comprise a Kringle 1 domain of human tPA. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 50% (e.g., 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) identical to SEQ ID NO: 4. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 50% identical to SEQ ID NO: 4. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 45% identical to SEQ ID NO: 4. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 40% identical to SEQ ID NO: 4. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 35% identical to SEQ ID NO: 4. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 30% identical to SEQ ID NO: 4. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 25% identical to SEQ ID NO: 4. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 20% identical to SEQ ID NO: 4. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 15% identical to SEQ ID NO: 4. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 10% identical to SEQ ID NO: 4. In some embodiments, the tPA variant comprises an amino acid sequence that is nogreater than 5% identical to SEQ ID NO: 4. In some embodiments, the tPA variant does not comprise the amino acid sequence of SEQ ID NO: 4.

[0051] In some embodiments, the tPA variant does not comprise an active catalytic domain of tPA. Such tPA variants are referred to as “enzymatically inactive tPA variants,’7which is used herein to denote all forms of tPA that are proteolytically inactive or have substantially reduced proteolytic activity relative to a corresponding wild type tPA protein (e.g., a wild type tPA protein of the same species). The proteolytic activity of a tPA variant solution can be measured by an in vitro assay converting plasminogen to plasmin by proteolytic cleavage (see. e.g, International Application Publication No. WO 2022 / 173998; Chandler et al., (2000) CLIN. CHEM. 46(1): 38-46) and characterized as activity unit in a given volume (e.g., U / mL) by fitting with a standard curve. The activity' unit in a given molar amount of protein (e.g., U / mol) can be calculated according to the concentration of the protein in the solution and its molecular weight. In some embodiments, the activity unit in a given molar amount of an enzymatically7inactive tPA no higher than 10%, 20%, 30%, 40%, or 50% of the activity7unit in a given molar amount of a corresponding wild ty pe tPA protein.

[0052] Non-limiting examples of enzymatically inactive tPA variants include active site mutants (e.g., substitutions (e.g., insertions or deletions), truncations or deletions) and catalytic domain mutants (e.g.. substitutions (e.g., insertions or deletions), truncations or deletions). For example, in some embodiments, the tPA variant comprises a catalytic domain of tPA (e.g, human tPA) having one or more amino acid substitutions resulting in a catalytically inactive catalytic domain. In some embodiments, the catalytic domain comprises a mutation at position 168 of SEQ ID NO: 6. In some embodiments, the mutation comprises a substitution (e.g, of Ala, Gly, or Thr) for the Ser at position 168 of SEQ ID NO: 6. In other embodiments, enzymatically inactive tPA can include variants with significant deletions of part of the catalytic domain of the tPA molecule. One example is a truncated catalytic domain that is missing a number of amino acids (e.g., 90-250, 90-200, 90-150, 90- 120, 90-100, 100-250, 100-200, 100-150, 100-120, 120-250, 120-200, 120-150, 150-250, 150-200, or 200-250 amino acids) from its carboxy terminal end.

[0053] In some embodiments, the tPA variant does not comprise a catalytic domain of tPA (e.g., human tPA). It is contemplated that the term “catalytic domain,” as used herein, includes full-length catalytic domains as well as functional fragments thereof that have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of an activity of a corresponding full-length catalytic domain (e.g., a human full-length catalytic domainhaving the amino acid sequence of SEQ ID NO: 6). In some embodiments, a functional fragment of the catalytic domain is a fragment that, when contained in the tPA variant, converts plasminogen to plasmin by proteolytic cleavage, relative to a corresponding tPA variant that does not contain the catalytic domain. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 50% (e.g., 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) identical to SEQ ID NO: 6. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 50% identical to SEQ ID NO: 6. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 45% identical to SEQ ID NO: 6. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 40% identical to SEQ ID NO: 6. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 35% identical to SEQ ID NO: 6. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 30% identical to SEQ ID NO: 6. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 25% identical to SEQ ID NO: 6. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 20% identical to SEQ ID NO: 6. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 15% identical to SEQ ID NO: 6. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 10% identical to SEQ ID NO: 6. In some embodiments, the tPA variant comprises an amino acid sequence that is no greater than 5% identical to SEQ ID NO: 6. In some embodiments, the tPA variant does not comprise the amino acid sequence of SEQ ID NO: 6.

[0054] In some embodiments, the tPA variant further comprises an EGF-like domain of tPA (e.g., human tPA). In some embodiments, the EGF-like domain of tPA comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%. at least 96%. at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 3. In some embodiments, the EGF-like domain of tPA comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 3. In some embodiments, the EGF- like domain of tPA comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 3. In some embodiments, the EGF-like domain of tPA comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 3. In some embodiments, the EGF- like domain of tPA comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3. In some embodiments, the EGF-like domain of tPA comprises an amino acidsequence that is at least 90% identical to SEQ ID NO: 3. In some embodiments, the EGF- like domain of tPA comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 3. In some embodiments, the EGF-like domain of tPA comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 3. In some embodiments, the EGF- like domain of tPA comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 3. In some embodiments, the EGF-like domain of tPA comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 3. In some embodiments, the EGF- like domain of tPA comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 3. In some embodiments, the EGF-like domain of tPA comprises the amino acid sequence of SEQ ID NO: 3.

[0055] It is understood that Chain A of a tPA protein comprises a tail region C-terminal to the Kringle 2 domain. In some embodiments, the tPA variant of the present disclosure further compnses the tail region, for example, of the same species as the Kringle 2 domain. In human tPA, the wild type tail region comprises the amino acid sequence of STCGLRQYSQPQFR (SEQ ID NO: 8). In some embodiments, the tail region incorporated in the tPA variant comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 8. In some embodiments, the tail region comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 8. In some embodiments, the tail region comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 8. In some embodiments, the tail region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, the tail region comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 8. In some embodiments, the tail region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 8. In some embodiments, the tail region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 8. In some embodiments, the tail region comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 8. In some embodiments, the tail region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 8. In some embodiments, the tail region comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 8. In some embodiments, the tail region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 8. In some embodiments, the tail region comprises the amino acid sequence of SEQ ID NO: 8.

[0056] Without wishing to be bound by theory', it is contemplated that a lysine-rich fragment of tPA catalytic domain, e.g, corresponding to human tPA fragment having the amino acid sequence of GQKDVPGVYTKVTNYLDWIRDNMR (SEQ ID NO: 9), may improve binding to LPR-1. Accordingly, in some embodiments, atPA variant of the present disclosure further comprises a catalytic domain fragment comprising an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%. at least 97%. at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises the amino acid sequence of SEQ ID NO: 9.

[0057] In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 9, wherein the catalytic domain fragment of tPA comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 9, a position corresponding to amino acid 11 of SEQ ID NO: 9, or at each position corresponding to amino acid 3 and amino acid 11 of SEQ ID NO: 9.

[0058] In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 70% (e.g., at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 9 and comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 75% identicalto SEQ ID NO: 9 and comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 9.

[0059] In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 9, wherein the catalytic domain fragment of tPA comprises a ly sine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 9.

[0060] In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 70% (e.g., at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 9, wherein the catalytic domain fragment of tPA comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 9. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9 and comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 9.

[0061] The present disclosure also provides tPA variants comprising and / or excluding corresponding tPA domains from other species, e.g., mammals such as primates, laboratory animals such as mice, and rats, and farm animals such as pigs and sheep. As shown in FIG. 1, mature ovine and murine tPA proteins are homologous to human tPA. Table 2 below shows the amino acid sequence identities between mature human tPA and its ovine and murine homologues, through the entire length and within selected domains, as assessed using the Clustal Omega program.Table 2: Human, ovine, and murine tPA amino acid sequence identities

[0062] As shown in Table 2, it is understood that tPA and its domains have as low as about 70% sequence identities among mammalian homologues. The cross-species experimental systems of Examples 1 and 2 below suggests that the cytokine function played by tPA is likely conserved among mammalian species. Accordingly, it is expected that tPA domains can retain their functions with as low as about 70% sequence identities.Ovine tPA variants

[0063] In some embodiments, the tPA variant is a variant of ovine (sheep) tPA. The amino acid sequence of a full length, wild type sheep tPA is set forth in SEQ ID NO: 20:MTNAMKTQFLCLLLCGAVFTWPSQETYRRLRRGARSYRVTCRDEKTQMTYL QHESWLRPLLRGNQVEHCRCNGGRAQCHSVPVRSCSEPWCFNGGTCQQALY SSDFVCQCPEGFMGKLCEIDTTATCYKDQGVAYRGTWSTAESGAECASWNS SGLAMKPYSGRRPDAVRLGLGNHNYCRNPDQDSKPWCYVFKAGKYISEFCS TPACTKVAEEDGDCYTGNGLAYRGTRSRTKSGFSCLPWNSVFLTSKIYTAWKSNARALGLGKHNHCRNPDGDAQPWCHVWKDRQLTWEYCDVPQCVTCGLR QYKRPQFRIKGGLFADITSHPWQAAIFVKNRRSPGQRFLCGGILISSCWVLSAA HCFQERYPPHHLKVVLGRTYRLVPGEEEQMFEVEKYIVHKEFDDDTYDNDIA LLHLKSNSLTCAQESGSVRTICLPDASLQLPDWTECELSGYGKHEASSPFFSER LKEAHVRLYPSSRCTSRHLFNRTVTNNMLCAGDTRSGGDHTNLHDACQGDS GGPLVCMKDNHMTLVGIISWGLGCGQKDIPGVYTKVTNYLDWIRDNMRL (SEQ ID NO: 20)

[0064] The amino acid sequence of a mature, wild type ovine tPA is set forth in SEQ ID NO: 33:SYRVTCRDEKTQMTYLQHESWLRPLLRGNQVEHCRCNGGRAQCHSVPVRSC SEPWCFNGGTCQQALYSSDFVCQCPEGFMGKLCEIDTTATCYKDQGVAYRGTWSTAESGAECASWNSSGLAMKPYSGRRPDAVRLGLGNHNYCRNPDQDSKP WCYVFKAGKYISEFCSTPACTKVAEEDGDCYTGNGLAYRGTRSRTKSGFSCL PWNSVFLTSKIYTAWKSNARALGLGKHNHCRNPDGDAQPWCHVWKDRQLT WEYCDVPQCVTCGLRQYKRPQFRIKGGLFADITSHPWQAAIFVKNRRSPGQR FLCGGILISSCWVLSAAHCFQERYPPHHLKVVLGRTYRLVPGEEEQMFEVEK YIVHKEFDDDTYDNDIALLHLKSNSLTCAQESGSVRTICLPDASLQLPDWTEC ELSGYGKHEASSPFFSERLKEAHVRLYPSSRCTSRHLFNRTVTNNMLCAGDTR SGGDHTNLHDACQGDSGGPLVCMKDNHMTLVGIISWGLGCGQKDIPGVYTK VTNYLDWIRDNMRL (SEQ ID NO: 33)

[0065] A wild-type, immature ovine tPA (e.g, having the amino acid sequence of SEQ ID NO: 20) comprises, from N-terminus to C-terminus, a signal peptide, a pro-peptide, a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a catalytic domain. A mature ovine tPA (e.g., having the amino acid sequence of SEQ ID NO: 33) comprises, from N-terminus to C-terminus, a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a catalytic domain. The mature ovine tPA can be cleaved, e.g., by plasmin or a kallikrein, into two polypeptide chains. Chain A includes, from N- terminus to C-terminus, the Finger domain, the EGF-like domain, the Kringle 1 domain, and the Kringle 2 domain. Chain B includes the catalytic domain that has protease activity. Exemplary sequences of these domains are provided in Table 2 below. Certain adjacent domains are connected by peptide linkers, which can be readily recognized by aligning the sequences of the domains with SEQ ID NO: 20.Table 2: SEQ ID NO: 20 sheep tPA domains

[0066] In some embodiments, a tP A variant disclosed herein comprises a Finger domain, a Kringle 2 domain, but not a Kringle 1 domain or a functional fragment thereof, of ovine tPA. In some embodiments, the tPA variant comprises a Finger domain, a Kringle 2 domain, but not a Kringle 1 domain of ovine tPA. Also contemplated are ovine tPA variants comprising a functional fragment of a Finger domain, a functional fragment of a Kringle 2 domain, but not a Kringle 1 domain or a functional fragment thereof, of ovine tPA.

[0067] In some embodiments, the tPA variant comprises a Finger domain of ovine tPA. It is contemplated that the Finger domain can be a full-length Finger domain of ovine tPA or a functional fragment thereof. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 21. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 21. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 21. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 21. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 21. In some embodiments, the Finger domain comprises an aminoacid sequence that is at least 95% identical to SEQ ID NO: 21. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 21. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 21. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 21. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 21. In some embodiments, the Finger domain comprises the amino acid sequence of SEQ ID NO: 21.

[0068] In some embodiments, the tPA variant comprises a Kringle 2 domain of ovine tPA. It is contemplated that the Kringle 2 domain can be a full-length Kringle 2 domain of ovine tPA or a functional fragment thereof. In some embodiments, the tPA variant comprises a Kringle 2 domain comprising an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%. at least 85%. at least 90%. at least 95%. at least 96%. at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 24. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 24. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 24. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 24. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 24. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 24. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 24. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 24. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 24. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 24. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 24. In some embodiments, the Kringle 2 domain comprises the amino acid sequence of SEQ ID NO: 24.

[0069] In some embodiments, the tPA variant does not comprise a Kringle 1 domain of ovine tPA. It is contemplated that the Kringle 1 domain can be a full-length Kringle 1 domain of ovine tPA or a functional fragment thereof. Such tPA variants can still contain a nonfunctional fragment of an ovine tPA Kringle 1 domain. In some embodiments, the tPAvariant does not comprise an amino acid sequence that is at least 70% (e.g., at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 23. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 70% identical to SEQ ID NO: 23. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 75% identical to SEQ ID NO: 23. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 80% identical to SEQ ID NO: 23. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 85% identical to SEQ ID NO: 23. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 23. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 95% identical to SEQ ID NO: 23. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 96% identical to SEQ ID NO: 23. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 97% identical to SEQ ID NO: 23. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 98% identical to SEQ ID NO: 23. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 99% identical to SEQ ID NO: 23. In some embodiments, the tPA variant does not comprise the amino acid sequence of SEQ ID NO: 23.

[0070] In some embodiments, the tPA variant does not comprise an active catalytic domain of ovine tPA. In some embodiments, the tPA variant comprises an ovine tPA catalytic domain having one or more amino acid substitutions resulting in an enzymatically inactive catalytic domain. In some embodiments, the catalytic activity of an enzymatically inactive ovine tPA is reduced by at least 70%, 80%, 90%, 95%, or 99%, relative to a corresponding wild type ovine tPA protein. In some embodiments, the catalytic domain comprises a mutation at position 168 of SEQ ID NO: 25. In some embodiments, the mutation comprises a substitution (e.g., of Ala, Gly, or Thr) for the Ser at position 168 of SEQ ID NO: 25.

[0071] In some embodiments, the tPA variant does not comprise a catalytic domain of ovine tPA. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 25. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 70% identical to SEQ ID NO: 25. In some embodiments, the tPA variant does not comprisean amino acid sequence that is at least 75% identical to SEQ ID NO: 25. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 80% identical to SEQ ID NO: 25. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 85% identical to SEQ ID NO: 25. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 25. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 95% identical to SEQ ID NO: 25. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 96% identical to SEQ ID NO: 25. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 97% identical to SEQ ID NO: 25. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 98% identical to SEQ ID NO: 25. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 99% identical to SEQ ID NO: 25. In some embodiments, the tPA variant does not comprise the amino acid sequence of SEQ ID NO: 25.

[0072] In some embodiments, the tPA variant further comprises an EGF-like domain of ovine tPA. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 70% (e.g., at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 22. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 22. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 22. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 22. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 22. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 22. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 22. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 22. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 22. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 22. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 22. In some embodiments, the EGF-like domain comprises the amino acid sequence of SEQ ID NO: 22.

[0073] In some embodiments, the tPA variant further comprises a tail region of ovine tPA.In ovine tPA, the wild type tail region comprises the amino acid sequence of VTCGLRQYKRPQFR (SEQ ID NO: 39). In some embodiments, the tail region incorporated in the tPA variant comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 39. In some embodiments, the tail region comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 39. In some embodiments, the tail region comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 39. In some embodiments, the tail region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 39. In some embodiments, the tail region comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 39. In some embodiments, the tail region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 39. In some embodiments, the tail region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 39. In some embodiments, the tail region comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 39. In some embodiments, the tail region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 39. In some embodiments, the tail region comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 39. In some embodiments, the tail region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 39. In some embodiments, the tail region comprises the amino acid sequence of SEQ ID NO: 39.

[0074] In some embodiments, the tPA variant further comprises a lysine-rich fragment of catalytic domain corresponding to ovine tPA. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to GQKDIPGVYTKVTNYLDWIRDNMR (SEQ ID NO: 44). In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 44. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 44. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 44. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 44. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 44. In some embodiments, the catalytic domainfragment comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 44. In some embodiments, the catalytic domain fragment comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the catalytic domain fragment comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 44. In some embodiments, the catalytic domain fragment comprises a lysine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 44. In some embodiments, the catalytic domain fragment comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 44.Mouse tPA variants

[0075] In some embodiments, the TPA variant is a variant of mouse tPA. the amino acid sequence of a full length, wild type mouse tPA is set forth in SEQ ID NO: 26:MKRELLCVLLLCGLAFPLPDQGIHGRFRRGARSYRATCRDEPTQTTYQQHQS WLRPMLRSSRVEYCRCNSGLVQCHSVPVRSCSEPRCFNGGTCQQALYFSDFV CQCPDGFVGKRCDIDTRATCFEEQGITYRGTWSTAESGAECINWNSSVLSLKP YNARRPNAIKLGLGNHNYCRNPDRDLKPWCYVFKAGKYTTEFCSTPACPKG KSEDCYVGKGVTYRGTHSLTTSQASCLPWNSIVLMGKSYTAWRTNSQALGL GRHNYCRNPDGDARPWCHVMKDRKLTWEYCDMSPCSTCGLRQYKRPQFRI KGGLYTDITSHPWQAAIFVKNKRSPGERFLCGGVLISSCWVLSAAHCFLERFP PNHLKVVLGRTYRVVPGEEEQTFEIEKYIVHEEFDDDTYDNDIALLQLRSQSK QCAQESSSVGTACLPDPNLQLPDWTECELSGYGKHEASSPFFSDRLKEAHVR LYPSSRCTSQHLFNKTVTNNMLCAGDTRSGGNQDLHDACQGDSGGPLVCMI NKQMTLTGIISWGLGCGQKDVPGVYTKVTNYLDWIHDNMKQ (SEQ ID NO: 26)

[0076] The amino acid sequence of a mature, wild type mouse tPA is set forth in SEQ ID NO: 35:SYRATCRDEPTQTTYQQHQSWLRPMLRSSRVEYCRCNSGLVQCHSVPVRSCS EPRCFNGGTCQQALYFSDFVCQCPDGFVGKRCDIDTRATCFEEQGITYRGTW STAESGAECINWNSSVLSLKPYNARRPNAIKLGLGNHNYCRNPDRDLKPWCY VFKAGKYTTEFCSTPACPKGKSEDCYVGKGVTYRGTHSLTTSQASCLPWNSI VLMGKSYTAWRTNSQALGLGRHNYCRNPDGDARPWCHVMKDRKLTWEYC DMSPCSTCGLRQYKRPQFRIKGGLYTDITSHPWQAAIFVKNKRSPGERFLCGG VLISSCWVLSAAHCFLERFPPNHLKVVLGRTYRVVPGEEEQTFEIEKYIVHEEFDDDTYDNDIALLQLRSQSKQCAQESSSVGTACLPDPNLQLPDWTECELSGYG KHEAS SPFFSDRLKE AHVRLYP S SRCTSQHLFNKTVTNNMLC AGDTRS GGNQ DLHDACQGDSGGPLVCMINKQMTLTGIISWGLGCGQKDVPGVYTKVTNYLD WIHDNMKQ (SEQ ID NO: 35)

[0077] A wild- type, immature mouse tPA (e.g., having the amino acid sequence of SEQ ID NO: 26) comprises, from N-terminus to C-terminus, a signal peptide, a pro-peptide, a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a catalytic domain. A mature mouse tPA (e.g. , having the amino acid sequence of SEQ ID NO: 35) comprises, from N-terminus to C-terminus, a Finger domain, an EGF-like domain, a Kringle1 domain, a Kringle 2 domain, and a catalytic domain. The mature mouse tPA can be cleaved, e.g., by plasmin or a kallikrein, into two polypeptide chains. Chain A includes, from N-terminus to C-terminus, the Finger domain, the EGF-like domain, the Kringle 1 domain, and the Kringle 2 domain. Chain B includes the catalytic domain that has protease activity . Exemplary sequences of these domains are provided in Table 3 below. Certain adjacent domains are connected by peptide linkers, which can be readily recognized by aligning the sequences of the domains with SEQ ID NO: 26.Table 3: SEQ ID NO: 26 mouse tPA domains

[0078] In some embodiments, a tPA disclosed herein comprises a Finger domain, a Kringle 2 domain, but not a Kringle 1 domain or a functional fragment thereof, of mouse tPA. In some embodiments, the tPA variant comprises a Finger domain, a Kringle 2 domain, but not a Kringle 1 domain of mouse tPA. Also contemplated are ovine tPA variants comprising a functional fragment of a Finger domain, a functional fragment of a Kringle 2 domain, but not a Kringle 1 domain or a functional fragment thereof, of mouse tPA.

[0079] In some embodiments, the tPA variant comprises a Finger domain of mouse tPA. It is contemplated that the Finger domain can be a full-length Finger domain of mouse tPA or a functional fragment thereof. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 27. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 27. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 27. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 27. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 27. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 27. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 27. In some embodiments, the Finger domain comprises an amino acid sequence that isat least 97% identical to SEQ ID NO: 27. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 27. In some embodiments, the Finger domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 27. In some embodiments, the Finger domain comprises the amino acid sequence of SEQ ID NO: 27.

[0080] In some embodiments, the tPA variant comprises a Kringle 2 domain of mouse tPA. It is contemplated that the Kringle 2 domain can be a full-length Kringle 2 domain of mouse tPA or a functional fragment thereof. In some embodiments, the tPA variant comprises a Kringle 2 domain comprising an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 30. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 30. In some embodiments, the Kringle 2 domain comprises an ammo acid sequence that is at least 75% identical to SEQ ID NO: 30. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 30. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 30. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 30. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 30. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 30. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 30. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 30. In some embodiments, the Kringle 2 domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 30. In some embodiments, the Kringle 2 domain comprises the amino acid sequence of SEQ ID NO: 30.

[0081] In some embodiments, the tPA variant does not comprise a Kringle 1 domain of mouse tPA. It is contemplated that the Kringle 1 domain can be a full-length Kringle 1 domain of mouse tPA or a functional fragment thereof. Such tPA variants can still contain a non-functional fragment of a mouse tPA Kringle 1 domain. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 70% (e.g.. at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 29. In some embodiments, the tPA variantdoes not comprise an amino acid sequence that is at least 70% identical to SEQ ID NO: 29. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 75% identical to SEQ ID NO: 29. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 80% identical to SEQ ID NO: 29. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 85% identical to SEQ ID NO: 29. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 29. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 95% identical to SEQ ID NO: 29. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 96% identical to SEQ ID NO: 29. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 97% identical to SEQ ID NO: 29. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 98% identical to SEQ ID NO: 29. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 99% identical to SEQ ID NO: 29. In some embodiments, the tPA variant does not comprise the amino acid sequence of SEQ ID NO: 29.

[0082] In some embodiments, the tPA variant does not comprise an active catalytic domain of mouse tPA. In some embodiments, the tPA variant comprises a mouse tPA catalytic domain having one or more amino acid substitutions resulting in an enzymatically inactive catalytic domain. In some embodiments, the cataly tic activity of an enzy matically inactive mouse tPA is reduced by at least 70%. 80%. 90%. 95%, or 99%, relative to a corresponding wild type mouse tPA protein. In some embodiments, the catalytic domain comprises a mutation at position 168 of SEQ ID NO: 31. In some embodiments, the mutation comprises a substitution (e.g., of Ala, Gly, or Thr) for the Ser at position 168 of SEQ ID NO: 31.

[0083] In some embodiments, the tPA variant does not comprise a catalytic domain of mouse tPA. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 31. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 70% identical to SEQ ID NO: 31. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 75% identical to SEQ ID NO: 31. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 80% identical to SEQ ID NO: 31. In some embodiments, the tPA variant does not comprise anamino acid sequence that is at least 85% identical to SEQ ID NO: 31. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 31. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 95% identical to SEQ ID NO: 31. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 96% identical to SEQ ID NO: 31. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 97% identical to SEQ ID NO: 31. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 98% identical to SEQ ID NO: 31. In some embodiments, the tPA variant does not comprise an amino acid sequence that is at least 99% identical to SEQ ID NO: 31. In some embodiments, the tPA variant does not comprise the amino acid sequence of SEQ ID NO: 31.

[0084] In some embodiments, the tPA variant further comprises an EGF-like domain of mouse tPA. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 28. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 28. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 28. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 28. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 28. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 28. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 28. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 28. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 28. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 28. In some embodiments, the EGF-like domain comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 28. In some embodiments, the EGF-like domain comprises the amino acid sequence of SEQ ID NO: 28.

[0085] In some embodiments, the tPA variant further comprises a tail region of mouse tPA. In mouse tPA, the wild type tail region comprises the amino acid sequence of STCGLRQYKRPQFR (SEQ ID NO: 40). In some embodiments, the tail region incorporatedin the tPA variant comprises an amino acid sequence that is at least 70% (e.g, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 40. In some embodiments, the tail region comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 40. In some embodiments, the tail region comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 40. In some embodiments, the tail region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 40. In some embodiments, the tail region comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 40. In some embodiments, the tail region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 40. In some embodiments, the tail region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 40. In some embodiments, the tail region comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 40. In some embodiments, the tail region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 40. In some embodiments, the tail region comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 40. In some embodiments, the tail region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 40. In some embodiments, the tail region comprises the amino acid sequence of SEQ ID NO: 40.

[0086] In some embodiments, the tPA variant further comprises a lysine-rich fragment of catalytic domain corresponding to mouse tPA. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to GQKDVPGVYTKVTNYLDWIHDNMK (SEQ ID NO: 45). In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 45. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 45. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 45. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 45. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 45. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 45. In some embodiments, the catalytic domain fragment comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the catalytic domain fragment comprises a lysine(K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 45. In some embodiments, the catalytic domain fragment comprises a lysine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 45. In some embodiments, the catalytic domain fragment comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 45.Fusion of tPA domains

[0087] A tPA variant of the present disclosure may comprise any Finger domain as disclosed herein, any Kringle 2 domain as disclosed herein, and optionally any EGF-like domain as disclosed herein. The tPA variant can further comprise any tail region and / or catalytic domain fragment as disclosed herein.

[0088] In some embodiments, a C-terminus of the Finger domain is linked, directly or indirectly, to an N-terminus of the Kringle 2 domain. In some embodiments, an N-terminus of the Finger domain is linked, directly or indirectly, to a C-terminus of the Kringle 2 domain. Polypeptide linkers are known in the art, and any such linker can be used to link the Finger domain to the Kringle 2 domain.

[0089] In some embodiments, the tPA variant further comprises an EGF-like domain, wherein a C-terminus of the Finger domain is linked, directly or indirectly, to an N-terminus of the EGF-like domain, and a C-terminus of the EGF-like domain is linked, directly or indirectly, to an N-terminus of the Kringle 2 domain.

[0090] In some embodiments, the C-terminus of the Finger domain is linked directly to the N-terminus of the EGF-like domain. In some embodiments, the C-terminus of the Finger domain is linked indirectly to the N-terminus of the EGF-like domain, such as through a polypeptide linker. Polypeptide linkers are known in the art, and any such linker is within the scope of the present disclosure. In some embodiments, the linker is a flexible peptide linker. In some embodiments, the linker is a rigid peptide linker. It is understood that the Finger domain and the EGF-like domain are adjacent to each other in certain wild type tPA proteins. Accordingly, in some embodiments, the Finger domain and the EGF-like domain are linked by an amino acid sequence linking these domains in a wild type tPA protein, for example, a wild type tPA protein of the same species as that of the Finger domain, the EGF-like domain, or both. In some embodiments, the C-terminus of the Finger domain is linked to the N- terminus of the EGF-like domain by an amino acid sequence of Lys-Ser (KS). In someembodiments, the C-terminus of the Finger domain is linked to the N-terminus of the EGF- like domain by an amino acid sequence of Arg-Ser (RS).

[0091] In some embodiments, the C-terminus of the EGF-like domain is linked directly to the N-terminus of the Kringle 2 domain. In some embodiments, the C-terminus of the EGF-like domain is linked indirectly to the N-terminus of the Kringle 2 domain, such as through a polypeptide linker. Polypeptide linkers are known in the art, and any such linker is within the scope of the present disclosure. In some embodiments, the linker is a flexible peptide linker. In some embodiments, the linker is a rigid peptide linker. In some embodiments, the linker is 3-50 (e.g, 3-40, 3-30, 3-20, 3-15, 3-12, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 5-40, 5-30, 5-20, 5- 15, 5-12, 5-10, 5-9, 5-8, 5-7, 5-6, 6-40, 6-30, 6-20, 6-15, 6-12, 6-10, 6-9, 6-8, 6-7, 7-40, 7-30, 7-20, 7-15, 7-12, 7-10, 7-9, 7-8, 8-40, 8-30, 8-20, 8-15, 8-12, 8-10, 8-9. 9-40. 9-30, 9-20. 9- 15, 9-12, 9-10, 10-40, 10-30, 10-20, 10-15, 10-12, 12-40, 12-30, 12-20. 12-15, 15-40, 15-30. or 15-20) amino acids in length. In some embodiments, the linker is about 3 to about 20 amino acids in length. In some embodiments, the linker is about 4 to about 20 amino acids in length. In some embodiments, the linker is about 5 to about 20 amino acids in length. In some embodiments, the linker is about 6 to about 20 amino acids in length. In some embodiments, the linker is about 7 to about 20 amino acids in length. In some embodiments, the linker is about 8 to about 20 amino acids in length. In some embodiments, the linker is about 9 to about 20 amino acids in length. In some embodiments, the linker is about 10 to about 20 amino acids in length. In some embodiments, the linker is about 11 to about 20 amino acids in length. In some embodiments, the linker is about 12 to about 20 amino acids in length. In some embodiments, the linker is about 13 to about 20 amino acids in length. In some embodiments, the linker is about 14 to about 20 amino acids in length. In some embodiments, the linker is about 15 to about 20 amino acids in length. In some embodiments, the linker is about 16 to about 20 amino acids in length. In some embodiments, the linker is about 17 to about 20 amino acids in length. In some embodiments, the linker is about 18 to about 20 amino acids in length. In some embodiments, the linker is about 19 to about 20 amino acids in length.

[0092] In some embodiments, the linker is about 3 to about 19 amino acids in length. In some embodiments, the linker is about 3 to about 18 amino acids in length. In some embodiments, the linker is about 3 to about 17 amino acids in length. In some embodiments, the linker is about 3 to about 16 amino acids in length. In some embodiments, the linker is about 3 to about 15 amino acids in length. In some embodiments, the linker is about 3 toabout 14 amino acids in length. In some embodiments, the linker is about 3 to about 13 amino acids in length. In some embodiments, the linker is about 3 to about 12 amino acids in length. In some embodiments, the linker is about 3 to about 11 amino acids in length. In some embodiments, the linker is about 3 to about 10 amino acids in length. In some embodiments, the linker is about 3 to about 9 amino acids in length. In some embodiments, the linker is about 3 to about 8 amino acids in length. In some embodiments, the linker is about 3 to about 7 amino acids in length. In some embodiments, the linker is about 3 to about 6 amino acids in length. In some embodiments, the linker is about 3 to about 5 amino acids in length. In some embodiments, the linker is about 3 to about 4 amino acids in length. In some embodiments, the linker is 3, 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.

[0093] In some embodiments, the C-terminus of the EGF-like domain is linked to the N- terminus of the Kringle 2 domain by an amino acid sequence of CCEIDTGNSD (SEQ ID NO: 7) or by an amino acid sequence of CCEIDTRATSEGNSD (SEQ ID NO: 59). In some embodiments, the C-terminus of the EGF-like domain is linked to the N-terminus of the Kringle 2 domain by an amino acid sequence of LCEIDAT (SEQ ID NO: 37) or by an amino acid sequence of LCEIDAEDGD (SEQ ID NO: 38).

[0094] In some embodiments, a tail region disclosed herein is linked directly to a C-terminus of the Kringle 2 domain, e.g, via a peptide bond. In some embodiments, a tail region is linked indirectly to a C-terminus of the Kringle 2 domain, e.g., by a peptide bond. In some embodiments, one or more additional amino acids (e.g., a proline residue) is linked to a C- terminus of the tail region, constituting a C-terminus of the tPA variant.

[0095] In some embodiments, the catalytic domain fragment of tPA is located at a C- terminus of the tPA variant. In some embodiments, the catalytic domain fragment of tPA is linked directly to a C-terminus of the tail region. In some embodiments, the catalytic domain fragment of tPA is linked indirectly to a C-terminus of the tail region, such as through a peptide linker as disclosed herein. In some embodiments, one or more additional amino acids (e.g., a proline residue) is linked to a C-terminus of the catalytic domain fragment, constituting a C-terminus of the tPA variant.

[0096] Specific tPA variants are also provided in the present disclosure, including human tPA variants comprising the amino acid sequences of SEQ ID NOs: 10-12 (mature proteins) and 14-16 (immature proteins) and ovine tPA variants comprising the amino acid sequencesof SEQ ID NO: 43 (immature proteins) and 46 (mature proteins). In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 10, 11, 12, 14, 15, 16, 43, or 46. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 10, 11, 12, 14, 15, or 16. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 10, 11, or 12. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 14, 15, or 16. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 43 or 46. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 43. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g., at least 75%. at least 80%. at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 46.

[0097] In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 10:SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSC SEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTGNSDCYFGNGSAYRG THSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKP WCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRP (SEQ ID NO: 10)In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 10. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 10. In some embodiments, the tPAvariant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 10.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 10. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 10. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 10.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 10. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 10. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 10.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 10. In some embodiments, the tPA variant comprises an amino acid sequence of SEQ ID NO: 10.

[0098] In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 11 :SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSC SEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATSEGNSDCYFGNGS AYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDG DAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRP (SEQ ID NO: 11)In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 11. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 11. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 11.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 11. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 11. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 1.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 11. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 11. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: I I.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 99%identical to SEQ ID NO: 11. In some embodiments, the tPA variant comprises an amino acid sequence of SEQ ID NO: 11.

[0099] In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g. at least 75%, at least 80%, at least 85%. at least 90%. at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 12:SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSC SEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTGNSDCYFGNGSAYRG THSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKP WCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRGQKDVPGVYTKVTNYLDWIRDNMRP(SEQ ID NO: 12)In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 12. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 12. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 12.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 12. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 12. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 12.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 12. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 12. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 12.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 12. In some embodiments, the tPA variant comprises an amino acid sequence of SEQ ID NO: 12.

[0100] In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g. at least 75%, at least 80%, at least 85%. at least 90%. at least 95%, at least 96%. at least 97%. at least 98%. at least 99%, or 100%) identical to SEQ ID NO: 14:MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKTQMIYQQ HQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALYF SDFVCQCPEGFAGKCCEIDTGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCD VPSCSTCGLRQYSQPQFRP (SEQ ID NO: 14)In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 14. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 14. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 14.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 14. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 14.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 14. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 14. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 14.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 14. In some embodiments, the tPA variant comprises an amino acid sequence of SEQ ID NO: 14.

[0101] In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 15:MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKTQMIYQQ HQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALYF SDFVCQCPEGFAGKCCEIDTRATSEGNSDCYFGNGSAYRGTHSLTESGASCLP WNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRP (SEQ ID NO: 15)In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 15. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 15. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 15.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 15. In some embodiments, the tPA variant comprises an amino acidsequence that is at least 90% identical to SEQ ID NO: 15. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 15. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 15. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 15. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 15. In some embodiments, the tPA variant comprises an amino acid sequence of SEQ ID NO: 15.

[0102] In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%. at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 16:MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKTQMIYQQ HQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALYF SDFVCQCPEGFAGKCCEIDTGNSDCYFGNGSAYRGTHSLTESGASCLPWNSM ILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCD VPSCSTCGLRQYSQPQFRGQKDVPGVYTKVTNYLDWIRDNMRP (SEQ ID NO: 16)In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 16. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 16. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 16. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 16. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 16. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 16. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 16.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 99%identical to SEQ ID NO: 16. In some embodiments, the tPA variant comprises an amino acid sequence of SEQ ID NO: 16.

[0103] In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g. at least 75%, at least 80%, at least 85%. at least 90%. at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 43:MTNAMKTQFLCLLLCGAVFTWPSQETYRRLRRGARSYRVTCRDEKTQMTYL QHESWLRPLLRGNQVEHCRCNGGRAQCHSVPVRSCSEPWCFNGGTCQQALY SSDFVCQCPEGFMGKLCEIDAEDGDCYTGNGLAYRGTRSRTKSGFSCLPWNS VFLTSKIYTAWKSNARALGLGKHNHCRNPDGDAQPWCHVWKDRQLTWEYCDVPQCVTCGLRQYKRPQFRP (SEQ ID NO: 43)In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 43. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 43. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 43.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 43. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 43. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 43.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 43. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 43. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 43.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 43. In some embodiments, the tPA variant comprises an amino acid sequence of SEQ ID NO: 43.

[0104] In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% (e.g. at least 75%, at least 80%, at least 85%. at least 90%. at least 95%, at least 96%. at least 97%. at least 98%. at least 99%, or 100%) identical to SEQ ID NO: 46:SYRVTCRDEKTQMTYLQHESWLRPLLRGNQVEHCRCNGGRAQCHSVPVRSC SEPWCFNGGTCQQALYSSDFVCQCPEGFMGKLCEIDAEDGDCYTGNGLAYRGTRSRTKSGFSCLPWNSVFLTSKIYTAWKSNARALGLGKHNHCRNPDGDAQP WCHVWKDRQLTWEYCDVPQCVTCGLRQYKRPQFRP (SEQ ID NO: 46)In some embodiments, the tPA variant comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 46. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 46. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 46.In some embodiments, the tPA variant comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 46. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 46. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 46. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 46. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 46. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 46. In some embodiments, the tPA variant comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 46. In some embodiments, the tPA variant comprises an amino acid sequence of SEQ ID NO: 46.

[0105] The tPA variant of the present disclosure, having truncation of one or more domains, can have a reduced size compared to a full length tPA protein. In some embodiments, the tPA variant comprises an amino acid sequence having a length of 125-400 amino acids, e.g.. 125-350, 125-300, 125-250, 125-200, 125-175, 125-150, 150-400, 150-350, 150-300, 150- 250, 150-200, 150-175, 175-400, 175-350, 175-300, 175-250, 175-200, 200-400, 200-350, 200-300, 200-250, 250-400, 250-350, 250-300, 300-400, 300-350, or 350-400 amino acids.

[0106] In some embodiments, the tPA variant disclosed herein comprises an amino acid sequence having a length of about 125 to about 400 amino acids, or any value or range inbetween. In some embodiments, a tPA variant comprises an amino acid sequence having a length of about 125 to about 400, about 130 to about 400, about 135 to about 400, about 140 to about 400, about 145 to about 400, about 150 to about 400, about 155 to about 400, about 160 to about 400, about 165 to about 400, about 170 to about 400, about 175 to about 400, about 180 to about 400, about 185 to about 400. about 190 to about 400, about 195 to about 400, about 200 to about 400. about 210 to about 400, about 220 to about 400, about 230 to about 400, about 240 to about 400, about 250 to about 400, about 260 to about 400, about 270to about 400, about 280 to about 400, about 290 to about 400, about 300 to about 400, about 325 to about 400, about 350 to about 400, or about 375 to about 400 amino acids, or any value or range in-between.

[0107] In some embodiments, the tPA variant comprises an amino acid sequence having a length of about 125 to about 375, about 125 to about 350, about 125 to about 325, about 125 to about 300, about 125 to about 290, about 125 to about 280, about 125 to about 270, about 125 to about 260, about 125 to about 250, about 125 to about 240, about 125 to about 230, about 125 to about 220, about 125 to about 210. about 125 to about 200, about 125 to about 195, about 125 to about 190, about 125 to about 185, about 125 to about 180, about 125 to about 175, about 125 to about 170, about 125 to about 165, about 125 to about 160, about 125 to about 155, about 125 to about 150, about 125 to about 145, about 125 to about 140, about 125 to about 135, or about 125 to about 130 amino acids, or any value or range in-between. In some embodiments, the tPA variant comprises an amino acid sequence having a length of about 130, 131, 132, 133, 134, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 189, 190, 194, 195, 200, 205, 210, 213, 215, 220, 224, 225, 229, 230, 235, 240, 245, 248, 250, 260, 270. 279, 280, 290, 300, 310, 314, 320, 325, 330, 340, 350, 360. 370, 375, 380, 390, or 400 amino acids, or any value or range in-between.

[0108] It is understood that additional tPA variants can be generated to have a cytokine activity, using one or more experimental methods as described herein (e.g.. in Examples 1-3 below). Some experimental methods can further assess the expression and secretion levels of tPA variants, which are factors for designing tPA variants suitable for delivery7in an expression vector. For example, in a first exemplary system, human cerebral microvascular endothelial cells or human trabecular meshwork cells can be cultured in vitro and transfected with plasmids encoding tPA variants operably linked to a universal (e.g., CMV) promoter or with a control plasmid. Transcription of MMP9, determined after transfection with the plasmids using methods known in the art (e.g., RT-PCR), indicates the ability7of the tPA variants to be expressed and secreted and to stimulate a signaling pathway that increases MMP9 expression. The expression and secretion of the tPA variants can also be measured by immunoassays, such as enzy me-linked immunosorbent assay (ELISA) and Western blot, using monoclonal anti-tPA antibodies that bind one of the epitopes in the tPA variants or antibodies that bind a tag fused with the tPA variants. Where the tag provides a signal (e.g., a fluorescent protein emits light upon excitation, or an enzyme catalyzing a chemiluminescent reaction), the expression and secretion of the tPA variants can also be measured usingcorresponding detection methods. Alternatively, in a second exemplary' system, cerebral microvascular endothelial cells or human trabecular meshwork cells can be cultured in vitro and exposed to same amounts of the tPA variant proteins or a control protein. The tPA variant proteins can be produced in a bacterial expression system and isolated using a tag (e.g, His tag). Eukaryotic expression systems can also be used. For example, tPA variant proteins can be produced by eukaryotic cells (e.g., HEK 293, HEK 293T. or CHO cells) transfected with plasmids that encode the tPA variants, and the concentrations of the secreted tPA variants can be measured, for example, using one of the assays described above in connection with the first exemplary system. The media of the eukary otic cells containing the secreted tPA variants, after adjustment for equal amounts, can be used as conditioned media to treat cerebral microvascular endothelial cells or human trabecular meshwork cells, without isolating the tPA variants from the media. Transcription of MMP9 in the cerebral microvascular endothelial cells or human trabecular meshwork cells can then be determined using methods known in the art. In this second exemplary' system, transcription o MMP9 indicates the cytokine activity of the tPA variants, without accounting for the expression and secretion levels of the tPA variants.Nucleic acid molecules

[0109] In some embodiments, the present disclosure provides nucleic acid molecules encoding atPA variant disclosed herein. In some embodiments, the encoded tPA variant is an immature tPA variant.

[0110] Exemplary nucleic acid molecules are provided in Table 4 below:Table 4: Exemplary nucleic acid (NA) molecules

[0111] The above referenced nucleic acid molecules are exemplary only and are not meant to be limiting in any way. The skilled artisan would recognize and appreciate that due to the degenerate nature of codons multiple nucleic acid molecules may be provided to encode the same amino acid sequence. Accordingly, in some embodiments, a nucleic acid molecule is provided, the nucleic acid molecule encodes a tPA variant disclosed herein. In some embodiments, the nucleic acid molecule encodes a tPA variant comprising an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 10. 11. 12. 14, 15, 16, 43, or 46. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 47, 48, 49, 50, 51, 52, 55, or 56. In some embodiments, the nucleic acid molecule comprises anucleotide sequence that is at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 47. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 48. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 49. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 50. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 50% (e.g.. at least 55%. at least 60%. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 51. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%. at least 90%. at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 52. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%. at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 55. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 56.

[0112] In some embodiments, the nucleic acid molecule encodes a mature tPA variant as disclosed herein, e.g., comprising an amino acid sequence that is at least 70% (e.g.. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 10, 11, or 12. In some embodiments, the nucleic acid molecule further encodes a signal peptide at the N-terminus of the maturetPA variant. The signal peptide can comprise a tPA signal peptide and optionally a tPA propeptide. In some embodiments, the signal peptide and the pro-peptide comprise an amino acid sequence that is at least 70% (e.g.. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 13. In some embodiments, the signal peptide and the pro-peptide comprise an amino acid sequence that is at least 70% identical to SEQ ID NO: 13. In some embodiments, the signal peptide and the pro-peptide comprise an amino acid sequence that is at least 75%. 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the signal peptide and the pro-peptide comprise an amino acid sequence of SEQ ID NO: 13. In some embodiments, the nucleic acid molecule encodes a tPA variant comprising an amino acid sequence that is at least 70% (e.g, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 14, 15, or 16. In some embodiments, the immature tPA variant comprises a heterologous signal peptide, e.g., a signal peptide known to mediate secretion of another protein.Gene therapy vectors

[0113] Vectors of the present disclosure useful for gene therapy can be any vector that can effectively increase tPA expression in the eye, including vectors that encode an enzymatically inactive tPA variant. Many vectors useful for transferring exogenous genes into target mammalian cells are available. The vectors may be episomal, e.g., plasmids or virus derived vectors such as cytomegalovirus vector, adenoviral vector, adeno-associated viral (AAV) vector, etc., or the vectors may be integrative, e.g., integrating the reprogramming gene into the target cell genome, through homologous recombination or random integration, e.g., retrovirus derived vectors such MMLV (Moloney Murine Leukemia Virus), HIV-1, ALV (Avian leukosis virus), or lentiviral vectors.

[0114] The vector can comprise any of the nucleic acid molecules disclosed herein encoding a tPA variant. In some embodiments, the vector further comprises a promoter operable linked to the nucleic acid molecule encoding the tPA variant. The promoter can be a ubiquitous promoter or a constitutive promoter. Non-limiting examples of constitutive and ubiquitous promoters within the scope of the present disclosure include RNA pol I promoter, RNA pol II promoter, RNA pol III promoter, cytomegalovirus (CMV), CAG (chicken betaactin promoter with CMV enhancer), and PGK (phosphoglycerate kinase 1) promoters. In certain embodiments, the promoter can be an inducible promoter that can control when a tPAvariant gene is expressed. Non-limiting examples of inducible promoters include tetracycline-regulated promoters (tet on or tet off), including but not limited to the TRE3GV promoter, and steroid-regulated promoters derived from glucocorticoid or estrogen receptors. Alternatively or in addition, the promoter can be a tissue specific promoter. Tissue specific promoters are known in the art, and any appropriate tissue specific promoter may be used to control the expression of tPA in the desired tissue. In some embodiments, the promoter is specific for cells of the eye, for example, a promoter active in trabecular meshwork cells.

[0115] Expression of tPA from a gene therapy vector can also be transient, that is, temporary’ expression of the tPA variant gene over a limited time span. Transient expression may be achieved by use of a non-integrative vector, where the vector is lost from the cell or cell population over time, or by use of an inducible promoter in an integrative or non-integrative vector that can be manipulated to stop or reduce expression of the reprogramming gene after a period of time. Notwithstanding, it is understood that certain non-integrative vectors can maintain expression for an extended period of time in a cell, e.g, anon-proliferative cell.

[0116] In some embodiments, the vectors disclosed herein further comprises a 5’ untranslated region (UTR) operably linked to the nucleic acid molecule encoding the tPA variant and / or to the promoter. In some embodiments, the 5’ UTR comprises a 5’ UTR sequence of a PLAT gene, for example, from the same species as the tPA from which the variant is derived. In some embodiments, a vector comprises a 5’ UTR sequence of a human PLAT gene operably linked to a nucleic acid encoding a human tPA variant. In some embodiments, the 5’ UTR sequence of a human PLAT gene comprises a nucleotide sequence at least 70% (e.g. , at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%. at least 99%. or 100%) identical toAGAGCTGAGATCCTACAGGAGTCCAGGGCTGGAGAGAAAACCTCTGCGAGGAA AGGGAAGGAGCAAGCCGTGAATTTAAGGGACGCTGTGAAGCAATC (SEQ ID NO: 58). In some embodiments, the 5’ UTR comprises a 5’ UTR sequence of the gene corresponding to the promoter. For example, in some embodiments, a vector comprises a promoter and a 5’ UTR sequence of the same eukaryotic gene.

[0117] In some embodiments, the vectors disclosed herein can further comprise markers to identify and / or select transduced cells. Examples of selectable markers include visual markers such as green fluorescent protein (GFP), red fluorescent protein (RFP), or fluorescein; epitope markers such as His, c-myc, GST, Flag, or HA tags;enzymatic / nutritional markers such as DHFR (dihydrofolate reductase); or antibiotic resistance markers such as neomycin, puromycin, blasticidin, or hygromycin.

[0118] Non-limiting examples of gene therapy vectors that are within the scope of the present disclosure include adeno-associated virus (AAV) vectors, including AAV2 vectors and self- complimentary AAV2 (scAAV2) vectors, lentivirus vectors, and non-viral vectors.

[0119] AAV is a small, nonenveloped icosahedral virus of the genus Dependoparvovirus and family Parvovirus. AAV has a single-stranded linear DNA genome of approximately 4.7 kb. AAV is capable of infecting both dividing and quiescent cells of several tissue types, w ith different AAV serotypes exhibiting different tissue tropism. Wild-type AAV genomes generally contain two 145 nucleotide ITRs, which contain signal sequences directing AAV replication, genome encapsidation and integration. In addition to the ITRs, three AAV promoters, p5, pl9, and p40, drive expression of two open reading frames encoding rep and cap genes. Two rep promoters, coupled with differential splicing of the single AAV intron, result in the production of four rep proteins (Rep 78, Rep 68, Rep 52. and Rep 40) from the rep gene. Rep proteins are responsible for genomic replication. The Cap gene is expressed from the p40 promoter, and encodes three capsid proteins (VP1, VP2, and VP3) which are splice variants of the cap gene. These proteins form the capsid of the AAV particle.

[0120] Because the cA-acting signals for replication, encapsidation, and integration are contained within the ITRs, some or all of the 4.3 kb internal genome may be replaced with foreign DNA, for example, an expression cassette for an exogenous gene of interest. Accordingly, in certain embodiments, the AAV vector comprises a genome comprising an expression cassette for an exogenous gene flanked by a 5’ ITR and a 3’ ITR. The ITRs may be derived from the same serotype as the capsid or a derivative thereof. Alternatively, the ITRs may be of a different serotype from the capsid, thereby generating a pseudotyped AAV. In certain embodiments, the ITRs are derived from AAV-2 or AAV-5.

[0121] At least one of the ITRs may be modified to mutate or delete the terminal resolution site, thereby allowing production of a self-complementary AAV vector. Self-complementary AAV (scAAV) are AAV wherein the coding region has been designed to form an intramolecular double-stranded DNA template. Accordingly, upon infection, the two complementary’ halves of scAAV will associate to form one double-stranded DNA unit that is ready for immediate nuclear transportation and transcription. Use of scAAV removes the rate-limiting step of traditional AAVs, which is second-strand synthesis by the infected cellmachinery. However, scAAV comes at a reduced packing capacity (2.4 kb for scAAV versus 4.7-6 kb for AAV).

[0122] AAV vectors may have single-stranded genomes that are 4.7 kb in size, or are larger or smaller than 4.7 kb. including oversized genomes that are as large as 5.2 kb. or as small as 3.0 kb. Thus, where the exogenous gene of interest to be expressed from the AAV vector is small, the AAV genome may comprise a stuffer sequence. Further, vector genomes may be substantially self-complementary' thereby allowing for rapid expression in the cell. In certain embodiments, the genome of a self-complementary AAV (scAAV) vector comprises from 5’ to 3’: a 5’ ITR; a first nucleic acid sequence comprising a promoter and / or enhancer operably linked to a coding sequence of a gene of interest; a modified ITR that does not have a functional terminal resolution site; a second nucleic acid sequence complementary or substantially complementary to the first nucleic acid sequence; and a 3‘ ITR. AAV vectors containing genomes of all types are suitable for use in the method of the present disclosure.

[0123] Non-limiting examples of AAV vectors include pAAV-MCS (Agilent Technologies), pAAVK-EFla-MCS (System Bio Catalog No. AAV502A-1), pAAVK-EFla-MCSl-CMV- MCS2 (System Bio Catalog No. AAV503A-1), pAAV-ZsGreenl (Clontech Catalog No. 6231), pAAV-MCS2 (Addgene Plasmid No. 46954), AAV-Stuffer (Addgene Plasmid No. 106248), pAAVscCBPIGpluc (Addgene Plasmid No. 35645), AAVSl_Puro_PGKl_3xFLAG_ Twin Strep (Addgene Plasmid No. 68375). pAAV-RAM- d2TTA::TRE-MCS-WPRE-pA (Addgene Plasmid No. 63931), pAAV-UbC (Addgene Plasmid No. 62806), pAAVSl-P-MCS (Addgene Plasmid No. 80488), pAAV-Gateway (Addgene Plasmid No. 32671), pAAV-Puro_siKD (Addgene Plasmid No. 86695), pAAVSl- Nst-MCS (Addgene Plasmid No. 80487), pAAVS 1 -Nst-C AG-DEST (Addgene Plasmid No. 80489), pAAVS 1-P-CAG-DEST (Addgene Plasmid No. 80490), pAAVf-EnhCB-lacZnls (Addgene Plasmid No. 35642), and pAAVSl-shRNA (Addgene Plasmid No. 82697). These vectors can be modified to be suitable for therapeutic use. For example, an exogenous gene of interest can be inserted in a multiple cloning site, and a selection marker (e.g, puro or a gene encoding a fluorescent protein) can be deleted or replaced with another (same or different) exogenous gene of interest. Further examples of AAV vectors are disclosed in U.S. Patent Nos. 5,871,982, 6,270,996, 7,238,526, 6,943,019, 6,953,690, 9,150,882, and 8,298,818, U.S. Patent Publication No. 2009 / 0087413, and PCT Publication Nos. WO2017075335A1, WO2017075338A2, and WO2017201258A1.

[0124] The rep and cap proteins can be provided in trans, for example, on a plasmid, to produce an AAV vector. A host cell line permissive of AAV replication must express the rep and cap genes, the ITR-flanked expression cassette, and helper functions provided by a helper virus, for example adenoviral genes Ela, Elb55K, E2a, E4orf6, and VA (see, Weitzman et al., Adeno-associated virus biology. Adeno-Associated Vims: Methods and Protocols, pp. 1-23, 2011). Methods for generating and purifying AAV vectors have been described in detail (see, e.g., Mueller et al.. (2012) Current Protocols in Microbiology, 14D.1.1-14D.1.21, Production and Discovery of Novel Recombinant Adeno-Associated Viral Vectors). Numerous cell types are suitable for producing AAV vectors, including HEK293 cells, COS cells, HeLa cells, BHK cells, Vero cells, as well as insect cells such as SF9 cells (see, e.g. U.S. Patent Nos. 6.156,303, 5.387,484, 5,741.683, 5,691,176. 5,688,676, and 8,163,543, U.S. Patent Publication No. 20020081721, and PCT Publication Nos. WOOO / 47757, WOOO / 24916, and WO96 / 17947). AAV vectors are typically produced in these cell types by one plasmid containing the ITR-flanked expression cassette, and one or more additional plasmids providing the additional AAV and helper vims genes. AAV particles may be purified, for example by affinity chromatography, iodixonal gradient, or CsCl gradient.

[0125] AAV includes numerous serologically distinguishable types including serotypes AAV-1 to AAV- 12, as well as more than 100 serotypes from nonhuman primates (see, e.g., Srivastava (2008) J. CELL BIOCHEM., 105(1): 17-24, and Gao et al. (2004) J. VIROL., 78(12), 6381-6388). The serotype of the AAV vector used in the present disclosure can be selected by a skilled person based on the efficiency of delivery, tissue tropism, and immunogenicity. For example, AAV-1, AAV-2, AAV -4, AAV-5, AAV-8, and AAV-9 can be used for delivery to the central nervous system; AAV-2, AAV-5, and AAV-8 can be used for deliver)' to the photoreceptor cells; AAV-1, AAV-2, AAV-4, AAV-5, and AAV-8 can be used for delivery to the retinal pigment epithelium. In certain embodiments, the AAV capsid protein comprises a sequence as disclosed in U.S. Patent No. 7,198,951, including but not limited to, AAV-2 (SEQ ID NO: 4 of U.S. Patent No. 7,198,951), AAV-1 (SEQ ID NO: 5 of U.S. Patent No. 7,198,951), AAV-3 (SEQ ID NO: 6 of U.S. Patent No. 7,198,951), AAV-8 (SEQ ID NO: 7 of U.S. Patent No. 7,198,951), and AAV-9 (SEQ ID NOs: 1-3 of U.S. Patent No. 7,198,951). AAV serotypes identified from rhesus monkeys, e.g., rh.8, rh.10, rh.39, rh.43, and rh.74, are also contemplated. Besides the natural AAV serotypes, modified AAV capsids have been developed for improving efficiency of delivery', tissue tropism, and immunogenicity. Exemplary' natural and modified AAV capsids are disclosed in U.S. PatentNos. 7,906,111, 9,493,788, and 7,198,951, and PCT Publication No. WO2017189964 A2. In some embodiments, the AAV vector is any AAV vector as provided for herein. In some embodiments, the AAV vector is an AAV-2, AAV-2.5, AAV-6, AAV-5, AAV-Anc80L65, or AAV-DJ serotype. In some embodiments, the AAV vector is a scAAV vector, for example, scAAV-2, scAAV-2.5, scAAV-6, scAAV-5, scAAV-Anc80L65, or scAAV-DJ serotype.

[0126] The AAV or scAAV vectors can comprise one or more mutations, e.g, Y444F, Y500F, and / or Y730F, in one or more capsid proteins (see, Petrs-Silva, Hilda et al. (2011) “Novel properties of tyrosine-mutant AAV2 vectors in the mouse retina " MOL. THER. 19, 2: 293-301). In some embodiments, the AAV or scAAV vector comprises capsid proteins VP1, VP2, and VP3 comprising amino acid sequences at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NOs: 17, 18, and 19 respectively:MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYK YLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQ ERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEP DSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMAT GSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNH LYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFR PKRLNFKLFN1QVKEVTQNDGTTT1ANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFS YTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYFLSRTNTPSGTTTQSRLQFSQA GASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEFSWTGATKYHLNGRDSL VNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTN PVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWA KIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYS TGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRFLTRNL (SEQ ID NO: 17)MAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLG QPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDR VITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCH FSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQV FTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYFLSR TNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSE FSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTN VDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPG MVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPA NPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSV NVDFTVDTNGVYSEPRPIGTRFLTRNL (SEQ ID NO: 18)MATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTY NNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNN WGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGS AHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGN NFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYFLSRTNTPSGTTTQSRLQ FSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEFSWTGATKYHLNG RDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEI RTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQG PIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFI TQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSE PRPIGTRFLTRNL (SEQ ID NO: 19)

[0127] In some embodiments, the AAV or scAAV vector comprises capsid proteins VP1, VP2, and VP3 comprising amino acid sequences at least 90% 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 17, 18, and 19, respectively. In some embodiments, the AAV or scAAV vector comprises capsid proteins VP1, VP2, and VP3 comprising amino acid sequences of SEQ ID NOs: 17, 18, and 19, respectively. In some embodiments, the amino acid sequence of the VP1 comprises the amino acid sequence of the VP2, which in turn comprises the amino acid sequence of the VP3.

[0128] In some embodiments, the AAV or scAAV vector comprises capsid proteins VP1, VP2, and VP3 comprising amino acid sequences at least 90% (e.g., at least 95%, at least 96%. at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NOs: 17, 18, and 19 respectively, provided that the sequences comprise the triple Y to F mutations shown in SEQ ID NOs: 17, 18, and 19 (Y to F mutations shown in bold and underlined relative to wild type AAV2 capsid proteins, mutations correspond to Y444F, Y500F, and Y730F of SEQ IDNO: 17). In some embodiments, the AAV or scAAV vector comprises capsid proteins VP1, VP2, and VP3 comprising amino acid sequences at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NOs: 17, 18, and 19 respectively, and comprise a phenylalanine (F) at one or more of amino acid positions 444, 500, 730 corresponding to SEQ ID NO: 17, in each of the capsid proteins. In some embodiments, the AAV or scAAV vector comprises capsid proteins VP1, VP2, and VP3 comprising amino acid sequences at least 90% (e.g.. at least 95%. at least 96%. at least 97%. at least 98%, at least 99%, or 100%) identical to SEQ ID NOs: 17, 18, and 19 respectively, and comprise a phenylalanine (F) at each of amino acid positions 444, 500, 730 corresponding to SEQ ID NO: 17, in each of the capsid proteins.

[0129] In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector is a pseudotyped lentiviral vector.

[0130] AAV (e.g., AAV-2) and lentivirus vectors can provide long term expression of proteins in the trabecular meshwork. Expression can be maintained for at least a period of week, months or years. ScAAV (e.g., scAAV-2) vectors have also been used for the longterm transfection of animals (up to 2 years) without adverse effects. Some of the tPA variants of the present disclosure have reduced sizes, which may be packaged in scAAV (e.g, scAAV-2).Pharmaceutical compositions

[0131] The present disclosure also provides a pharmaceutical composition comprising a tPA variant, nucleic acid molecule, or vector and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a tPA variant as disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a nucleic acid molecule encoding a tPA variant as disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a vector (e.g., AAV vector) comprising a nucleic acid molecule encoding a tPA variant as disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a cell that produces the tPA variant (e.g. , comprising a nucleic acid or vector encoding the tPA variant) and a pharmaceutically acceptable carrier.

[0132] A pharmaceutically acceptable carrier is within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers suitable for the present disclosure include buffers, solvents, excipients, dispersion media, coatings, isotonic and absorption delaying agents, and the like, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or w ater / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g.. Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23 d ed. 2020).

[0133] In certain embodiments, a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH. osmolarity, viscosity, clarity, color, isotonicity', odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight poly peptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or w etting agents (such as pluronics, PEG. sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); diluents; excipients and / or pharmaceuticaladjuvants. Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0134] In certain embodiments, a pharmaceutical composition may contain nanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles see, Anselmo et al. (2016) BIOENG. TRANSL. MED. 1: 10-29).

[0135] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled- delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g, porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L- glutamate, ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.

[0136] Pharmaceutical compositions containing a tPA variant protein, nucleic acid, vector and / or viral particle disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intracameral injection, intravitreal injection, topical application, or by an implantable device.

[0137] Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

[0138] Ingredients which may be desirable to use in the ophthalmic preparations of the pharmaceutical compositions disclosed herein include preserv atives, co-solvents, buffers, viscosity building agents and penetration enhancers. Viscosity building agents, such ashydroxymethyl cellulose, hydroxyethyl cellulose, methylcellulose, polyvinylpyrrolidine, a polymer matrix such as CAPA4101 or the like, may be added to the compositions of the present invention to improve the retention of the compound in the conjunctival sac or surrounding area. In order to prepare sterile ophthalmic ointment formulations, the tPA variants or vectors disclosed herein may be combined with a preservative in an appropriate vehicle, such as white petroleum, mineral oil or liquid lanolin. Sterile ophthalmic gel formulations may be prepared by suspending the tPA therapeutic agent in a hydrophilic base prepared from the combination of, for example, carbopol-940, or the like, according to the methods know n in the art for other ophthalmic formulations.

[0139] In some embodiments, a use of a tPA variant as disclosed herein is provided. In some embodiments, the use of the tPA variant as disclosed herein is in the preparation of a medicament. In some embodiments, a use of a vector comprising a nucleic acid molecule encoding atPA variant as disclosed herein is provided. In some embodiments, the use of the vector is in the preparation of a medicament.Methods of treatment

[0140] Intraocular pressure (IOP). the fluid pressure within the eye, can be measured in units of millimeters of mercury (mmHg) or kilopascals (kPa). Normal intraocular pressure is typically considered to be between 10 mmHg and 20 mmHg. The average value of intraocular pressure is 15.5 mmHg with fluctuations of about 2.75-3.50 mmHg. Elevated intraocular pressure (above 21 mmHg or 2.8 kPa) is the most important and only modifiable risk factor for glaucoma.

[0141] Open angle glaucoma, e.g, steroid-induced glaucoma, is caused by IOP elevation resulting from a decrease in aqueous humor outflow' facility'. Although the exact mechanism remains unclear, increased extracellular matrix (ECM) deposition in the trabecular meshwork (TM) has been reported and is consistently detected in both glaucomatous human specimens and animal models of the disease.

[0142] The ECM structure of the conventional outflow pathw ay is dynamic and continually remodeled by matrix metalloproteinases (MMPs). MMPs are zinc endopeptidases that are secreted in their zymogen (pro-MMP) form for subsequent activation via proteolytic cleavage. The expression and activity of certain MMPs, such as MMP-2, MMP-9 MMP-13, and MMP-1, are reduced in cases of primary open angle glaucoma and in animal models of ocular hypertension. Conversely, administration of exogenous MMPs, during anteriorsegment organ culture perfusion experiments in vitro or in animal models in vivo, increases outflow facility’.

[0143] MMP regulation occurs via cytokine-dependent transcriptional control and via proteolytic post-translational activation. tPA functions as a cytokine by promoting intracellular signaling cascades and gene expression changes following interactions with cell surface receptors, such as low-density-lipoprotein receptor-related protein 1 (LRP-1) and N- methyl-D-aspartate receptor (NMD AR). By this mechanism, tPA enhances MMP transcriptional expression in brain, retinal, lung and renal tissues.

[0144] Previous studies have found that steroids cause a reduction in tPA at the TM and that exogenous administration of tPA can prevent and reduce steroid-induced IOP elevation in sheep and prevent steroid-induced reduction of outflow facility in mice. On the other hand, deletion of the gene encoding tPA (Plat) in mice causes a significant reduction in outflow facility. This effect is associated with a reduction in Mmp-9 expression in angle ring tissues of tPA deficient mice. Furthermore, over-expression of tPA in steroid treated mice results in increased expression of Mmp-2, Mmp-9 and Mmp-13 in angle ring tissue.

[0145] Without being bound to any particular theory, the tPA variants of the present disclosure are beneficial for the treatment of IOP and IOP related conditions through preservation of critical protein domain contacts necessary' for tPA’s role in regulating IOP, such as the Finger domain and the Kringle 2 domain. Elimination of protein domains or critical residues responsible for tPA’s function as a serine protease is also contemplated.

[0146] It is understood that the tPA variant may play a cytokine role by binding LPR-1 via its Finger domain and binding NMD AR via its Kringle 2 domain. For a more in depth review of tPA protein binding partners, see Mulder, M et al. (1997) BLOOD COAGULATION & FIBRIOLYSIS vol. 8, 2: 124-33; Fukao, H et al. (2000) LIFE SCIENCES vol. 66, 25: 2473- 87; Parmer, R. et al. (2020) J. NEUR. DIS. & STROKE vol. 7,1: 1153; Grobmyer, S. R. et l. (1993) J. BIOL. CHEM. vol. 268, 18: 13291-300; and Stockinger, H. et (1992) THROMBOSIS RES. vol. 67, 5: 589-99.

[0147] Regarding its role as a serine protease. tPA has enzymatic activity and can degrade many cell and extracellular matrix proteins. For this reason, it is tightly regulated both at the transcriptional level as well as through specific protein inhibitors (serpins) as well as binding proteins. Overproduction of tPA or exogenous supplementation can overwhelm the inhibitory action of these proteins, allowing tPA to degrade potentially useful proteins thatcan adversely affect the eye tissue physiology. The enzymatic activity of tPA has been well characterized and is dependent on the presence of an active site serine in position 478. Conversion of the active site serine-478 to an alanine reduces and / or removes the protein's enzymatic activity but still allows tPA to bind to other protein partners. Similarly, removal or all or most of the catalytic domain of tPA would also accomplish this goal.

[0148] Accordingly, the methods and compositions described herein can be used to treat lOP-associated conditions. Non-limiting examples of lOP-associated conditions include, but are not limited to, ocular hypertension, glaucoma, including primary glaucomas such as open angle glaucoma, certain types of closed angle glaucoma and developmental glaucoma that have partially open angle, and secondary glaucoma, such as but not limited to steroid-induced glaucoma, pigmentary glaucoma and pseudoexfoliation glaucoma.

[0149] In some embodiments, administration of a tPA variant, vector, or pharmaceutical composition as disclosed herein improves aqueous humor outflow in the eye. Aqueous humor is the clear, watery fluid that fills the complex space in the front of the eye which is bounded at the front by the cornea and at the rear by the front surface or face of the vitreous humor. Production, circulation, and drainage of aqueous humor into and out of the anterior chamber of the eye maintains the IOP at a relatively constant level. The trabecular meshwork is a sponge-like tissue located near the cornea and iris that functions to drain the aqueous humor from the eye. The trabecular meshwork offers a certain resistance to the outflow of aqueous humor that is needed to maintain a steady-state IOP. The inverse of this resistance is trabecular outflow facility, a measure of the conductivity of the trabecular meshwork. In glaucomatous eyes the resistance to aqueous humor outflow is increased due to an increase in different forms of extracellular material deposited within the meshwork, which decreases outflow facility'.

[0150] Provided herein is a method of increasing outflow facility in a subject in need thereof. The method comprises administering to the subject an effective amount of a tPA variant as disclosed herein, thereby increasing outflow facility in the subject. This can be achieved by administering an effective amount of a vector encoding a tPA variant or a pharmaceutical composition disclosed herein. In some embodiments, the subject has open angle glaucoma, for example, juvenile open angle glaucoma. The tPA variant or pharmaceutical composition comprising the same or the vector encoding the tPA variant or pharmaceutical composition comprising the same can be administered by intracameral injection, intravitreal injection,topical application, an implantable device, or an implantation of cells that produce the tPA variant.

[0151] Also provided herein is a method of treating elevated intraocular pressure in a subj ect in need thereof. The method comprises administering to the subject an effective amount of a tPA variant as disclosed herein, thereby treating the elevated intraocular pressure in the subject. This can be achieved by administering an effective amount of a vector encoding a tPA variant or pharmaceutical composition disclosed herein. In some embodiments, the subject has open angle glaucoma, for example, juvenile open angle glaucoma. The tPA variant or pharmaceutical composition comprising the same or the vector encoding the tPA variant or pharmaceutical composition comprising the same can be administered by intracameral injection, intravitreal injection, topical application, an implantable device, or an implantation of cells that produce the tPA variant.

[0152] Also provided is a method of treating chronic elevated intraocular pressure in a subject in need thereof is provided. The method comprises administering to the subject an effective amount of a tPA variant as disclosed herein, thereby treating the chronic elevated intraocular pressure in the subject. This can be achieved by administering an effective amount of a vector encoding a tPA variant or pharmaceutical composition disclosed herein. In some embodiments, the subject has open angle glaucoma, for example, juvenile open angle glaucoma. The tPA variant or pharmaceutical composition compnsing the same or the vector encoding the tPA variant or pharmaceutical composition comprising the same can be administered by intracameral injection, intravitreal injection, topical application, an implantable device, or an implantation of cells that produce the tPA variant.

[0153] Also provided is a method of treating an lOP-associated condition in a subject in need thereof. The method comprises administering to the subject an effective amount of a tPA variant as disclosed herein, thereby treating the lOP-associated condition in the subject. This can be achieved by administering an effective amount of a vector encoding a tPA variant or a pharmaceutical composition disclosed herein. In some embodiments, the subject has open angle glaucoma, for example, juvenile open angle glaucoma. The tPA variant or pharmaceutical composition comprising the same or the vector encoding the tPA variant or pharmaceutical composition comprising the same can be administered by intracameral injection, intravitreal injection, topical application, an implantable device, or an implantation of cells that produce the tPA variant. Non-limiting examples of lOP-associated conditions include, but are not limited to, ocular hypertension, glaucoma, including primary glaucomassuch as closed angle glaucoma and open angle glaucoma, developmental glaucoma, and secondary glaucoma, such as but not limited to steroid-induced glaucoma, pigmentary glaucoma and pseudoexfoliation glaucoma.Routes of administration

[0154] The tPA variants, vectors, or pharmaceutical compositions of the present disclosure can be administered to an eye of a patient as solutions, suspensions, or emulsions (dispersions). For example, the composition can be delivered topically to the eye in the form of drops, sprays, or gels. It can also be absorbed into contact lens or other non-biodegradable or biodegradable material that is placed on the cornea or conjunctiva. Alternatively, the tPA variants, vectors, or pharmaceutical compositions can be administered by injection (e.g., intracameral, intravitreal, intraorbital, subconjunctival, supraciliary and / or sub-tenon injection). The tPA variants, vectors, or pharmaceutical compositions can also be administered by means of an implantable device, which can be attached, for example, to a subconjunctival, anterior chamber or vitreous region of the eye. For administration to a patient, the agent or composition is prepared with pharmaceutically acceptable ophthalmological carriers, excipients, or diluents.

[0155] Proteins like tPA variants are preferably administered intraocularly, as their penetration of the ocular wall is limited. Intracameral injections (injections into the anterior chamber) are easier to perform, but proteins injected there are cleared fairly rapidly. Proteins injected intravitreally are eliminated in large part (- 70%) through the anterior chamber. Because diffusion in the vitreous is delayed, proteins injected there have a longer duration of action. Accordingly, in some embodiments, the tPA variants, vectors, or pharmaceutical compositions disclosed herein are administered to the patient via intravitreal injection.

[0156] Other sites and modes of administration include topical administration, administration via iontophoresis; implantation of cells that are genetically engineered to constantly produce atPA variant disclosed herein; implantation of slow release device; and subconjunctival administration. A review of methods for administration of ophthalmic drugs can be found in Kompella, el al.. (2010) THER. DELIV. 1:435-456.

[0157] Also contemplated is delivery of a tPA variant by implantation of cells that are genetically engineered to constantly produce the tPA variant within the eye. Such engineered cells may, for example, reside within a permeable device that allows diffusion of their protein products in the eye (such as Encapsulated Cell Technology, available fromNeurotech Pharmaceuticals). Such a device or cells can be implanted surgically in the posterior or anterior segment of the eye and provide for extended administration of specific doses of tPA.

[0158] Also contemplated is delivery of a tPA variant by implantation of slow release devices within the eye. A tPA variant can be formulated in a slowly biodegradable substrate, for example, poly(lactic-co-gly colic) acid (PLGA) or polylactic acid (PLA), which can be implanted in the anterior (or posterior) segments surgically and allowed to release the tPA variant over long periods of time (up to, or even more than, 2 years). The device can also reside outside the eye (e.g, in the subconjunctival space) and connect with the anterior chamber (AC) via a small tube. In such case discharge of the medication can be controlled externally, and the device can be refillable.

[0159] Subconjunctival administration (injection through the conjunctiva) is also contemplated herein.

[0160] Generally, a therapeutically effective amount of active component, for example, a tPA variant or a vector disclosed herein, can be determined by a person of medical skills according to the type and extent of disease or indication to be treated, the overall health of the patient, the form of the active component, the pharmaceutical formulation, and the route of administration. tPA has been used in acute situations by intracameral injection usually at a dosages of 10-25 pg (Kim, M. et al., (1998) Ophthalmic Surg Lasers 29(9): 762-766, Wu, T. et al.. (2009) Eye (Land) 23(1): 101-107). For intravitreal use, tPA has been used to dissolve sub-macular hemorrhages at a dosage of 30-100 pg (Chen, C. et al., (2007) Retina 27(3) : 321 - 328. In some embodiments, the therapeutically effective amount of a tPA variant is in the range of 0. 1 ng to 10 pg per eye. Turnover of aqueous humor is approximately 120 minutes. Assuming a linear model of elimination, in one hour about 50% of the administered tPA variant remains in the aqueous humor. In some embodiments, the therapeutically effective amount of a tPA variant administered by a slow-release formulation or device is in the range of 10 pg to 5 ng per hour for each eye, or in the range of 0. 1 ng to 100 ng per day for each eye.

[0161] In the case of viral delivery of a nucleic acid encoding a tPA variant, a therapeutically effective amount of a vector encoding the tPA variant is in the range of 105to 1015vector genomes per eye.

[0162] Dosing frequency can vary, depending on factors such as route of administration, dosage amount, the rate of degradation of the active component, and the disease being treated. The tPA variants, vectors, or pharmaceutical compositions can be administered for at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 years. The tPA variants, vectors, or pharmaceutical compositions can be administered on a recurrent or repeated basis, such as on a daily, weekly, bi-weekly, monthly, bi-monthly, or on an annual basis, to provide a reduction in IOP over periods of time such as 1 day to 4 weeks, 1 to 12 months, or a year or more. Less frequent administration of a composition that provides extended release or extended expression of a tPA variant is contemplated. This recurrent basis differs from the frequency at which tPA is administered to treat acute fibrin build-up, such as administration of a single or limited number of intraocular tPA injections, in the immediate post-operative period following ocular surgery, to reverse fibrin accumulation. The approaches described herein can be performed in the absence of apparent fibrin accumulation, as a long-term solution to the problem of chronic elevated IOP.

[0163] The tPA variant, vector, or pharmaceutical composition disclosed herein can be administered to the subject for as long as the condition and / or elevated IOP persists, in any manner that provides extended administration of the tPA therapeutic agent and / or long-term reduction of IOP. In some embodiments, the IOP is reduced to within normal levels of 10-20 mmHg. In some embodiments, the IOP is reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mmHg or more in the subject, relative to the IOP of the subject prior to treatment. For example, treatment of a glaucoma patient can include lowering IOP and / or preventing, reducing, or ameliorating eye pain, optic nerve damage, retinal cell damage, or retinal cell loss. Although lowering IOP is preferably lowering IOP to within normal levels of 10-20 mmHg, any lowering of IOP, such as by 1-10 mmHg, 10-20 mmHg, or 20 mmHg or more in a subject, relative to before treatment was commenced, is considered to be effective. In some embodiments, the reduction of IOP is maintained for 1 day to 4 weeks, 1 to 12 months, or a year or more.

[0164] Depending upon the circumstances, the methods and compositions provided herein can be used to improve ocular outflow facility, prevent, reduce or ameliorate eye pain, reduce optic nerve damage, retinal cell damage, or retinal cell loss, prevent the advancement of an lOP-associated condition; cause regression of an lOP-associated condition and / or enhance orimprove the therapeutic effect(s) of additional treatment(s) administered to ameliorate an lOP-associated condition.EXAMPLES

[0165] The following examples are illustrative, but not limiting, of the compounds, compositions, particles, polypeptides, and methods described herein.Example 1: tPA variants upregulate MMP9 in cerebral microvascular endothelial cells

[0166] Variants of tPA were designed and tested for their ability to upregulate MMP9 expression, which was known to be associated with increase of outflow faci 1 i ty (FIG. 2A). Ovine tPA Variant 1, after removal of its signal peptide and pro-domain (SEQ ID NO: 13), has the amino acid sequence of Chain A of mature ovine tPA, followed by a proline residue at the C-terminus. Ovine tPA Variant 2 includes fragments of tPA Variant 1. with removal of the Finger domain and the EGF-like domain. Ovine tPA Variant 3 includes fragments of tPA Variant 1, with removal of the Kringle 1 domain. The amino acid sequences of the tPA variants designed are shown in Table 5 below:Table 5: Exemplary amino acid sequences of tPA variants

[0167] Exemplary nucleic acid sequences of the tPA variants designed are show n in Table 6 below; Due to the degenerate nature of codons, the skilled artisan will readily recognize other nucleic acid sequences that encode the ovine tPA variants and can be expressed in ovine tissues.Table 6: Exemplary nucleic acid sequences of tPA variants

[0168] The tPA variants were inserted into self-complementary AAV (scAAV) genomes under the control of a CMV promoter, and packaged into AAV2 capsids that include triple Y- to-F mutations (Petrs-Silva, H. et al. (2011) MOL. THER. vol. 19,2: 293-301) to generate AAV vectors. Cerebral microvascular endothelial cells were transfected with the AAV vectors and MM P transcription was assessed. An scAAV vector encoding a GFP transgene was used as a negative control. As shown in FIG. 3, transfection of Ovine tPA Variant 3 resulted in an increase in average MMP9 transcription over the negative control. These results suggest that the Kringle 1 domain has a negative impact on the ability of tPA to upregulate MMP9 transcription.

[0169] It is expected that human tPA variants, e.g., those having amino acid sequences of SEQ ID NOs: 10-12 (FIG. 2B), can also be assessed using this experimental system.Example 2: Ovine tPA Variant 3 reduced intraocular pressure and increased outflow facility in a mouse model

[0170] To test the ability of tPA variants to affect outflow' facility, a steroid-induced mouse model of outflow' facility reduction was utilized.

[0171] Briefly. C57BL / 6 mice were injected periocularly in both eyes with dexamethasone (50pL, 4mg / ml) to induce outflow facility reduction. Periocular injections of dexamethasone w ere repeated on a w eekly basis to maintain a reduced outflow facility for the duration of the study. Concurrently with the initial dexamethasone periocular injection, an scAAV construct delivering Ovine tPA Variant 3 transgene or a control scAAV construct delivering a GFP transgene were administered via intracameral injection (2pL, 1-3 x 1012vg / ml) incontralateral eyes. Previous work has demonstrated robust GFP expression approximately 3 weeks after intracameral injection of scAAV-GFP. Therefore, 3 weeks was elected as the end-point for the mouse study. IOP was measured weekly until study end-point, at which time the animals were sacrificed, eyes were enucleated and outflow facility’ was measured. The results of the study are shown in FIG. 4.

[0172] As shown in FIG. 4A, dexamethasone inj ection resulted in an increase of IOP by approximately 5 mmHg that was sustained for the duration of the study in control treated animals. The eyes treated with the scAAV delivering Ovine tPA Variant 3 (scAAV - PLATvar) exhibited a return to baseline IOP at three weeks post vector injection. The reduction in IOP at three w eeks correlates with the outflow facility data presented in FIG. 4B. which shows that the eyes treated with scAAV delivering Ovine tPA Variant 3 (scAAV- PLATvar) exhibited increased outflow^ facility. These results demonstrate that Ovine tPA Variant 3, a variant without the catalytic domain or the Kringle 1 domain, is able to successfully modulate IOP and increase outflow facility in a steroid-induced mouse model of outflow facility reduction.

[0173] It is expected that human tPA vanants, e.g., those having amino acid sequences of SEQ ID NOs: 10-12 (FIG. 2B), can also be assessed by this animal model.Example 3: Ovine tPA Variant 3 reduced intraocular pressure in a sheep model

[0174] The ability of select tPA variants to modulate IOP was assessed in a sheep model of steroid induced elevated IOP. Sheep were treated with Prednisolone acetate 0.5% three times daily for the duration of the study to induce and maintain IOP elevation. 10 days after initiation of steroid treatment, each animal (N=6) received intracameral injections of either an scAAV construct delivering Ovine tPA Variant 3 transgene or a control scAAV construct delivering a GFP transgene (50pL, 1-3 x 1012 vg / mL) in contralateral eyes. IOP was monitored biweekly for the duration of the study.

[0175] As shown in FIG. 5, a significant reduction in IOP was noted in eyes treated with the scAAV delivering the Ovine tPA Variant 3 (scAAV -PL ATvar) as compared to eyes treated with the control vector. No significant clinical adverse events (e.g., media clarity, fundus changes, evidence of inflammation) w ere observed in any of the eyes.

[0176] These examples and results demonstrate that Ovine tPA Variant 3, a variant without the catalytic domain or the Kringle 1 domain, is able to successfully reduce steroid induced IOP in sheep over a prolonged period of time.

[0177] It is expected that human tPA variants, e.g., those having amino acid sequences of SEQ ID NOs: 10-12 (FIG. 2B), can also be assessed by this animal model.INCORPORATION BY REFERENCE

[0178] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0179] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the disclosure described herein. Various structural elements of the different embodiments and various disclosed method steps may be utilized in various combinations and permutations, and all such variants are to be considered forms of the disclosure. The scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. A tissue plasminogen activator (tPA) variant comprising a Finger domain and a Kringle 2 domain of tPA, wherein the tPA variant does not comprise a Kringle 1 domain.

2. The tPA variant of claim 1, wherein the Finger domain of tPA comprises an amino acid sequence at least 70% (e.g, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 2.

3. The tPA variant of claim 1 or 2, wherein the Kringle 2 domain of tPA comprises an amino acid sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 5.

4. The tPA variant of any one of claims 1-3, wherein the tPA variant does not comprise the amino acid sequence of SEQ ID NO: 4.

5. The tPA variant of any one of claims 1-3, wherein the tPA variant comprises an amino acid sequence no greater than 50% (e.g., 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) identical to SEQ ID NO: 4.

6. The tPA variant of any one of claims 1-5, wherein the tPA variant does not comprise an active catalytic domain of tPA.

7. The tPA variant of claim 6, wherein the tPA variant does not comprise the amino acid sequence of SEQ ID NO: 6.

8. The tPA variant of claim 6, wherein the tPA variant comprises an amino acid sequence no greater than 50% (e.g., 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) identical to SEQ ID NO: 6.

9. The tPA variant of claim 6, wherein the tPA variant comprises a mutation (e.g. , a substitution of Ala) at a position corresponding to amino acid 168 of SEQ ID NO: 6.

10. The tPA variant of any one of claims 1-9, further comprising an EGF-like domain of tPA.

11. The tPA variant of claim 10, wherein the EGF-like domain of tPA comprises an amino acid at least 70% (e.g, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 3.

12. The tPA variant of any one of claims 1-11, comprising a Finger domain, a Kringle 2 domain, and an EGF-like domain of tPA, wherein a C-terminus of the Finger domain is linked, directly or indirectly, to an N-terminus of the EGF-like domain, and a C-terminus of the EGF-like domain is linked, directly or indirectly, to an N-terminus of the Kringle 2 domain.

13. The tPA variant of claim 12, wherein the C-terminus of the Finger domain is linked to the N-terminus of the EGF-like domain by an amino acid sequence of Lys-Ser.

14. The tPA variant of claim 12 or 13. wherein the C-terminus of the EGF-like domain is linked to the N-terminus of the Kringle 2 domain by a peptide linker 3-20 amino acids in length (e.g, consisting of the amino acid sequence of SEQ ID NO: 7 or 59).

15. The tPA variant of any one of claims 1-14, further comprising a tail region comprising an amino acid sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 8, wherein the tail region is linked to a C-terminus of the Kringle 2 domain.

16. The tPA variant of claim 15, wherein the tail region comprises the amino acid sequence of SEQ ID NO: 8 linked to the C-terminus of the Kringle 2 domain by a peptide bond.

17. The tPA variant of any one of claims 1-16, further comprising a catalytic domain fragment of tPA comprising an amino acid sequence at least 70% (e.g, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 9, wherein the catalytic domain fragment of tPA comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 9.

18. The tPA variant of claim 17, wherein the catalytic domain fragment of tPA is linked at a C-terminus of the tPA variant.

19. The tPA variant of any one of claims 1-18, comprising an amino acid sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:

10. 11, or 12.

20. The tPA variant of claim 19, comprising the amino acid sequence of SEQ ID NO: 10.

21. The tPA variant of claim 19, comprising the amino acid sequence of SEQ ID NO: 11.

22. The tPA variant of claim 19, comprising the amino acid sequence of SEQ ID NO: 12.

23. The tPA variant of any one of claims 1-22, wherein the length of the tPA variant is in the range of 125-400 amino acids.

24. The tPA variant of claim 23, wherein the length of the tPA variant is in the range of 125-250 amino acids.

25. A vector comprising a nucleic acid sequence encoding the tPA variant of any one of claims 1-24.

26. The vector of claim 25, wherein the nucleic acid sequence encodes a signal peptide at an N-terminus of the tPA variant.

27. The vector of claim 26, wherein the signal peptide comprises an amino acid sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 13.

28. The vector of claim 27, wherein the nucleic acid sequence encodes an amino acid sequence at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 14, 15, or 16.

29. The vector of any one of claims 25-28, further comprising a promoter operably linked to the nucleic acid sequence encoding the tPA variant.

30. The vector of claim 29, wherein the promoter is active in trabecular meshwork cells.

31. The vector of any one of claims 25-30, wherein the vector is a viral vector.

32. The vector of claim 31 , wherein the viral vector is an adeno-associated virus (AAV) vector.

33. The vector of claim 32, wherein the AAV vector is a self-complementary AAV vector.

34. The vector of claim 32 or 33, wherein the AAV vector comprises capsid proteins VP1, VP2, and VP3 comprising amino acid sequences at least 90% e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NOs: 17, 18, and 19, respectively.

35. Use of the tPA variant of any one of claims 1-24 or the vector of any one of claims 25-34 in the preparation of a medicament.

36. A method of increasing outflow facility7in a subject in need thereof, the method comprising administering to the subject an effective amount of the tPA variant of any one of claims 1-24 or the vector of any one of claims 25-34.

37. A method of treating chronic elevated intraocular pressure in a subject in need thereof, the method comprising administering to the subject an effective amount of the tPA variant of any one of claims 1-24 or the vector of any one of claims 25-34.

38. The method of claim 36 or 37, wherein the subject has open angle glaucoma.

39. The method of claim 38, wherein the subject has juvenile open angle glaucoma.

40. The method of any one of claims 36-39, wherein the tPA variant or vector is administered by intracameral injection, intravitreal injection, topical application, an implantable device, and an implantation of cells that produce the tPA variant.

41. The tPA variant of any one of claims 1 - 24, wherein said variant upregulates the expression of matrix metalloproteinases (MMPs).

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