Tissue plasminogen activator variants and methods of use thereof

Abstract

Provided herein are tissue plasminogen activator (tPA) variant proteins and vectors (e.g., AAV vectors) encoding the tPA variant proteins. 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

Docket No: 100-2369WO01TISSUE PLASMINOGEN ACTIVATOR VARIANTS AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Provisional Patent Application No. 63 / 774,470, filed on March 19, 2025, the entireties of which are hereby incorporated herein by this reference.FI ELD OF THE DISCLOSURE

[0001] The present disclosure relates to tissue plasminogen activator (tPA) variant proteins and gene therapy vectors encoding such variant proteins useful in treating intraocular pressure (IOP)-associated conditions, such as glaucoma, and use thereof,SEQUENCE LISTINGThe instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said.xml copy, created on March 17, 2026, is named 100-2369WO01, and is 210,601 bytes in size.BACKGROUND

[0002] 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.

[0003] Tissue plasminogen activator (tPA) has been found to reduce steroid-induced IOP elevation in animal models. As components of the fibrinolytic system, tPA and urokinase-type 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.Docket No: 100-2369WO01

[0004] tPA 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 (NMDAR) It has been reported that an S478A substitution, which abrogates the catalytic activity of tPA, does not affect its cytokine activity.

[0005] 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

[0006] Applicant has previously developed methods and compositions for treating an intraocular pressure (IOP)-associated condition using variant tPA, see U. S. Patent No.10,946,076 and International Application Nos. PCT / US2022 / 016043 and PCT / US2024 / 047456. The present di sclosure is based upon Applicant’s further development of additional 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.

[0007] 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 unexpected effects on the model systems provided for assaying IOP.

[0008] These tPA variants that Applicant describes offer a number of advantageous properties as opposed to naturally occurring tPA and previous tPA. First, the tPA variants have high cytokine activity, which increases (i.e., upregulates) the expression of matrix metalloproteinases (MMPs) that can improve outflow facility. Second, the tPA variants lack catalytic activity, 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.., the catalytic domain) reduces the sizes of the tPA variants, thereby enabling delivery of nucleic acidsDocket No: 100-2369WO01encoding certain tPA variants by viral delivery vectors having size limits, such as a self- complementary AAV (scAAV) vector. Such delivery methods are particularly suitable for treating chronic ocular diseases.

[0009] Accordingly, the disclosure provides a catalytically inactive tPA (e.g., human tPA) variant that comprises two or more of a Finger domain, an EGF-like domain, a Kringle 1 domain, and a Kringle 2 domain. In certain embodiments, the tPA variant includes at least one of the Finger domain and the EGF-like domain. In certain embodiments, when a complete domain is not included, fragments thereof can be included, except that the tPA variants cannot contain (i) a Finger domain fragment that retain all four cysteine residues (corresponding to positions 1, 31, 33, and 40 of SEQ ID NO: 2) or (ii) a Kringle 1 domain fragment. In certain embodiments, when the tPA variant includes a deletion of the EGF-like domain, it also contains a deletion of the Kringle 1 domain. A tail region or segment may also be included in the tPA variant. In addition, the tPA variant may contain a catalytically inactive fragment of a catalytic domain, such as a lysine-rich fragment (e.., having an amino acid sequence at least 80% identical to SEQ ID NO: 9, 47, or 115) or a fragment in an N-terminal portion of the catalytic domain (e.g., having an amino acid sequence at least 80% identical to SEQ ID NO: 110).

[0010] In one aspect, the disclosure provides a catalytically inactive tPA (e.g., human tPA) variant that comprises a Finger domain, an optional EGF-like domain, a deletion of a Kringle 1 domain, an optional Kringle 2 domain, and an optional tail region; provided that the tPA variant includes at least one of an EGF-like domain and a Kringle 2 domain, and that the tPA variant does not comprise the amino acid sequences of SEQ ID NO: 43, SEQ ID NO: 46, or SEQ ID NO: 49. In some embodiments, the tPA variant further comprises a catalytically inactive fragment of a catalytic domain. The catalytically inactive fragment of the catalytic domain can have an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 110, SEQ ID NO: 115, or SEQ ID NO: 47. In some embodiments, the tPA variant comprises a Finger domain, an EGF-like domain, a deletion of a Kringle 1 domain, a Kringle 2 domain, a tail region, and a catalytically inactive fragment of a catalytic domain. The catalytically inactive fragment of the catalytic domain can have an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 110 or SEQ ID NO: 115. In some embodiments, the tPA variant comprises a Finger domain, an EGF-like domain, a deletion of a Kringle 1 domain, a Kringle 2 domain, aDocket No: 100-2369WO01deletion of a tail region, and a catalytically inactive fragment of a catalytic domain. The catalytically inactive fragment of the catalytic domain can have an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 47. The tPA variant comprising a Finger domain, an optional EGF-like domain, a deletion of a Kringle 1 domain, an optional Kringle 2 domain, and an optional tail region may comprise an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 91, 92, 113, 114, 118, 119, 125, 126, 129, 130, 133, 134, and 176.

[0011] In another aspect, the disclosure provides a catalytically inactive tPA (e.g., human tPA) variant that comprises a fragment of a Finger domain or a deletion of a Finger domain, an EGF-like domain, a Kringle 1 domain, and a Kringle 2 domain. In some embodiments, the fragment of a Finger domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NO: 50, 54, 57, and 60. In some embodiments, the tPA variant comprises a deletion of the Finger domain, an EGF-like domain, a Kringle 1 domain, and a Kringle 2 domain. In some embodiments, the fragment of a Finger domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NO: 50, 57, and 60. In some embodiments, the tPA variant comprises a deletion of the Finger domain, an EGF-like domain, a Kringle 1 domain, and a Kringle 2 domain. In some embodiments, the tPA variant further comprises a tail region. In some embodiments, the tPA variant comprises a deletion of a catalytic domain. The tPA variant comprising a fragment of a Finger domain or a deletion of a Finger domain, an EGF-like domain, a Kringle 1 domain, and a Kringle 2 domain may comprise an amino acid sequence at least 80% (e.g, at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 51, 53, 55, 56, 58, 59, 61, 62, 63, and 64. The tPA variant comprising a fragment of a Finger domain or a deletion of a Finger domain, an EGF-like domain, a Kringle 1 domain, and a Kringle 2 domain may comprise an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 51, 53, 58, 59, 61, 62, 63, and 64.

[0012] In another aspect, the disclosure provides a catalytically inactive tPA (e.g., human tPA) variant that comprises a Finger domain, a fragment of an EGF-like domain or a deletion ofDocket No: 100-2369WO01an EGF-like domain, an optional Kringle 1 domain, and a Kringle 2 domain. In some embodiments, the tPA variant comprises a Finger domain, a fragment of an EGF-like domain, a Kringle 1 domain, and a Kringle 2 domain. In some embodiments, the fragment of the EGF-like domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NO: 65, 68, and 71. In some embodiments, the tPA variant comprises a Finger domain, a deletion of an EGF-like domain, a deletion of a Kringle 1 domain, and a Kringle 2 domain. In some embodiments, the tPA variant further comprises a tail region. In some embodiments, the tPA variant comprises a deletion of a catalytic domain. The tPA variant comprising a Finger domain, a fragment of an EGF-like domain or a deletion of an EGF-like domain, an optional Kringle I domain, and a Kringle 2 domain may comprise an amino acid sequence at least 80% e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 70, 72, 73, 133, and 134.

[0013] In another aspect, the disclosure provides a catalytically inactive tPA (e.g., human tPA) variant that comprises a Finger domain, an EGF-like domain, a Kringle 1 domain, and a fragment of a Kringle 2 domain or a deletion of a Kringle 2 domain. In some embodiments, the tPA variant comprises a Finger domain, an EGF-like domain, a Kringle 1 domain, and a deletion of a Kringle 2 domain. In some embodiments, the tPA variant comprises a Finger domain, an EGF-like domain, a Kringle 1 domain, and a fragment of a Kringle 2 domain. In some embodiments, the fragment of the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 93, 96, 99, 102, and 105. In some embodiments, the tPA variant further comprises a tail region. In some embodiments, the tPA variant comprises a deletion of a catalytic domain. The tPA variant comprising a Finger domain, an EGF-like domain, a Kringle 1 domain, and a fragment of a Kringle 2 domain or a deletion of a Kringle 2 domain may comprise an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 108, 109, 127, and 128.

[0014] In another aspect, the disclosure provides a catalytically inactive tPA (e.g., human tPA) variant that comprises a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, an optional tail region, and a catalytically inactive fragment of a catalytic domain. InDocket No: 100-2369WO01some embodiments, the catalytically inactive fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%. at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 110, 115, and 47. In some embodiments, the catalytically inactive fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 110. In some embodiments, the catalytically inactive fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 115. In some embodiments, the catalytically inactive fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 47. The tPA variant comprising a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, an optional tail region, and a catalytically inactive fragment of a catalytic domain may comprise an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 111, 112, 116, 117, 123, and 124.

[0015] In another aspect, the disclosure provides a catalytically inactive tPA (e.g., human tPA) variant that comprises a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, a deletion of a tail region, and an optional catalytically inactive fragment of a catalytic domain. In some embodiments, the tPA variant comprises a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, a deletion of a tail region, and a catalytically inactive fragment of a catalytic domain. In some embodiments, the catalytically inactive fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g, at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NO: 47. In some embodiments, the tPA variant comprises a Finger domain, an EGF- like domain, a Kringle 1 domain, a Kringle 2 domain, a deletion of a tail region, and a deletion of a catalytic domain. The tPA variant comprising a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, a deletion of a tail region, and an optional catalytically inactive fragment of a catalytic domain may comprise an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NO: 120, 121, 123, and 124.Docket No: 100-2369WO01

[0016] As provided for herein, a tPA variant of the present disclosure may comprise one or more of a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a tail region. The Finger domain can comprise an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 2. In some aspects, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 3. In some aspects, the Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 4. In some aspects, the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g, at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 5. In some aspects, the tail region comprises an amino acid sequence at least 80% (e.g, at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 8.

[0017] A number of specific tPA variants have been designed and tested that have amino acid sequences of SEQ ID NOs: 53, 56, 59, 62, 64, 67, 70, 73, 75, 78, 81, 84, 87, 90, 92, 95, 98, 101, 104, 107, 109, 112, 114, 117, 119, 121, 124, 126, 128, 130, 132, 134, 175, and 176 (mature protein sequences). Also disclosed herein are tPA variants having an amino acid sequence at least 80% (e.g, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 53, 56, 59, 62, 64, 67, 70, 73, 75, 78, 81, 84, 87, 90, 92, 95, 98, 101, 104, 107, 109, 112, 114, 117, 119, 121, 124, 126, 128, 130, 132, 134, 175, or 176. In some embodiments, the tPA variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 59, 62, 64, 67, 70, 73, 92, 95, 98, 101, 104, 107, 109, 112, 114, 117, 119, 121, 124, 126, 128, 130, 134, 175, and 176. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 53, 59, 62, 64, 67, 70, 73, 92, 95, 98, 101, 104, 107, 109, 112, 114, 117, 119, 121, 124, 126, 128, 130, 134, 175, or 176.

[0018] 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.

[0019] Also contemplated herein are nucleic acid molecules encoding the tPA variants provided for herein. In some embodiments, the nucleic acid molecules are operably linked to aDocket No: 100-2369WO01promoter sequence. Tn some embodiments, the nucleic acid molecule further comprises a 5’ UTR sequence In some embodiments, the 5’ UTR sequence comprises a nucleotide sequence having at least 70% (e.g, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to SEQ ID NO: 52. The nucleic acid molecules provided for herein may be incorporated into a vector, such as a viral vector or a non-viral vector.

[0020] 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 an immature tPA variant. For example, the immature tPA variant can have an amino acid sequence at least 80% (e.g., 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, which includes the signal peptide and a pro-domain of tPA cleaved from mature tPA. The tPA variants having amino acid sequences of SEQ ID NOs: 53, 56, 59. 62, 64, 67, 70, 73, 75, 78, 81, 84, 87, 90, 92, 95, 98, 101, 104, 107, 109, 112, 114, 117, 119, 121, 124, 126, 128, 130, 132, 134, 175, and 176, can therefore further include the signal peptide and pro-domain of tPA, resulting in immature protein sequences set forth in SEQ ID NOs: 51, 55, 58, 61, 63, 66, 69, 72, 74, 77, 80, 83, 86, 89, 91, 94, 97, 100, 103, 106, 108, 111, 113, 116, 118, 120, 123, 125, 127, 129, 131, and 133, respectively. Immature tPA variants having an amino acid sequence at least 80% (i.e., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NOs: 51, 55, 58, 61, 63, 66, 69, 72, 74, 77, 80, 83, 86, 89, 91, 94, 97, 100, 103, 106, 108, 111, 113, 116, 118, 120, 123, 125, 127, 129, 131, and 133 are also contemplated. The vector can further include a promoter, e.g., a promoter active in trabecular meshwork cells, operably linked to the nucleic acid molecule encoding the tPA variant,

[0021] The vector disclosed herein can be a viral vector, such as an AA V 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 80% (e.g., 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 NOs: 12, 13, and 14, respectively.Docket No: 100-2369WO01

[0022] The tPA variant or the vector disclosed herein can be used as a medical treatment or in the preparation of a medi cament. 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 a tPA 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

[0023] FIG, 1A-1E illustrate exemplary tPA variant constructs of the present disclosure. FIG. 1A illustrates Variants 0, 1, 2, 3, 4, 5, 11, 12, 13, 14, and 15. FIG. IB illustrates Variants 0, 21, 22, 23, 24, 31, 32, 33, 34, 35, and 36. FIG. 1C illustrates Variants 0, 41, 42, 43, 44, 45, and 46. FIG. ID illustrates Variants 0, 51, 51-K1, 52, 52-K1, 53, 54, and 54-K1. FIG. IE illustrates Variants 0, 101, 103, 115, and 124. The lengths of the domains are not drawn to scale.

[0024] FIG. 2 illustrates MMP9 gene expression in human cerebral microvascular endothelial cells transfected with plasmids encoding tPA variants.DETAILED DESCRIPTION

[0025] The present disclosure is based, in part, upon development of 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.Docket No: 100-2369WO01

[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 valiant 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 the protein domain encompasses any fragment still capable of binding the protein partner.another example, wherein a protein domain binds a protein partner and the binding to the protein partner initiates a signaling cascade, a “functi onal 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.., 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 query sequence 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 analysisDocket No: 100-2369WO01using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (see Karlin et al., (1990) PROC. NATL. ACAD. SCI. 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.Docket No: 100-2369WO01

[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 enzymatic 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 (NMDAR). 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.Human tPA DomainsDocket No: 100-2369WO01

[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 atPA 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:MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKTQMI YQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTC QQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAE CTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKA GKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIG KVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDV PSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGG ILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHK EFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECEL SGYGKHEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGD TRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPG VYTKVTNYLDWIRDNMRP (SEQ ID NO: 1)

[0041] In some embodiments, the amino acid sequence of a mature, human tP A is set forth in SEQ ID NO: 27:SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPV KSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGIS YRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPD RDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESG ASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVL KNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAK HRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEE EQKFEVEK YI VHKEFDDDTY DNDI ALLQLK SD S SRC AQE S S V VRTVCLPP ADLQLPDWTECEL SGYGKHEAL SPF YSERLKEAHVRL YP S SRCT SQHLLN RT VTDNMLC AGDTRSGGPQ ANLHDACQGDSGGPL VCLN DGRMTLVGIIS WGLGCGQKDVPGVYTKVTNYLDWIRDNMRP (SEQ ID NO: 27)Docket No: 100-2369WO01wherein SEQ ID NO: 27 is the mature amino acid sequence of SEQ ID NO: 1 where the signal peptide and pro-peptide sequences of SEQ ID NO: 11 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: Human tPA DomainsChain Domain Sequence SEQ ID NO Signal peptide MDAMKRGLCCVLLLCGAVFVSPSQEIHARF 11 & pro-peptide RRGARFinger domain VICRDEKTQMIYQQHQSWLRPVLRSNRVEY 9 CWCNSGRAQCHSVPVKS EGF-like CSEPRCFNGGTCQQALYFSDFVCQCPEGFA 3 domain GKCCEIDKringle 1 TRATCYEDQGISYRGTWSTAESGAECTNW 4 Chain A domain NSS ALAQK PYSGRRPD A IRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEKringle 2 GNSDCYFGNGSAYRGTHSLTESGASCLPWN 5 domain SMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTTail CGLRQYSQPQFR 8 Catalytic IKGGLFADIASHPWQAAIFAKHRRSPGERFL 6 domain CGGILISSCWILSAAHCFQERFPPHHLTVILG RTYRVVPGEEEQKFEVEKYIVHKEFDDDTYChain B DNDIALLQLK SD S SRC AQES S V VRT VCLPPADLQL PDW TECELSGYGKHE AL SPF YSERLK E AHVRLYP S SRCT SQHLLNRT VTDNMLC AGDTRSGGPQANLHDACQGDSGGPLVCLNDGDocket No: 100-2369WO01RMTLVGIISWGLGCGQKDVPGVYTKVTNY LDWIRDNMRP

[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 (which includes 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., et al., (2012) J. PHARM PHARMACOL.64(8): 1025-1039).

[0044] In some embodiments, a tPA variant of the present disclosure comprises a Finger domain or a fragment thereof, an EGF-like domain or a fragment thereof, a Kringle 1 domain or a fragment thereof, and / or a Kringle 2 domain or a fragment thereof, of tPA (e.g., human tPA). In some embodiments, a tPA variant of the present disclosure may comprise a deletion of one or more of the Finger domain, the EGF-like domain, the Kringle 1 domain, or the Kringle 2 domain, provided that the tPA variant does not comprises a deletion of both the Finger domain and the EGF-like domain or a deletion of both the Finger domain and the Kringle 1 domain.

[0045] In the context of the present disclosure, unless specifically stated otherwise, a “deletion” of a domain refers to any embodiment wherein the deleted domain retains no greater than 10 amino acids (including no amino acid) of the corresponding full-length wild-type domain. Accordingly, a tPA variant which comprises a deletion of a given domain may have no residual amino acids from the given domain as compared to the full length domain, or may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous or non-contiguous amino acids of the full-length wild¬ type domain.

[0046] In some embodiments, the tPA variant comprises a Finger domain of human tPA. The term “Finger domain,” as used herein, includes a full-length wild-type Finger domain andDocket No: 100-2369WO01variants thereof having an amino acid sequence at least 80% (e.g., 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. It is contemplated that a Finger domain may 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 wild-type Finger domain (e.g., a human Finger domain having the amino acid sequence of SEQ ID NO: 2). It is further contemplated that an activity of a Finger domain can be measured in an isolated polypeptide consisting of just the Finger domain, or can be measured in a larger protein that further incorporates other domains, e.g., the other domains and regions of tPA. Without wishing to be bound by theory, it is believed that the Finger domain interacts with LRP-1 and other receptors such as NMDAR. Accordingly, in some embodiments, a Finger domain, when contained in a tPA variant, allows the tPA variant to bind LRP-1 and / or NMDAR, relative to a corresponding tPA variant that contains a deletion of a Finger domain. In some embodiments, the tPA variant comprising a 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 (SEQ ID NO: 2) binds LRP-1, as measured by an in vitro binding assay such as Surface Plasmon Resonance (SPR) assay. In some embodiments, the tPA variant comprising a Finger domain binds NMDAR 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 (SEQ ID NO: 2) binds NMDAR, as measured by an in vitro binding assay such as SPR assay.

[0047] In some embodiments, the tPA variant comprises a fragment of a Finger domain of human tPA. A fragment of a Finger domain comprises an amino acid sequence of at least 11 contiguous or non-contiguous amino acids of SEQ ID NO: 2 but is less than 80% identical to SEQ ID NO: 2. In some embodiments, the fragment of the Finger domain does not include any heterologous sequence that is absent from SEQ ID NO: 2. In some embodiments, the fragment of the Finger domain comprises no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous or non¬ contiguous heterologous amino acids absent from SEQ ID NO: 2. In some embodiments, the fragment of the Finger domain does not comprise all four cysteine residues corresponding to positions 1, 31, 33, and 40 of SEQ ID NO: 2 In some embodiments, the fragment of the Finger domain comprises only one, two, or three of the cysteine residues corresponding to positions I, 31, 33, and 40 of SEQ ID NO: 2. In some embodiments, the fragment of the Finger domainDocket No: 100-2369WO01comprises none of the cysteine residues corresponding to positions 1, 31, 33, and 40 of SEQ ID NO: 2.

[0048] In some embodiments, the tPA variant comprises an EGF-like domain of human tPA. The term ‘'EGF-like domain,” as used herein, includes a full-length wild-type EGF-like domain and variants thereof having an amino acid sequence at least 80% e.g., 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 certain embodiments, the EGF-like domain comprises a tyrosine residue at a position corresponding to Tyrl7 of SEQ ID NO: 3 or Tyr67 of SEQ ID NO: 27. It is contemplated that an EGF-like domain may 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 wild-type EGF- like domain (e.g., a human EGF-like domain having the amino acid sequence of SEQ ID NO: 3). It is further contemplated that an activity of the EGF-like domain can be measured in an isolated polypeptide consisting of just the EGF-like domain, or can be measured in a larger protein that further incorporates other domains, e.g., the other domains and regions of tPA. Without wishing to be bound by theory', it is believed that the EGF-like domain acts in concert with the Finger domain to bind LRP-1 and other receptors such as NMDAR. Accordingly, in some embodiments, a EGF-like domain, when contained in a tPA variant, allows the tPA variant to bind and / or NMDAR, relative to a corresponding tPA variant that contains a deletion of an EGF- like domain. In some embodiments, the tPA variant comprising a EGF-like 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 EGF-like domain (SEQ ID NO: 3) binds LRP-1, as measured by an in vitro binding assay such as SPR assay. In some embodiments, the tPA variant comprising a EGF-like domain binds NMDAR 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 EGF-like domain (SEQ ID NO: 3) binds NMDAR, as measured by an in vitro binding assay such as SPR assay.

[0049] In some embodiments, the tPA variant comprises a fragment of an EGF-like domain of human tPA. A fragment of an EGF-like domain comprises an amino acid sequence of at least 11 contiguous or non-contiguous amino acids of SEQ ID NO: 3 but is less than 80% identical to SEQ ID NO: 3. In some embodiments, the fragment of the EGF-like domain does not includeDocket No: 100-2369WO01any heterologous sequence that is absent from SEQ ID NO: 3 In some embodiments, the fragment of the EGF-like domain comprises no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous or non-contiguous heterologous amino acids absent from SEQ ID NO: 3.

[0050] In some embodiments, the tPA variant comprises a Kringle 1 domain of human tPA. The terra “Kringle I domain,” as used herein, includes a full-length wild-type Kringle 1 domain and variants thereof having an amino acid sequence at least 80% (e.g., 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: 4. Without wishing to be bound by theory, it is contemplated that the Kringle 1 domain may bind other tPA receptors that may have competing actions with LRP-1, and such other tPA receptors may allow binding of Plasminogen Activator Inhibitor-1 (PAI-1), which enhances degradation of tPA and / or inhibits tPA activity'. Notwithstanding, the presence of a complete Kringle 1 domain does not necessarily abrogates tP ’s cytokine activity.

[0051] In some embodiments, the tPA variant comprises a fragment of a Kringle 1 domain of human tPA. A fragment of a Kringle 1 domain comprises an amino acid sequence of at least 11 contiguous or non-contiguous amino acids of SEQ ID NO: 4 but is less than 80%> identical to SEQ ID NO: 4. In some embodiments, the fragment of the Kringle 1 domain does not include any heterologous sequence that is absent from SEQ ID NO: 4. In some embodiments, the fragment of the Kringle 1 domain comprises no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous or non-contiguous heterologous amino acids absent from SEQ ID NO: 4.

[0052] In some embodiments, the tPA variant comprises a Kringle 2 domain of human tPA. The term “Kringle 2 domain,” as used herein, includes a full-length wild-type Kringle 2 domain or a variant thereof having an amino acid sequence at least 80% (e.g., 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. It is contemplated that a Kringle 2 domain may 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 wild-type Kringle 2 domain (e.g., a human Kringle 2 domain having the amino acid sequence of SEQ ID NO: 5). It is further contemplated that an activity of a Kringle 2 domain can be measured in an isolated polypeptide consisting of just the Kringle 2 domain, or can be measured in a larger protein that further incorporates other domains, e.g., the other domains and regions of tPA. Without wishing to be bound by theory, it is believed that the Kringle 2 domain interactsDocket No: 100-2369WO01with LRP-1 and / or other receptors such as NMDAR Accordingly, in some embodiments, a Kringle 2 domain, when contained in a tPA variant, allows the tPA variant to bind LRP-1 and / or NMDAR, relative to a corresponding tPA variant that contains a deletion of Kringle 2 domain. In some embodiments, the tPA variant comprising a Kringle 2 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 Kringle 2 domain binds LRP-1, as measured by an in vitro binding assay such as Surface Plasmon Resonance (SPR) assay. In some embodiments, the tPA variant comprising a Kringle 2 domain binds NMDAR 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 Kringle 2 domain binds NMDAR, as measured by an in vitro binding assay such as Surface Plasmon Resonance (SPR) assay.

[0053] In some embodiments, the tPA variant comprises a fragment of a Kringle 2 domain of human tP A. A fragment of a Kringle 2 domain comprises an amino acid sequence of at least 11 contiguous or non-contiguous amino acids of SEQ ID NO: 5 but is less than 80% identical to SEQ ID NO: 5. In some embodiments, the fragment of the Kringle 2 domain does not include any heterologous sequence that is absent from SEQ ID NO: 5. In some embodiments, the fragment of the Kringle 2 domain comprises no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous or non-contiguous heterologous amino acids absent from SEQ ID NO: 5.

[0054] 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 comprises 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 CGLRQYSQPQFR (SEQ ID NO: 8). In some embodiments, the tail region incorporated in the tPA variant comprises an amino acid sequence that is at least 80% (e.g., 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.

[0055] 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,” which 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 tPADocket No: 100-2369WO01protein 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 etal., (2000) CLIN. CHEM. 46(l):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 enzymatically inactive tPA no higher than 10%, 20%, 30%, 40%, or 50% of the activity unit in a given molar amount of a corresponding wild type tPA protein

[0056] 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), such as those involved in the catalytic activity.

[0057] In some embodiments, the tPA variant does not comprise a complete catalytic domain of tPA (e.g., human tPA) or a functional fragment thereof that has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the catalytic activity of a corresponding full-length catalytic domain (e.g., a human full-length catalytic domain having the amino acid sequence of SEQ ID NO: 6), where the catalytic activity can be measured by conversion of plasminogen to plasmin by proteolytic cleavage In some embodiments, the tPA variant comprises a deletion of a catalytic domain. In some embodiments, the tPA variant comprises a fragment of a catalytic domain with 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.Docket No: 100-2369WO01

[0058] 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) or GQKDVPGVYTKVTNYLDWIRDNMRP (SEQ ID NO: 47), may improve binding to LRP-1. Accordingly, in some embodiments, a tPA variant of the present disclosure further comprises a catalytic domain fragment comprising an amino acid sequence that is at least 80% (e.g, 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 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises the amino acid sequence of SEQ ID NO: 9 or 47.

[0059] In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% (e.g., 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 or 47, a position corresponding to amino acid 11 of SEQ ID NO: 9 or 47, or at each position corresponding to amino acid 3 and amino acid 11 of SEQ ID NO: 9 or 47.

[0060] In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% (e.g., 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 or 47 and comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 9 or 47 and comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 9 or 47 and comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acidDocket No: 100-2369WO01sequence that is at least 90% identical to SEQ ID NO: 9 or 47 and comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9 or 47 and comprises a lysine (K) residue at a position corresponding to amino acid 3 of SEQ ID NO: 9 or 47.

[0061] In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% (e.g, 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 or 47, wherein the catalytic domain fragment of tPA comprises a lysine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 9 or 47 and comprises a lysine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 9 or 47 and comprises a lysine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9 or 47 and comprises a lysine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9 or 47 and comprises a lysine (K) residue at a position corresponding to amino acid 11 of SEQ ID NO: 9 or 47.

[0062] In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% (e.g, 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 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 9 or 47 and comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 9 or 47 and comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least22Docket No: 100-2369WO0190% identical to SEQ ID NO: 9 or 47 and comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 9 or 47. In some embodiments, the catalytic domain fragment comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 9 or 47 and comprises a lysine (K) residue at each position corresponding to amino acids 3 and 11 of SEQ ID NO: 9 or 47.

[0063] Without wishing to be bound by theory, another fragment of the catalytic domain, having the amino acid sequence set forth in SEQ ID NO: 110, also includes multiple lysine residues and may facilitate LRP-1 binding. Accordingly, in some embodiments, a tPA variant of the present disclosure further comprises a fragment of a catalytic domain comprising an amino acid sequence that is at least 80% (e.g., 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: 110. In some embodiments, the fragment of catalytic domain does not have catalytic activity of converting plasminogen to plasmin by proteolytic cleavage.

[0064] In some embodiments, one or more additional amino acids (e.g., a proline residue) is linked to a C-terminal domain, constituting a C-terminus of the tPA variant. Where the C-terminal domain is a catalytic domain or a C-terminal fragment thereof (e.g., the lysine-rich fragment) that already includes a proline at the C-terminus, no such additional proline residue needs to be added.Fusion of Human tPA domains

[0065] A tPA variant of the present disclosure may comprise two or more of the tPA domains as disclosed herein. To facilitate proper folding of the fusion proteins, N-terminal and / or C-terminal fragments of certain domains may be retained. For example, where a tPA variant does not include a complete EGF-like domain, N-terminal and / or C-terminal fragments of an EGF-like domain may still be present in the fusion protein to connect the Finger domain and Kringle 1 domain in a proper orientation. However, in some embodiments the entirety of a domain is deleted. In such embodiments, where there are no retained or residual amino acids from the deleted domain, the domain which is N-terminal to the deleted domain is directly fused to the domain which is C-terminal of the deleted domain.

[0066] Heterologous linkers can also be used. Peptide linkers are known in the art, and any such linker is within the scope of the present disclosure In some embodiments, the linker is aDocket No: 100-2369WO01flexible 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. 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 to about 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, theDocket No: 100-2369WO01linker 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.

[0067] Specific fusion proteins are summarized in Table 2 and described in more detail below. In this table, indicates deletion of a domain and SEQ ID NOs in parentheses indicate fragments of the domains. Without wishing to be bound by theory, it is contemplated that the small fragments may serve as linkers for connecting adjacent domains.Docket No: 100-2369WO01Table 2: tPA Domain Fusion ProteinsSEQ ID NOtPA variant Signal Pro Finger EGF- Kringle Kringle Tail Catalytic Complete Complete Seq Segment like 1 2 protein mature protein V ariant 0 7 10 2 3 4 5 8 11 32 33 Variant 1 7 10 2 3 4 5 8 36 37 Variant 2 7 10 - - 4 5 8 40 41 Variant 3 7 10 2 3 5 8 - 42 43 Variant 4 7 10 9 3 - 5 8 45 46 Variant 5 7 10 2 3 5 8 47 48 49 Variant 11 7 10 (50) 3 4 5 8 - 51 53 Variant 12 7 10 (54) 3 4 5 8 - 55 56 Variant 13 7 10 (57) 3 4 5 8 - 58 59 Variant 14 7 10 (60) 3 4 5 8 - 61 62 Variant 15 7 10 - 3 4 5 8 - 63 64 Variant 21 7 10 2 (65) 4 5 8 - 66 67 Variant 22 7 10 2 (68) 4 5 8 69 70 Variant 23 7 10 2 (71) 4 5 8 - 72 73 V ariant 24 7 10 2 4 5 8 - 74 75 Variant 31 7 10 2 3 (76) 5 8 77 78Docket No: 100-2369WO01SEQ ID NOtPA variant Signal Pro Finger EGF- Kringle Kringle Tail Catalytic Complete Complete Seq Segment like 1 2 protein mature protein Variant 32 7 10 2 3 (79) 5 8 80 81 Variant 33 7 10 2 3 (82) 5 8 - 83 84 Variant 34 7 10 2 3 (85) 5 8 - 86 87 Variant 35 7 10 2 3 (88) 5 8 - 89 90 Variant 36 7 10 2 3 5 8 91 92 Variant 41 7 10 2 3 4 (93) 8 - 94 95 V ariant 42 7 10 2 3 4 (96) 8 - 97 98 Variant 43 7 10 2 3 4 (99) 8 100 101 Variant 44 7 10 2 3 4 (102) 8 - 103 104 V ariant 45 7 10 2 3 4 (105) 8 - 106 107 Variant 46 7 10 2 3 4 - 8 108 109 Variant 51 7 10 2 3 4 5 8 (110) 111 112 Variant 51 -KI 7 10 2 3 5 8 (110) 113 114 Variant 52 7 10 2 3 4 5 8 (115) 116 117 Variant 52-K1 7 10 2 3 5 8 (115) 118 119 Variant 53 7 10 2 3 4 5 - 120 121 Variant 54 7 10 2 3 4 5 (47) 123 124Docket No: 100-2369WO01SEQ ID NOtPA variant Signal Pro Finger EGF- Kringle Kringle Tail Catalytic Complete Complete Seq Segment like 1 2 protein mature protein Variant 54-K1 7 10 2 3 5 - (47) 125 126 Variant 101 7 10 2 3 4 8 - 127 128 Variant 103 7 10 2 3 - - 8 - 129 130 V ariant 115 7 10 3 5 8 - 131 132 Variant 124 7 10 2 - 5 8 133 134Docket No: 100-2369WO01

[0068] The sequences set forth in the SEQ ID NOs identified in Table 2 are provided in Table 1, Table 3, or Table 7.Table 3: tPA Domain Variants and FragmentsDomain Variant Amino Acid Sequence SEQ ID NO or FragmentHuman tPA signal MDAMKRGLCCVLLLCGAVFVSPSQ 7 sequenceHuman tPA pro EIHARFRRGARSYQ 10 segmentFinger domain DEKTQMIYQQHQSWLRPVLRSNRVES 50 fragment inVariant 11Finger domain VICRDEKTQMIYCWCNSGRAQCHSVPVKS 54 fragment inVariant 12Finger domain VICRCNSGRAQCHSVPVKS 57 fragment inVariant 13Finger domain VICWCNSGRAQCHSVPVKS 60 fragment inVariant 14EGF-like domain CSEPRCFNGGTCQQALYFSDFVCQ 65 fragment inVariant 21EGF-like domain QCPEGFAGKCCEID 68 fragment inVariant 22EGF-like domain C SEPRPEGF AGKCCEID 71 fragment inVariant 23Kringle 1 domain TRATCYEDQGISYRGTWSTAESGAECTNYCRNPD 76 fragment in RDSKPWCYVFKAGKYSSEFCSTPACSEVariant 31Docket No: 100-2369WO01Domain Variant Amino Acid Sequence SEQ ID NO or FragmentKringle 1 domain TRATCYEDQGISS TPACSE 79 fragment inVariant 32Kringle 1 domain TRATCYEDQGISYRGTWSTAESGAECTNKAGKYS 82 fragment in SEFCSTPACSEVariant 33Kringle 1 domain KPWCYVFKAGKYSSEFCSE 85 fragment inVariant 34Kringle 1 domain TRATCTNWN SS AL AQKPWCSE 88 fragment inVariant 35Kringle 2 domain GNSDCYFGNGSAYRGTHSLTESGASCLPYCRNPD 93 fragment in GD AKPWCHVLKNRRL TWEYCD VPSCS TVariant 41Kringle 2 domain GNSDCYFGNGSADVPSCST 96 fragment inVariant 42Kringle 2 domain GNSDCYFGNGSAYRGTHSLTESGASCLPLKNRRL 99 fragment in TWEYCD VPSC STVariant 43Kringle 2 domain KPWCHVLKNRRLTWEYCD 102 fragment inVariant 44Kringle 2 domain GNSDCYFGNGSAYRGT 105 fragment inVariant 45Lysine-rich GQKDVPGVYTKVTNYLDWIRDNMRP 47 fragment ofcatalytic domainCatalytic domain IKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGI 110 fragment in LISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRC AQES S V VRTVCLPDocket No: 100-2369WO01Domain Variant Amino Acid Sequence SEQ ID NO or FragmentVariants 51 and51-K1Lysine-rich CGQKDVPGVYTKV TN YLDWIRDNMRP 115 fragment ofcatalytic domainwith cysteine

[0069] In some embodiments, a tPA variant of the present disclosure comprises the domains or fragments thereof of one reference tPA variant identified in Table 2. In some embodiments, each of these domains in the tPA variant comprises an amino acid sequence at least 80% (e.g., 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 the sequence identified in Table 2 for the corresponding reference tPA variant. In some embodiments, the tPA variant further comprises the fragment(s) of tPA domain(s), if present, of the corresponding reference tPA variant. In certain embodiments, the domains in the tPA variants are linked from N-terminus to C-terminus, corresponding to the order in Table 2 from left to right. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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 the full sequence of the corresponding reference tPA variant, as identified in Table 2.Constructs corresponding to Variants 11 and 13-15

[0070] In some embodiments, a tPA variant of the present disclosure comprises a deletion of a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a tail segment. In other embodiments, a tPA variant of the present disclosure comprises a fragment of a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a tail segment. The fragment of the Finger domain can be 11-30 (e.g., 11-25, 11-20, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30) amino acids in length In some embodiments, the fragment of the Finger domain does not comprise all four cysteine residues corresponding to positions 1, 31, 33, and 40 of SEQ ID NO: 2. In some embodiments, the fragment of the Finger domain comprises only one, two, or three of the cysteine residues corresponding to positions 1, 31, 33, and 40 ofDocket No: 100-2369WO01SEQ ID NO: 2. In some embodiments, the fragment of the Finger domain comprises none of the cysteine residues corresponding to positions 1, 31, 33, and 40 of SEQ ID NO: 2. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 tail segment comprises an amino acid sequence at least 80% e.g., 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the fragment of the Finger domain, the EGF-like domain, the Kringle 1 domain, the Kringle 2 domain, and the tail segment from N-terminus to C-terminus.

[0071] Corresponding to Variant 11, a tPA variant of the present disclosure can comprise a fragment of a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a tail segment. In some embodiments, the fragment of the Finger domain comprises an amino acid sequence at least 80% (e.g., 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 EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the fragment of theDocket No: 100-2369WO01Finger domain, the EGF-like domain, the Kringle 1 domain, the Kringle 2 domain, and the tail segment from N-terminus to C-terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g, 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, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 53.

[0072] Corresponding to Variant 13, a tPA variant of the present disclosure can comprise a fragment of a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a tail segment. In some embodiments, the fragment of the Finger domain comprises an amino acid sequence at least 80% (e.g., 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: 57. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the fragment of the Finger domain, the EGF-like domain, the Kringle 1 domain, the Kringle 2 domain, and the tail segment from N-terminus to C -terminus. In some embodiments, the tPA variant comprises anDocket No: 100-2369WO01amino acid sequence at least 80% (e.g, 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: 58. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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 In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 59.

[0073] Corresponding to Variant 14, a tPA variant of the present disclosure can comprise a fragment of a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a tail segment. In some embodiments, the fragment of the Finger domain comprises an amino acid sequence at least 80% (e.g., 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: 60. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the fragment of the Finger domain, the EGF-like domain, the Kringle 1 domain, the Kringle 2 domain, and the tail segment from N-terminus to C -terminus In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 61. In someDocket No: 100-2369WO01embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g, 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 62.

[0074] Corresponding to Variant 15, a tPA variant of the present disclosure can comprise an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a tail segment. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the Finger domain. In some embodiments, the tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the EGF-like domain, the Kringle 1 domain, the Kringle 2 domain, and the tail segment from N-terminus to C-terminus, optionally with a serine (S) residue linked to the N-terminus of the EGF-like domain. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 63. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical toDocket No: 100-2369WO01SEQ ID NO: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 64.Constructs corresponding to Variants 21-23

[0075] In some embodiments, a tPA variant of the present disclosure comprises a Finger domain, a fragment of an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a tail segment. The fragment of the EGF-like domain can be 10-25 (e.g., 11-20, 11-15, 15-25, 15-20, or 20-25) amino acids in length. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the Kringle 2 domain comprises an amino acid sequence at least 80% e.g., 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 tail segment comprises an amino acid sequence at least 80% (e.g, 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the fragment of the EGF-like domain, the Kringle 1 domain, the Kringle 2 domain, and the tail segment from N-terminus to C-terminus,

[0076] Corresponding to Variant 21, a tPA variant of the present disclosure can comprise a Finger domain, a fragment of an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the fragment of the EGF-like domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at leastDocket No: 100-2369WO0190%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 65. In some embodiments, the Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the fragment of the EGF-like domain, the Kringle 1 domain, the Kringle 2 domain, and the tail segment from N-terminus to C-terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 66. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g, 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: 67.

[0077] Corresponding to Variant 22, a tPA variant of the present disclosure can comprise a Finger domain, a fragment of an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the fragment of the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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: 68. In some embodiments, the Kringle 1 domain comprises an amino acidDocket No: 100-2369WO01sequence at least 80% (e.g., 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: 4. In some embodiments, the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the fragment of the EGF-like domain, the Kringle 1 domain, the Kringle 2 domain, and the tail segment from N-terminus to C -terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 69. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 70.

[0078] Corresponding to Variant 23, a tPA variant of the present disclosure can comprise a Finger domain, a fragment of an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the fragment of the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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: 71. In some embodiments, the Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, theDocket No: 100-2369WO01Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the fragment of the EGF-like domain, the Kringle 1 domain, the Kringle 2 domain, and the tail segment from N-terminus to C-terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 72. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 73.Constructs corresponding to Variant 36

[0079] Corresponding to Variant 36, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 2 domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 2 domain comprises an amino acid sequence at least 80% (e.g, 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 tail segment comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%,Docket No: 100-2369WO01at least 99%, or 100%) identical to SEQ ID NO: 8. In some embodiments, the tPA variant comprises a deletion of the Kringle 1 domain. In some embodiments, the tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 2 domain, and the tail segment from N-terminus to C-terminus, optionally with a serine and a glutamate (SE) connecting the EGF-like domain and the Kringle 2 domain. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 91. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 92.Constructs corresponding to Variants 41-46

[0080] In some embodiments, a tPA variant of the present disclosure comprises a Finger domain, an EGF-like domain, a Kringle 1 domain, a deletion of a Kringle 2 domain, and a tail segment. In other embodiments, a tPA variant of the present disclosure comprises a Finger domain, an EGF-like domain, a Kringle 1 domain, a fragment of a Kringle 2 domain, and a tail segment. The fragment of the Kringle 2 domain can be 11-70 (e.g., 11-65, 11-60, 11-55, 11-50, 11-45, 11-40, 11-35, 11-30, 11-25, 11-20, 11-15, 15-70, 15-65, 15-60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-70, 20-65, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-70, 25-65, 25-60, 25-55, 25-50, 25-45, 25-40, 25-35, 25-30, 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-60, 50-55, 55-70, 55-65, 55-60, 60-70, 60-65, or 65-70) amino acids in length. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g, 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 IDDocket No: 100-2369WO01NO: 2. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 1 domain, the fragment of the Kringle 2 domain, and the tail segment from N-terminus to C -terminus.

[0081] Corresponding to Variant 41, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 1 domain, a fragment of a Kringle 2 domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4 In some embodiments, the fragment of the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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: 93. In some embodiments, the tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 1 domain, the fragment of the Kringle 2 domain, and the tail segment from N-terminus to C-terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 94. In someDocket No: 100-2369WO01embodiments, the tPA variant further comprises a signal sequence, a pro segment, or a combination thereof In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the tPA variant further comprising the signal sequence and the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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: 95.

[0082] Corresponding to Variant 41, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 1 domain, a fragment of a Kringle 2 domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the fragment of the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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: 93. In some embodiments, the tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 1 domain, the fragment of the Kringle 2 domain, and the tail segment from N-terminus to C -terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 94. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or aDocket No: 100-2369WO01combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 95.

[0083] Corresponding to Variant 42, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 1 domain, a fragment of a Kringle 2 domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the fragment of the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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: 96. In some embodiments, the tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant, comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 1 domain, the fragment of the Kringle 2 domain, and the tail segment from N-terminus to C-terminus In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 97. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atDocket No: 100-2369WO01least 98%, at least 99%, or 100%) identical to SEQ ID NO: 7 In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 98.

[0084] Corresponding to Variant 43, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 1 domain, a fragment of a Kringle 2 domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g, 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: 4. In some embodiments, the fragment of the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g, 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: 99. In some embodiments, the tail segment comprises an amino acid sequence at least 80% (e.g, 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 1 domain, the fragment of the Kringle 2 domain, and the tail segment from N-terminus to C -terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 100. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., at least 85%, at leastDocket No: 100-2369WO0190%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 10. In some embodiments, the immature tPA variant further comprises an amino acid sequence at least 80% (e.g., 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: 101.

[0085] Corresponding to Variant 44, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 1 domain, a fragment of a Kringle 2 domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the fragment, of the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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: 102. In some embodiments, the tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 1 domain, the fragment of the Kringle 2 domain, and the tail segment from N-terminus to C -terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% e.g., 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: 103. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acidDocket No: 100-2369WO01sequence at least 80% (e.g., 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: 104.

[0086] Corresponding to Variant 45, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 1 domain, a fragment of a Kringle 2 domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g, 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the fragment of the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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: 105. In some embodiments, the tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 1 domain, the fragment of the Kringle 2 domain, and the tail segment from N-terminus to C -terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 106. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 107.Docket No: 100-2369WO01

[0087] Corresponding to Variant 46, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 1 domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the Kringle 2 domain. In some embodiments, the tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 1 domain, and the tail segment from N-terminus to C-terminus, optionally with a threonine (T) residue connecting the Kringle 1 domain and the tail segment. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 108. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 109.Additional tPA variant constructs

[0088] Corresponding to Variant 51, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, a tail segment,Docket No: 100-2369WO01and a fragment of a catalytic domain, wherein the fragment of the catalytic domain does not have catalytic activity of converting plasminogen to plasmin by proteolytic cleavage. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 tail segment comprises an amino acid sequence at least 80% (e.g., 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 fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., 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: 110. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 1 domain, the Kringle 2, the tail segment, and the fragment of the catalytic domain from N-terminus to C-terminus. In some embodiments, a cleavage site between chain A and chain B, having the amino acids of QFR-I (QFR at the C-terminus of the tail segment and I at the N-terminus of SEQ ID NO: 110), is mutated to avoid or reduce truncation of the tPA variant in vitro or in vivo. For example, a substitution or deletion can be made at the arginine (R) residue or the isoleucine (I) residue. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 111. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at leastDocket No: 100-2369WO0199%, or 100%) identical to SEQ ID NO: 10 In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 112.

[0089] Corresponding to Variant 51-K1, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 2 domain, a tail segment, and a fragment of a catalytic domain, wherein the fragment of the catalytic domain does not have catalytic activity of converting plasminogen to plasmin by proteolytic cleavage. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 tail segment comprises an amino acid sequence at least 80% (e.g., 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 fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., 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: 110. In some embodiments, the tPA variant comprises a deletion of the Kringle 1 domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 2, the tail segment, and the fragment of the catalytic domain from N-terminus to C- terminus. In some embodiments, a cleavage site between chain A and chain B, having the amino acids of QFR-I (QFR at the C -terminus of the tail segment and I at the N-terminus of SEQ ID NO: 110), is mutated to avoid or reduce truncation of the tPA variant in vitro or in vivo. For example, a substitution or deletion can be made at the arginine (R) residue or the isoleucine (I) residue. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 113. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., at leastDocket No: 100-2369WO0185%, 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g, 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 114.

[0090] Corresponding to Variant 52, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, a tail segment, and a fragment of a catalytic domain, wherein the fragment of the catalytic domain does not have catalytic activity of converting plasminogen to plasmin by proteolytic cleavage. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 tail segment comprises an amino acid sequence at least 80% (e.g., 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 fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., 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: 115. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 1 domain, the Kringle 2, the tail segment, and the fragment of the catalytic domain from N-terminus to C-terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 116. In some embodiments, an immature tPA variant further comprisesDocket No: 100-2369WO01a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 117.

[0091] Corresponding to Variant 52-K1, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 2 domain, a tail segment, and a fragment of a catalytic domain, wherein the fragment of the catalytic domain does not have catalytic activity of converting plasminogen to plasmin by proteolytic cleavage. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 tail segment comprises an amino acid sequence at least 80% (e.g, 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 fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., 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: 115. In some embodiments, the tPA variant comprises a deletion of the Kringle 1 domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 2, the tail segment, and the fragment of the catalytic domain from N-terminus to C-terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 118. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In someDocket No: 100-2369WO01embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 119.

[0092] Corresponding to Variant 53, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 1 domain, and a Kringle 2 domain. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the tail segment. In some embodiments, the tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 1 domain, and the Kringle 2 from N-terminus to C-terminus, optionally with a proline (P) residue added to the C-terminus of the Kringle 2 domain. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 120. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g, 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: 7. In some embodiments, the pro segment sequence comprises an amino acidDocket No: 100-2369WO01sequence at least 80% (e.g., 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, In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 121.

[0093] Corresponding to Variant 54, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 1 domain, a Kringle 2 domain, and a fragment of a catalytic domain, wherein the fragment of the catalytic domain does not have catalytic activity of converting plasminogen to plasmin by proteolytic cleavage. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g, 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 tPA variant comprises a deletion of the tail segment. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 1 domain, the Kringle 2, and the fragment of the catalytic domain from N-terminus to C -terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 123. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acidDocket No: 100-2369WO01sequence at least 80% (e.g., 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, In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 124.

[0094] Corresponding to Variant 54-K1, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 2 domain, and a fragment of a catalytic domain, wherein the fragment of the catalytic domain does not have catalytic activity of converting plasminogen to plasmin by proteolytic cleavage. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., 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 fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the Kringle 1 domain. In some embodiments, the tPA variant comprises a deletion of the tail segment. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 2, and the fragment of the catalytic domain from N-terminus to C-terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 125. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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:Docket No: 100-2369WO0110 In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 126.

[0095] Corresponding to Variant 101, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, a Kringle 1 domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g., 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 Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., 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: 4. In some embodiments, the tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the Kringle 2 domain. In some embodiments, the tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, the Kringle 1 domain, and the tail segment from N-terminus to C-terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 127. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof. In some embodiments, the signal sequence comprises an amino acid sequence at least. 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 128.Docket No: 100-2369WO01

[0096] Corresponding to Variant 103, a tPA variant of the present disclosure can comprise a Finger domain, an EGF-like domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g, 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. In some embodiments, the EGF-like domain comprises an amino acid sequence at least 80% (e.g, 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 tail segment comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the Kringle 1 domain. In some embodiments, the tPA variant comprises a deletion of the Kringle 2 domain. In some embodiments, the tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the EGF-like domain, and the tail segment from N-terminus to C -terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g, 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: 129. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g, 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. In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 130.

[0097] Corresponding to Variant 124, a tPA variant of the present disclosure can comprise a Finger domain, a Kringle 2 domain, and a tail segment. In some embodiments, the Finger domain comprises an amino acid sequence at least 80% (e.g., 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 In some embodiments, the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at leastDocket No: 100-2369WO0199%, or 100%) identical to SEQ ID NO: 5. In some embodiments, the tail comprises an amino acid sequence at least 80% (e.g., 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 tPA variant comprises a deletion of the EGF-like domain. In some embodiments, the tPA variant comprises a deletion of the Kringle 1 domain. In some embodiments, the tPA variant comprises a deletion of the catalytic domain. In some embodiments, the tPA variant comprises the Finger domain, the Kringle 2 domain, and the tail segment from N-terminus to C-terminus. In some embodiments, the tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 133. In some embodiments, an immature tPA variant further comprises a signal sequence, a pro segment, or a combination thereof In some embodiments, the signal sequence comprises an amino acid sequence at least 80% (e.g., 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: 7. In some embodiments, the pro segment sequence comprises an amino acid sequence at least 80% (e.g., 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, In some embodiments, the immature tPA variant comprises an amino acid sequence at least 80% (e.g., 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: 134.

[0098] Where a tPA variants described herein includes a tail segment, also contemplated is a corresponding tPA variant without the tail segment, and vice versa.

[0099] It is discovered that certain fragments of a catalytic domain does not abrogate the activity of a tPA variant in upregulating MMP9 expression. Accordingly, where a tPA variant comprises a deletion of the catalytic domain, another tPA variant comprising a fragment of a catalytic domain is also contemplated. In some embodiments, the fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., 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 fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., 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: 110. In some embodiments, the fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., at leastDocket No: 100-2369WO0185%, 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: 115.

[0100] 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.

[0101] 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 270 to 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.

[0102] 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,Docket No: 100-2369WO01220, 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 inbetween.

[0103] 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 Example 1 below). Some experimental methods can further assess the expression and secretion levels of tPA variants, which are factors for designing tPA variants suitable for delivery in 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 ability of 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 enzyme-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 using corresponding 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 eukaryotic 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 methodsDocket No: 100-2369WO01known in the art. In this second exemplary system, transcription of MMP9 indicates the cytokine activity of the tPA variants, without accounting for the expression and secretion levels of the tPA variants.tPA Variants of Other Species

[0104] The present disclosure also provides tPA variants comprising corresponding tPA domains and fragments thereof from other species, e.g., mammals such as primates, laboratory animals such as mice, and rats, and farm animals such as pigs and sheep.

[0105] 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: 15:MTNAMKTQFLCLLLCGAVFTWPSQETYRRLRRGARSYRVTCRDEKTQM TYLQHESWLRPLLRGNQMiHCRCNGGRAQCHSVPVRSCSEPWCFNGGTC QQALYSSDFVCQCPEGFMGKLCEIDTTATCYKDQGVAYRGTWSTAESGA ECASWNSSGLAMKPYSGRRPDAVRLGLGNHNYCRNPDQDSKPWCYVFK AGKYISEFCSTPACTKVAEEDGDCYTGNGLAYRGTRSRTKSGFSCLPWNSVFLTSKIYTAWKSNARALGLGKHNHCRNPDGDAQPWCHVWKDRQLTW EYCDVPQCVTCGLRQYKRPQFRIKGGLFADITSHPWQAAIFVKNRRSPGQ RFLCGGILISSCWVLSAAHCFQERYPPHHLKVVLGRTYRLVPGEEEQMFE VEKYIVFIKEFDDDTYDNDIALLFILKSNSLTCAQESGSVRT1CLPDASLQLP DWTECELSGYGKHEAS SPFF SERLKEAHVRLYPS SRCTSRHLFNRTVTNN MLCAGDTRSGGDHTNLHD ACQGDSGGPL VCMKDNI IMTL VGIISWGLGC GQKDIPGVYTKVTNYLDWIRDNMRL (SEQ ID NO: 15)

[0106] The amino acid sequence of a mature, wild type ovine tPA is set forth in SEQ ID NO: 28:SYRVTCRDEKTQMTYLQFIESWLRPLLRGNQMiHCRCNGGRAQCHSVPV RSCSEPWCFNGGTCQQALYSSDFVCQCPEGFMGKLCEIDTTATCYKDQG VAYRGTWSTAESGAECASWNSSGLAMKPYSGRRPDAVRLGLGNFINYCR NPDQDSKPWCYVFKAGKYISEFCSTPACTKVAEEDGDCYTGNGLAYRGT RSRTKSGFSCLPWNSVFLTSKIYTAWKSNARALGLGKHNHCRNPDGDAQ PWCHVWKDRQLTWEYCDVPQCVTCGLRQYKRPQFRIKGGLFADITSHPDocket No: 100-2369WO01WQAATFVKNRRSPGQRFLCGGILISSCWVLSAAHCFQERYPPHHLKVVLG RTYRLVPGEEEQMFEVEKYlVIIKEFDDDTYDNDIALLin SNSLTCAQES GSVRTICLPDASLQLPDWTECELSGYGKHEASSPFFSERLKEAHVRLYPSS RCTSRHLFNRTVTNNMLCAGDTRSGGDHTNLHDACQGDSGGPLVCMKD NHMTLVGIISWGLGCGQKDIPGVYTKVTNYLDWIRDNMRL (SEQ ID NO:28)

[0107] A wild-type, immature ovine tPA (e.g., having the amino acid sequence of SEQ ID NO: 15) 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: 28) 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 4. Certain adjacent domains are connected by peptide linkers, which can be readily recognized by aligning the sequences of the domains with SEQ ID NO: 15.Table 4: Sheep tPA DomainsChain Domain Sequence SEQ ID NO Signal peptide MTNAMKTQFLCLLLCGAVFTWPSQETYRR 29 & pro-peptide LRRGARFinger domain CRDEKTQMTYLQHESWLRPLLRGNQVEHC 16 RCNGGRAQCHSVPV EGF-like CSEPWCFNGGTCQQALYSSDFVCQCPEGFM 17 domain GKKringle 1 ATCYKDQGVAYRGTWSTAESGAECASWNS 18 Chain A domain SGLAMKPYSGRRPDAVRLGLGNHNYCRNP DQDSKPWCYVFKAGKYISEFCSTPACTKringle 2 CYTGNGLAYRGTRSRTKSGFSCLPWNSVFL 19 domain TSKIYTAWKSNARALGLGKHNHCRNPDGDAQP WCHVWKDRQLTWE YCD VPQC VTCGLRQYKRPQFR 34TailDocket No: 100-2369WO01Chain Domain Sequence SEQ ID NO Catalytic IKGGLFADITSHPWQAAIFVKNRRSPGQRFL 20 domain CGGILISSCWVLSAAHCFQERYPPHHLKVVL GRTYRLVPGEEEQMFEVEKYIVHKEFDDDT YDNDIALLHLKSNSLTCAQESGSVRTICLPD ASLQLPDWTECELSGYGKHEASSPFFSERLK EAHVRLYPSSRCTSRHLFNRTVTNNMLCAG DTRSGGDHTNLHDACQGDSGGPLVCMKDNChain BHMTLVGIISWGLGCGQKDIPGVYTKVTNYL DWIRDNMRLLysine-rich GQKDIPGVYTKVTN YLDWIRDNMR 38 fragment ofcatalyticdomain

[0108] 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: 21:MKRELLCVLLLCGLAFPLPDQGIHGRFRRGARSYRATCRDEPTQTTYQQH Q S WLRPMLRS S RVE YC RCNSGLVQC H SVP VR SCSEPRCFNGGTCQQAL Y FSDFVCQCPDGFVGKRCDIDTRATCFEEQGITYRGTWSTAESGAECINWN SSVLSLKPYNARRPNAIKLGLGNFINYCRNPDRDLKPWCYVFKAGKYTTE FCSTPACPKGKSEDCYVGKGVTYRGTHSLTTSQASCLPWNSIVLMGKSYT AWRTNSQALGLGRHNYCRNPDGDARPWCHVMKDRKLTWEYCDMSPCS TCGLRQYKRPQFR1KGGLYTDITSHPWQAAIFVKNKRSPGERFLCGGVLIS SCWVLSAAHCFLERFPPNHLKVVLGRTYRVVPGEEEQTFEIEKYIVHEEFD DDTYDNDIALLQLRSQSKQCAQESSSVGTACLPDPNLQLPDWTECELSGY GKHEASSPFFSDRLKEAHVRLYPSSRCTSQHLFNKTVTNNMLCAGDTRSG GNQDLHDACQGDSGGPLVCMINKQMTLTGIISWGLGCGQKDVPGVYTK VTNYLDWIHDNMKQ (SEQ ID NO: 21)

[0109] The amino acid sequence of a mature, wild type mouse tPA is set forth in SEQ ID NO: 30:Docket No: 100-2369WO01SYRATCRDEPTQTTYQQHQSWLRPMLRSSRVEYCRCNSGLVQCHSVPVR SCSEPRCFNGGTCQQALYFSDFVCQCPDGFVGKRCDIDTRATCFEEQGITY RGTW STAESGAECINWNSSVTSLKPYNARRPNAIKLGLGNHNYCRNPDRDLK DLKPWCYVFKAGKYTTEFCSTPACPKGKSEDCYVGKGVTYRGTHSLTTS QASCLPWNSIVLMGKSYTAWRTNSQALGLGRHNYCRNPDGDARPWCHV MKDRKLTWEYCDMSPCSTCGLRQYKRPQFRIKGGLYTDITSHPWQAAIF VKNKRSPGERFLCGGVLISSCWVLSAAHCFLERFPPNHLKVVLGRTYRVV PGEEEQTFEIEKYIVHEEFDDDTYDNDIALLQLRSQSKQCAQESSSVGTAC LPDPNLQLPDWTECELSGYGKHEASSPFFSDRLKFAHVRLYPSSRCTSQH LFNKTVTNNMLCAGDTRSGGNQDLHDACQGDSGGPLVCMINKQMTLTG IISWGLGCGQKDVPGVYTKVTNYLDWIHDNMKQ (SEQ ID NO: 30)

[0110] A wild-type, immature mouse tPA (e.g., having the amino acid sequence of SEQ ID NO: 21) 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: 30) 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 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 5. Certain adjacent domains are connected by peptide linkers, which can be readily recognized by aligning the sequences of the domains with SEQ ID NO: 21.Table 5: Mouse tPA DomainsChain Domain Sequence SEQ ID NO Signal peptide MKRELLCVLLLCGLAFPLPDQGIHGRFRRG 31 & pro-domain ARFinger domain CRDEPTQTTYQQHQ S WLRPMLRS SRVEYCR 22 Chain ACNSGLVQCHSVPV23-308EGF-like CSEPRCFNGGTCQQALYFSDFVCQCPDGFV 23 domain GK24Kringle 1 ATCFEEQGITYRGTW STAESGAEC1NWN S SDocket No: 100-2369WO01Chain Domain Sequence SEQ ID NO domain VLSLK PYNARRPNAIKLGLGNHNYCRNPDRDLKPWC YVFKAGKYTTEFC STPACPKringle 2 CYVGKGVTYRGTHSLTTSQASCLPWNSIVL 25 domain MGKSYTAWRTNSQALGLGRHNYCRNPDG DARPWCHVMKDRKLTWEYCDMSPCTail STCGLRQYKRPQFR 35 Catalytic IKGGLYTDITSHPWQAAIFVKNKRSPGERFL 26 domain CGGVLISSCWVLSAAHCFLERFPPNHLKVV LGRTYRVVPGEEEQTFEIEKYIVHEEFDDDDT YDNDIALLQLRSQSKQCAQESSSVGTACLP DPNLQLPDWTECELSGYGKHEASSPFFSDR LKEAHWLYPSSRCTSQHLFNKTVTNNMLCChain B AGDTRSGGNQDLHDACQGDSGGPLVCMIN309-559 KQMTLTGIISWGLGCGQKDVPGVYTKVTN YLDWIHDNMKQLysine-rich GQKDVPGVYTKVTNYLDWIHDNMK 39 fragment ofcatalyticdomain

[0111] Mature ovine and murine tPA proteins are homologous to human tPA. Table 6 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 6: Human, ovine, and murine tPA amino acid sequence identitiesSequence Identity to Human tPARegionOvine tPA Murine tPAMature tPA 82.6% 80.2%Chain A 78.4% 78.0%Finger domain 79.1% 81.4%EGF-like domain 90.6% 93.8%Kringle 1 domain 87.1% 82.4%Kringle 2 domain 73.2% 72.0%Docket No: 100-2369WO01Sequence Identity to Human tPARegionOvine tPA Murine tPAChain B (catalytic domain) 86.9% 82.9%

[0112] As shown in Table 4, it is understood that tPA and its domains have as low as about 70% sequence identities among mammalian homologues. It has been found 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. The skilled artisan, based on the disclosure provided for herein, would readily be able to make and use sheep and mouse tPA variants in accordance with the exemplary human tPA variants provided for herein. Where a human tPA variant is disclosed, tPA variants of other species (e.g., sheep or mouse) are also contemplated that have corresponding domains and, where applicable, fragments of domains. The corresponding fragments can be identified by aligning the sequence of the human tPA dom ain with the sequence of the same domain from the other species.Nucleic Acids and Gene Therapy Vectors

[0113] In some embodiments, the present disclosure provides nucleic acid molecules encoding a tPA variant disclosed herein. In some embodiments, the encoded tPA variant is an immature tPA variant. The immature tPA variant can further comprise a signal peptide and / or a pro-peptide at the N-tenninus. In some embodiments, the signal sequence 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: 7. In some embodiments, the pro segment sequence 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: 10. In certain embodiments, the immature tPA variant comprises, at the N-terminus, 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: 11. Exemplar}' immature tPA variant sequences are provided in the “tPA Variants” section including Table 2 above. InDocket No: 100-2369WO01some embodiments, the immature tPA variant comprises a heterologous signal peptide, e.g., a signal peptide known to mediate secretion of another protein.

[0114] 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.

[0115] 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), GAG (chicken beta-actin 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 tPA variant 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.

[0116] 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-integrativeDocket No: 100-2369WO01vector 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., a non-proliferative cell.

[0117] 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 sequence is located between the promoter and the nucleic acid sequence encoding the tPA variant. 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 to AGAGCTGAGATCCTACAGGAGTCCAGGGCTGGAGAGAAAACCTCTGCGAGGAAAG GGAAGGAGCAAGCCGTGAATTTAAGGGACGCTGTGAAGCAATC (SEQ ID NO: 52). 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. A nucleotide sequence of an exemplary gene construct comprising the 5’ UTR sequence linked to a nucleic acid encoding Variant 124 is:AGAGCTGAGATCCTACAGGAGTCCAGGGCTGGAGAGAAAACCTCTGCGAGGAAAG GGAAGGAGCAAGCCGTGAATTTAAGGGACGCTGTGAAGCAATCATGGACGCCATGA AGAGGGGACTGTGCTGCGTGCTGCTGCTGTGCGGAGCCGTGTTCGTGAGCCCATCCC AGGAGATCCACGCAAGGTTTAGGAGAGGAGCACGCTCCTACCAGGTGATCTGTAGG GATGAGAAGACCCAGATGATCTATCAGCAGCACCAGAGCTGGCTGAGGCCCGTGCT GCGGTCCAACAGAGTGGAGTACTGCTGGTGTAATTCTGGCCGGGCACAGTGCCACA GCGTGCCTGTGAAGTCCGGCAACTCTGACTGTTACTTCGGCAATGGCTCCGCCTATA GAGGCACCCACTCTCTGACAGAGAGCGGCGCCTCCTGCCTGCCTTGGAACTCTATGA TCCTGATCGGCA. AGGTGTACACAGCACAGAATCCAAGCGCCCAGGCCCTGGGACTG GG. AAAGCAC / XACTATTGCCGGAATCCAGACGGCGATGCCAAGCCCTGGTGTCACGT GCTGAAGAACAGGCGCCTGACCTGGGAGTACTGTGATGTGCCCTCTTGCAGCACATG TGGCCTGCGGCAGTATTCTCAGCCCCAGTTTAGACCTTGA (SEQ ID NO: 174)Docket No: 100-2369WO01

[0118] The sequence of SEQ ID NO: 174 is exemplary only. Any of the tPA nucleic acid disclosed herein may further comprise the disclosed 5’ UTR

[0119] 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, puroraycin, blasticidin, or hygromycin.

[0120] Non-limiting examples of gene therapy vectors that are within the scope of the present disclosure include adeno-associated vims (AAV) vectors, including AAV2 vectors and self-complimentary AAV2 (scAAV2) vectors, lentivirus vectors, and non-viral vectors.

[0121] 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, with 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, pl 9, 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.

[0122] Because the cis-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 ITRsDocket No: 100-2369WO01may 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,

[0123] 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 cell machinery.However, scAAV comes at a reduced packing capacity (2.4 kb for scAAV versus 4.7-6 kb for AAV).

[0124] 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 AAA7vector 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 modi fied 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.

[0125] Non-limiting examples of AAV vectors include pAAV-MCS (Agilent Technologies), pAAVK-EFla-MCS (System Bio Catalog No. AAV502A-1), pAAVK-EFla-MCS1-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),AAVS1 Puro PGK1 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 (AddgeneDocket No: 100-2369WO01Plasmid No. 32671), pAAV-Puro siKD (Addgene Plasmid No 86695), pAAVSl-Nst-MCS (Addgene Plasmid No. 80487), pAAVSl -Nst-CAG-DEST (Addgene Plasmid No, 80489), pAAVSl-P-CAG-DEST (Addgene Plasmid No. 80490), pAAVf-EnhCB-lacZnls (Addgene Plasmid No. 5642), 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.

[0126] 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 eta]., Adeno-associated virus biology. Adeno-Associated Virus: Methods and Protocols, pp. 1-23, 2011). Methods for generating and purifying AA V 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 virus genes. AAV particles may be purified, for example by affinity chromatography, iodixonal gradient, or CsCl gradient.

[0127] 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 aDocket No: 100-2369WO01skilled 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 delivery 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. Patent Nos. 7,906,111, 9,493,788, and 7,198,951, and PCT Publication No. WO2017189964A2. 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, 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

[0128] 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 80% (e.g., 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 NOs: 12, 13, and 14, respectively:\l A ADG Y LPDWLEDTL SEGIRQW WKLKPGP PPPKP AERHKDD SRGL VLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHAD AEFQERLKEDTSFGGNLGRAVFQAK. KRVLEPLGLVEEPVKTAPGK. KRPVEH SP VEPDS S SGTGK AGQQP ARKRLNFGQTGD AD S VPDPQPLGQPP A AP S GLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTS TRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSDocket No: 100-2369WO01PRDWQRLTNNNWGFRPKRLNFKLFNIQV’KEVTQNDGTTTTANNLTSTVQV FTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSF YCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYL YFLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTS ADNNN S EF S WT GAI KY HLN GRD SL VNPGPAM ASHKDDEEKFFPQ SG VLI FGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGL KHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENS KRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRFLTR-NL (SEQ ID NO: 12)MAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWEICDSTW MGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFD FNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIA NNLTSTVQVFTDSE YQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNN GSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRL MNPLIDQ YL YFLSR TNTP SGTTTQSRLQF SQ AG ASDIRDQSRNWLPGPC Y RQQRVSKTSADNNNSEFSWTGATKYHLNGRDSLVNPGPAMASHKDDEE KFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTN LQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHP SPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIE WELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRFLTR NL (SEQ ID NO: 13)MATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWAL PTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEY F P SQMLRTGN NFTF S YTFED VPFH S S YAHSQ SLD RLMNPLIDQ YL YFLSRT NTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSDocket No: 100-2369WO01EFSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGS EKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNT QGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQI LIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEI QYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRFLTRNL (SEQ ID NO: 14)

[0129] In some embodiments, the AAV or scAAV vector comprises capsid proteins VP1, VP2, and VP3 comprising amino acid sequences at least 85%, 90%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 12, 13, and 14, respectively. In some embodiments, the AAV or scAAV vector comprises capsid proteins VP1, VP2, and VP3 comprising amino acid sequences of SEQ ID NOs: 12, 13, and 14, 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.

[0130] In some embodiments, the AAV or scAAV vector comprises capsid proteins VP1, VP2, and VP3 comprising amino acid sequences at least 80% (e.g., 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 NOs: 12, 13, and 14 respectively, provided that the sequences comprise the triple Y to F mutations shown in SEQ ID NOs: 12, 13, and 14 (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 ID NO: 17). In some embodiments, the AAV or scAAV vector comprises capsid proteins VPI, VP2, and VP3 comprising amino acid sequences at least 80% (e.g., 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 NOs: 12, 13, and 14 respectively, and comprise a phenylalanine (F) at one or more of amino acid positions 444, 500, 730 corresponding to SEQ ID NO: 12, in each of the capsid proteins. In some embodiments, the AAV or scAAV vector comprises capsid proteins VPI, VP2, and VP3 comprising amino acid sequences at least 80% (e.g., 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 I D NOs: 12, 13, and 14 respectively, and comprise a phenylalanine (F ) at each of amino acid positions 444, 500, 730 corresponding to SEQ ID NO: 12, in each of the capsid proteins.

[0131] In some embodiments, the vector is a lentiviral vector. In some embodiments, the lenti viral vector is a pseudotyped lentiviral vector.Docket No: 100-2369WO01

[0132] 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 long-term 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

[0133] 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.

[0134] 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 excessive toxicity, 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 water / 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 (23rd ed. 2020).Docket No: 100-2369WO01

[0135] 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 polypeptides; saltforming 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 wetting 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 pharmaceutical adjuvants. 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.

[0136] 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).Docket No: 100-2369WO01

[0137] 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.

[0138] 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.

[0139] 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.

[0140] Ingredients which may be desirable to use in the ophthalmic preparations of the pharmaceutical compositions disclosed herein include preservatives, co-solvents, buffers, viscosity building agents and penetration enhancers. Viscosity building agents, such as hydroxymethyl 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 known in the art. for other ophthalmic formulations.Docket No: 100-2369WO01

[0141] 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 a tPA variant as disclosed herein is provided. In some embodiments, the use of the vector is in the preparation of a medicament.Methods of treatment

[0142] 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.

[0143] 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.

[0144] The ECM structure of the conventional outflow pathway 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 anterior segment organ culture perfusion experiments in vitro or in animal models in vivo, increases outflow facility.

[0145] 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-1 ipoprotein receptor-related protein 1 (LRP-1 ) and N-methyl-D-aspartate receptor (NMDAR). By this mechanism, tPA enhances MMP transcriptional expression in brain, retinal, lung and renal tissues.Docket No: 100-2369WO01

[0146] 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.

[0147] 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.

[0148] It is understood that the tPA variant may play a cytokine role by binding LRP-1 via its Finger domain and binding NMDAR 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 etal. (2000) LIFE SCIENCES vol. 66, 25: 2473-87; Panner, R. et al. (2020) J. NEUR. DIS. & STROKE vol. 7,1: 1153; Grobmyer, S. R. etal. (1993) J. BIOL. CHEM. vol. 268, 18: 13291-300; and Stockinger, H. etr? / .(1992) THROMBOSIS RES. vol. 67, 5: 589-99.

[0149] 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 that can 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.Docket No: 100-2369WO01

[0150] 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.

[0151] 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 1OP 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.

[0152] 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.

[0153] Also provided herein is a method of treating elevated intraocular pressure 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 elevated intraocular pressure in theDocket No: 100-2369WO01subject 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.

[0154] 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 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.

[0155] 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 examplejuvenile 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 glaucomas such 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.Docket No: 100-2369WO01Routes of administration

[0156] 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

[0157] 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.

[0158] Other sites and modes of administration include topical administration, administration via iontophoresis; implantation of cells that are genetically engineered to constantly produce a tPA 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, et al., (2010) THER. DELIV. 1:435-456.

[0159] 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 from Neurotech 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.Docket No: 100-2369WO01

[0160] 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-glycolic) 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.

[0161] Subconjunctival administration (injection through the conjunctiva) is also contemplated herein.

[0162] 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.

[0163] 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.

[0164] 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,Docket No: 100-2369WO013, 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.

[0165] 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 admini stration 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.

[0166] 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 IOP-associated condition; cause regression of an lOP-associated condition and / or enhance or improve the therapeutic effect(s) of additional treatment(s) administered to ameliorate an lOP-associated condition.Docket No: 100-2369WO01EXAMPLES

[0167] 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

[0168] V ariants of tPA were designed and tested for their ability to upregulate MMP9 expression, which is known to be associated with increase of outflow facility (FIG. ] A-1E). The amino acid sequences of the tPA variants designed and tested are shown in Table 7.Table 7: Exemplary amino acid sequences of tPA variantsConstruct Amino Acid Sequence SEQ ID NOImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 32 VarO QM1YQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGT\VSTAESGAECTNV'7NSSALAQKPYSGRRPDAIRLGLGNIINYCRNP DRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHS LTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGD AKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADI ASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHH LTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKS DSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYS ERLKEAHVRIAPSSRCTAQHLIAIRTVTDNMLCAGDTRSGGPQANL HDACQGDSGGPLVCLN DGRMI ’LVGIISWGLGCGQKD VPGVYTKVT NYLDWIRDNMRPMature SYQVICRDEKTQMIY QQHQSWLRPVLRSNRVEY CWCNSGRAQCHS 33 VarO VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYF GNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGK HNYCRNPDGDAKPWCHVLKNRRLTWEYCD VPSCSTCGLRQY SQP QFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSA AHCFQERFPPHHLTV1LGRTYRVVPGEEEQKFEVEKYIVHKEFDDDT YDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSG YGKHEALSPFYSERLXEAHVRLYPSSRCTAQHLLNRTVTDNMLCA GDTRSGGPQANLHDACQGDSGGPEVCLNDGRMTLVGIISWGLGCG QKD VPGVYTKV TNYLDWIRDNMRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 36 Vari QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG WSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNFINYCRNP DRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSM1LIGKVYTAQNPSAQALGLGKHNYCRNPDGDDocket No: 100-2369WO01Construct Amino Acid Sequence SEQ ID NO AKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRPMature SYQVICRDEKTQM1YQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 37 Vari VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYF GNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGK HNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQP QFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQTRATCYE 40 Var2 DQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGN IINYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNG SAYRGTHSLTESGASCLPUTSSMILIGKWTAQNPSAQALGLGKHbA^ CRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRPMature SYQ TRATCYEDQGISYRG TW STAESGAEC TNWN SSALAQKPYSGR 41 Var2 RPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSE GNSDCYFGNGSAYRGTHSLTESGASCLPWNSM1LIGKVYTAQNPSA QALGLGKHNYCRNPDGDAKPWCIWLKNRRLTWEYCDVPSCSTCG LRQYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIT1ARFRRGARSYQVICRDEKT 42 Var3 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCE1DTGNSDCYFGNGSAYR GTHSLTESGASCLPWNSMIL1GKVYTAQNPSAQALGLGKHNYCRNP DGDAKPWCHVTKNRRLTWEYCDVPSCSTCGLRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 43 Var3 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTGNS DCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQAL GLGKHNYCRNPDGDAKPWCHVLKNRRI. TWEYCDVPSCSTCGLRQ YSQPQFRPImmature MDAMKRGI. CCVLUXGAVFVSPSQEIHARFRRGARSYQVICRDEKT 45 Var4 QM1YQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATSEGNSDCYFGN GSAYRGTHSLTESGASCAPWNSMILIGKVYTAQNPSAQALGLGKIIN YCRNPDGDAKPWCIWLKNRRLTWEYCDVPSCSTCGLRQYSQPQFR PMature SYQVICRDFXTQMIYQQHQSWLRPVLRSNRVF. YCWCNSGRAQCHS 46 Var4 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATS EGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPS AQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTC GLRQYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 48 Var5 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCFISVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTGNSDCYFGNGSAYR GTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRL1AVEYCDVPSCSTCGLRQYSQPQFRGQKDDocket No: 100-2369WO01Construct Amino Acid Sequence SEQ ID NO VPGVYTKVTNYLDWIRDNMRPMature SYQVICRDEKTQM1YQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 49 Var5 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTGNS DCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQAL GLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQ YSQPQFRGQKD VPGVYTKVTNYLDWIRDNMRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQDEKTQM1 51 Vari 1 YQQHQSWLRPVLRSNRVESCSEPRCFNGGTCQQALYFSDFVCQCPE GFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQ KPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCS TPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYT AQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVP SCSTCGLRQYSQPQFRPMature SYQDEKTQMIYQQHQSWLRPVLRSNRVESCSEPRCFNGGTCQQAL 53 Vari 1 YFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAE CTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYV FKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLP WNSMILIGKVYTAQNPSAQALGLGKIINYCRNPDGDAKPWCETVLK NRRLTWEYCDVPSCSTCGLRQYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 55 Vari 2 QMIYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALYFSDFVCQ CPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSA LAQKPYSGRRPD AIRLG LGNF1NYCRNPDRDSKPWCYVFKAGKYSS EFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGK VYTAQNPSAQALGLGKIINYCRNPDGDAKPWCHVLKNRRLTWEYC DVPSCSTCGLRQYSQPQFRPMature SYQVICRDEKTQMIYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQ 56 Vari 2 ALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQG1SYRGTWSTAESG AECrNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWC YVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASC LPWNSMILIGKVYTAQNPSAQALGLGKIINYCRNPDGDAKPWCirVL KNRRLTWEYCDVPSCSTCGLRQYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRCNSG 58 Vari 3 RAQCHSVPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCE IDTRATCYEDQGISYRGTW STAESGAECTNWN S SALAQKPY SGRRP DAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGN SDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQA LGLGKI INYCRNPDGD AKPWCITV LKNRRLTW EYCDVPSCSTCGLR QYSQPQFRPMature SYQVICRCNSGRAQCIISVPVKSCSEPRCFNGGTCQQALYFSDFVCQ 59 Vari 3 CPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSA LAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSS EFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMIL1GKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDocket No: 100-2369WO01Construct Amino Acid Sequence SEQ ID NO DVPSCSTCGLRQYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQV1CWCNS 61 Vari 4 GRAQCHSVPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCC EIDTRATCYEDQGISYRGTWSTAESGAECTNV'TSSSALAQKPYSGRR PDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEG NSDCYFGNGSAYRG'IHSLTESGASCLPWNSMILIGKVYTAQNPSAQ ALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGL RQYSQPQFRPMature SYQVICWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALYFSDFVCQ 62 Vari 4 CPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSA LAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSS EFCSTPACSFGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMTLIGK VYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYC DVPSCSTCGLRQYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQSCSEPRCF 63 Vari 5 NGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGT WSTAESG AECTNWNSS ALAQKPYSGRRPD AIRLGI GNI INYCRNPD RDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSL TESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDA KPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRPMature SYQSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATC 64 Vari 5 YEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGL GNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFG NGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKH NYCRNPDGDAKPWCIIVLKNRRLTWEYCDVPSCSTCGLRQYSQPQF RPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 66 Var21 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQTRATCYEDQGISYRGTWSTAESGAECTN WNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKA GKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNS MILIGKVYTAQNPSAQALGLGKIINYCRNPDGDAKPWCIIVLKNRRL TWEYCDVPSCSTCGLRQYSQPQFRPMature SYQVICRDFKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 67 Var21 VPVKSCSEPRCFNGGTCQQALYFSDFVCQTRATCYEDQGISYRGTW STAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDR DSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLT ESGASCLPWNSMILIGKWTAQNPSAQALGLGKFINYCRNPDGDAK PWCHVLKNRRI-TV'EYCDVPSCSTCGLRQYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIFIARFRRGARSYQVICRDEKT 69 Var22 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSQCPEGF AGKCCEIDTRATCYEDQGISYRGl’WSTAESGAECTNWNSSALAQKP YSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTP ACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKIINYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCDocket No: 100-2369WO01Construct Amino Acid Sequence SEQ ID NO STCGLRQYSQPQFRPMature SYQVICRDEKTQM1YQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 70 Var22 VPVKSQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECT NWNSSALAQKPYSGRRPDAIRLGLGNIINYCRNPDRDSKPWCYVFK AGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWN SM1LIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRR LTWEYCD VPSC STCGLRQY SQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 72 Var23 QM1YQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRP EGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALA QKPYSGRRPDAIRLGLGNIINYCRNPDRDSKPWCYVFKAGKYSSEF CSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKV YTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCD VPSCSTCGLRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 73 Var23 VPVKSCSEPRPEGFAGKCCEIDTRATCYEDQG1SYRGTWSTAESGAE CTNVkTsSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYV FKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTFISLTESGASCLP WNSMILIGKWTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLK NRRLTWEY CD VPSC STCGLRQYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 74 Var24 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSDTRATC YEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGL GNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFG NGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKH NVTRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQF RPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 75 Var24 VPVKSDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPY SGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPA CSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQN PSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCS TCGLRQYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 77 Var31 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG TWSTAESGAECTNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSE GNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSA QALGLGKHNYCRNPDGDAKPWCIWLKNRRLTWEYCDVPSCSTCG LRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 78 Var31 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGTWSTAESGAECTNYCRNPDRDSKPWCYVFKAGKY SSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCFWLKNRRLWEDocket No: 100-2369WO01Construct Amino Acid Sequence SEQ ID NO YCD VPSC STCGLRQY SQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQV1CRDEKT 80 Var32 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISSTPA CSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMTLIGKVYTAQN PSAQALGLGKHNYCRNPDGDAKI’WCHVLKNRRLIWEYCDVPSCS TCGLRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 81 Var32 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCE1DTRAT CYEDQGISSTPACSEGNSDCYFGNGSAYRGTFTSLTESGASCLPWNS MILIGKWTAQNPSAQALGLGKIIFA^CRNPDGDAKPWCHVLKNRRL TWEYCDVPSCSTCGLRQYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 83 Var33 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG TWSTAESGAECTNKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGT HSLTESGASCLPWNSMILTGKVYTAQNPSAQALGLGKIINYCRNPDG DAKPWCIIVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 84 Var33 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGTWSTAESGAECTNKAGKYSSEFCSTPACSEGNSDC YFGNGSAYRGTHSLTESGASCLPWNSMIL1GKVYTAQNPSAQALGL GKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYS QPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 86 Var34 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFV’CQCPEGFAGKCCEIDKPWCYVFKAGKYSSE FCSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSNnLIGKVYTAQ NPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSC STCGLRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 87 Var34 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDKPWC YVFKAGKYSSEFCSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNS MILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRL TWEYCD VPSC STCGLRQY SQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 89 Var35 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCTNWNSSALAQ KPWCSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYT AQNPSAQALGLGKHNYCRNPDGDAKPWCFIVLKNRRLTWEYCDVP SCSTCGLRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 90 Var35 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCTNWNSSALAQKPWCSEGNSDCYFGNGSAYRGTHSLTESGASCLPDocket No: 100-2369WO01Construct Amino Acid Sequence SEQ ID NO WNSM1LIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLK NRRLTWEYCDVPSCSTCGLRQYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIFIARFRRGARSYQVICRDEKT 91 Var36 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCIISVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDSEGNSDCYFGNGSAY RGTHSLTESGASCLPWNSNflLIGKVYTAQNPSAQALGLGKHNYCRN PDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 92 Var36 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCE1DSEGNS DCYFGNGSAYRGTIISLTESGASCLPWNSMILIGKVYTAQNPSAQAL GLGKFINYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQ YSQPQFRPImmature MDAMKRGLCCVELECGAVFVSPSQEIHARFRRGARSYQVICRDEKT 94 Var41 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG WSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNP DRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTIIS LTESGASCLPYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCG LRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 95 Var41 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQG1SYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYF GNGSAYRGTHSLTESGASCLPYCRNPDGDAKPWCHVLKNRRLTWE YCDVPSCSTCGLRQYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIFIARFRRGARSYQVICRDEKT 97 Var42 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG TWSTAESGAECTNWNSSALAQKPYSGRRPDA1RLGLGNHNYCRNP DRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSADVPSCS TCGLRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 98 Var42 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGTWSTAESGAECTNWSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYF GNGSAD VPSCSTCGLRQY SQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 100 Var43 QM1YQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG TWSTAESGAECTNV-7NSSALAQKPYSGRRPDATRLGLGNIIN\TRNP DRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHS LTESGASCLPLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 101VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATDocket No: 100-2369WO01Construct Amino Acid Sequence SEQ ID NOVar43 CYEDQGISYRGWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNFINYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYF GNGSAYRGTHSLTESGASCLPLKNRRLTWEYCDVPSCSTCGLRQYS QPQFRPImmature MDAMKRGLCCVLELCGAVFVSPSQEIFLARFRRGARSYQVICRDEKT 103 Var44 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG TWSTAESGAECTNWNSSALAQKPYSGRRPDA1RLGLGNHNYCRNP DRDSKPWCYVFKAGKYSSEFCSTPACSEKPWCIIVLKNRRLTWEYC DCGLRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 104 Var44 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEKPWCHVL KNRRLTWEYCDCGLRQYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 106 Var45 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG WSTAESGAECTNWSSAIAQKPYSGRRPDAIRLGLGNHNYCRNP DRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTC GLRQYSQPQFRPMature SYQVICRDEKTQM1YQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 107 Var45 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCE1DTRAT CYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNFINYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYF GNGSAYRGTCGLRQYSQPQFRPImmature MDAMKRGLCCVI, LLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 108 Var46 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG WSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNP DRDSKPWCYVFKAGKYSSEFCSTPACSETCGLRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 109 Var46 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGTWSTAESGAECTNWSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSETCGLRQYS QPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 111 Var51 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG TWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNP DRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHS LTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKFINYCRNPDGD AKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADI ASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDocket No: 100-2369WO01Construct Amino Acid Sequence SEQ ID NO DSSRCAQESSVVRTVCLPMature SYQVICRDEKTQM1YQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 112 Var51 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYF GNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGK HNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQP QFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDТ YDNDIALLQLKSDSSRCAQESSVVRTVCLPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 175 Var51 with VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGB chain LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYF cleaved GNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGK HNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQP QFRImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 113 Var51-Kl QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDGNSDCYFGNGSAYRG THSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPD GDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLF ADIASFIPWQAAIFAKTIRRSPGERFLCGGILISSCWILSAAHCFQERFP PHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALL QLKSDS SRC AQES SVVRTVCLPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 114 Var51-Kl VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDGNSD CYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALG LGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWI LSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFD DDTYDNDIALLQLK SD SSRC AQES SVVR TVCLPMature 176 SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVar51-KlVPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDGNSDwith B CYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGchain LGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQY cleaved SQPQFRImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 116 Var52 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG WSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNFINYCRNP DRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHS LTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRCGQKDVPGDocket No: 100-2369WO01Construct Amino Acid Sequence SEQ ID NO VYTKVTNYLDWIRDNMRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 117 Var52 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYF GNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGK HNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQP QFRCGQKDVPGVYTKVTNYLDWIRDNMRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIEIARFRRGARSYQVICRDEKT 118 Var52-K1 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDGNSDCYFGNGSAYRG THSLTESGASCLPWNSMILIGKWTAQNPSAQALGLGKHNYCRNPD GDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRCGQKD VPGVYTKVTNYLDWIRDNMRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 119 Var52-Kl VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDGNSD CYFGNGSAYRGTHSLTESGASCLPWNSMILIGKWTAQNPSAQALG LGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQY SQPQFRCGQKDVPGVYTKVTNYLDWIRDNMRPImmature MDAMKRGLCCVLELCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 120 Var53 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG TWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNP DRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHS LTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGD AKPWCHVLKNRRLTWEYCDVPSCSTPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 121 Var53 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYF GNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGK HNYCRNPDGDAKPWCHVTKNRRLTWEYCDVPSCSTPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 123 Var54 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG TWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNELNYCRNP DRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHS LTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGD AKPWCHVLKNRRLTWEYCDVPSCSTGQKDVPGVYTKVTNYLDWI RDNMRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 124 Var54 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKDocket No: 100-2369WO01Construct Amino Acid Sequence SEQ ID NO HNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTGQKDVPGVY TKVTNYLDWIRDNMRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIFIARFRRGARSYQVICRDEKT 125 Var54-Kl QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPD GDAKPWCHVLKNRRLTWEYCDVPSCSTGQKDVPGVYTKVTNYLD WIRDNMRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 126 Var54-Kl VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDGNSD CYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALG LGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTGQKDVPGVYTKVTNYLDWIRDNMRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 127 Var101 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRG WSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNP DRDSKPWCYVFKAGKYSSEFCSTPACSECGLRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 128 VarlOl VPVKSCSEPRCFNGGTCQQAEYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLG LGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSECGLRQYS QPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 129 Var103 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRC FNGGTCQQALYFSDFVCQCPEGFAGKCCEIDCGLRQYSQPQFRPMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 130 Var103 VPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDCGLR QYSQPQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQCSEPRCFN 131 Var115 GGTCQQALYFSDFVCQCPEGFAGKCCEIDGNSDCYFGNGSAYRGT HSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDG DAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRPMature SYQCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDGNSDCY 132 Var115 FGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLG KHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQ PQFRPImmature MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKT 133 Var124 QMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSGNSDC YFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGL GKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYS QPQFRPDocket No: 100-2369WO01Construct Amino Acid Sequence SEQ ID NOMature SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHS 134 Var124 VPVKSGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTA QNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRPIn the Table above “mature” indicates constructs after cleavage of signal and pro segment sequences, and “immature” indicates constructs containing signal and pro segment sequences.

[0169] Exemplary nucleic acid sequences of the tPA variants designed and tested are shown in Table 8. Due to the degenerate nature of codons, the skilled artisan will readily recognize other nucleic acid sequences that encode the human tPA variants and can be expressed in human tissues.Table 8: Exemplary nucleic acid sequences of tPA variantsConstruct Nucleic Acid Sequence SEQ ID NOVarO ATGGATGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 135 CGGAGCCGTGTTCGTGAGCCCATCCCAGGAGATCCACGCACGGT TTCGGAGAGGCGCCAGAAGCTACCAGGTGATCTGCAGGGACGA GAAGACCCAGATGATCTATCAGCAGCACCAGAGCTGGCTGAGGC CTGTGCTGCGGTCCAACAGAGTGGAGTACTGCTGGTGTAATTCT GGCCGGGCACAGTGTCACAGCGTGCCTGTGAAGTCTTGCAGCGA GCC AAGAI GTTTCAACGGCGGC AC ATGCCAGCAGGCCCTG TATT TCTCCGATTTCGTGTGCCAGTGTCCTGAGGGCTTTGCCGGCAAGT GCTGTGAGATCGATACCCGGGCCACATGTTACGAGGACCAGGGC ATCTCCTATAGAGGAACCTGGTCTACAGCAGAGAGCGGAGCAGA GTGCACCAACTGGAATAGCTCCGCCCTGGCCCAGAAGCCTTACT CTGGCAGGCGCCCAGATGCAATCAGGCTGGGACTGGGCAACCAC AATTAT TGTCGGAATCCCGATAGAGACTC TAAGCCT TGGTGCTAC GTGTTCAAGGCCGGCAAGTATTCTAGCGAGTTTTGCAGCACCCC CGCCTGTTCCGAGGGCAACTCTGACTGTTACTTCGGCAATGGCTC CGCCTATAGGGGCACCCACTCTCTGACAGAGAGCGGCGCCTCCT GCCTGCCATGGAACTCTATGATCCTGATCGGCAAGGTGTACACA GCCCAGAATCCAAGCGCCCAGGCCCTGGGACTGGGAAAGCACA ACTATTGTCGCAATCCAGATGGAGACGCAAAGCCATGGTGCCAC GTGCTGAAGAACCGGAGACTGACCTGGGAGTACTGCGATGTGCC CTCCTGCTCTACATGTGGCCTGCGGCAGTATAGCCAGCCTCAGTT CAGAATCAAGGGCGGCCTGTTTGCAGACATCGCATCCCACCCAT GGCAGGCAGCCATCTTCGCAAAGCACAGGCGCTCTCCAGGAGAG CGGTTTCTGTGCGGAGGAATCCTGATCTCCTCTTGTTGGATCCTG TCCGCCGCCCACTGC TTCCAGGAGAGATTTCCCCCTCACCACC TG ACCGTGATCCTGGGCAGGACATACCGCGTGGTGCCAGGCGAGGAGGAGCAGAAGTTCGAGGTGGAGAAGTACATCGTGCACAAGGAGDocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO TTTGACGATGACACCTATGATAATGACATCGCCCTGCTGCAGCTG AAGAGCGACAGCTCCAGGTGTGCCCAGGAGTCTAGCGTGGTGCG CACCGTGTGCCTGCCACCAGCAGATCTGCAGCTGCCAGACTGGA CAGAGTGTGAGCTGTCCGGATACGGAAAGCACGAGGCCCTGAGC CCTTTTTATrCCGAGCGGCTGAAGGAGGCACACGTGAGGCTGTA CCCATCCTCTCGCTGTACCGCCCAGCACCTGCTGAACAGGACCGT GACAGATAATATGCTGTGCGCAGGAGACACACGCAGCGGAGGA CCACAGGCCAACCTGCACGATGCATGTCAGGGAGACTCCGGAGG ACCTCTGGTGTGCCTGAATGATGGCAGAATGACCCTGGTGGGAA TCATCAGCTGGGGACTGGGATGCGGACAGAAGGACGTGCCTGGC GTGTACACCAAGGTGACAAACTATCTGGATTGGATCAGGGACAA TATGCGCCCATGAVari ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 136 CGGAGCCGTGTTCGTGTCCCCATCTCAGGAGATCCACGCAAGGT TTAGGAGAGGAGCACGCAGCTACCAGGTGATCTGTAGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGTCTTGGCTGAGGCC TGTGCTGAGGAGCAACAGGGTGGAGTACTGCTGG TGTAAT TCCG GCCGGGCACAGTGCCACTCTGTGCCAG1GAAGAGCTGCTCCGAG CCCAGATGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTACTT CTCCGACTTCGTGTGCCAGTGTCCTGAGGGCTTTGCCGGCAAGTG CTGTGAGATCGACACCAGGGCCACATGCTACGAGGATCAGGGCA TCTCTTATAGGGGAACCTGGAGCACAGCAGAGTCCGGAGCCGAG TGTACCAACTGGAATAGCTCCGCCCTGGCACAGAAGCCATACTC CGGCAGGCGCCCCGACGCAATCAGGCTGGGCCTGGGCAACCACA ATTATTGCCGGAATCCTGACAGAGATAGCAAGCCATGGTGTTAC GTGTTCAAGGCCGGCA AGTATTCTAGCG AG 111 1 GCTCCACCCC A GCCTGTTCTGAGGGCAACAGCGATTGCTACTTCGGCAATGGAAG CGCCTATAGGGGAACCCACTCCCTGACAGAGTCCGGAGCCTCTT GTCTGCCTTGGAACTCCATGATCCTGATCGGCAAGGTGTACACA GCCCAGAATCCATCTGCCCAGGCCCTGGGACTGGGAAAGCACAA CTATTGCAGAAATCCCGACGGCGATGCCAAGCCTTGGTGTCACG TGCTGAAGAACCGGAGACTGACCTGGGAGTACTGCGATGTGCCC TCTTGCAGCACATGTGGCCTGCGGCAGTATAGCCAGCCCCAGTTT AGACCTTGAVar2 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 137 CGGAGCCGTGTTCGTGTCCCCATCTCAGGAGATCCACGCCAGAT TTCGGAGAGGCGCCCGGTCCTACCAGACCAGAGCCACATGCTAT GAGGACCAGGGCATCTCTTACAGGGGAACCTGGTCCACAGCAGA GTCTGGAGCCGAGTGTACCAACTGGAATAGCTCCGCCCTGGCCC AGAAGCCATATAGCGGCAGGCGCCCCGATGCAATCAGGCTGGGC CTGGGCAACCACAATTACTGCAGGAACCCTGACCGCGATTCTAA GCCATGGTGTTACGTGTТCAAGGCCGGCAAGTACТCTAGCGAGT TTTGCTCCACCCCTGCCTGTAGCGAGGGCAACTCCGACTGCTATT TCGGCAATGGATCTGCCTACAGGGGAACCCACAGCCTGACAGAG TCTGGCGCCAGCTGTCTGCCATGGAACAGCATGATCCTGATCGG CAAGGTGTATACAGCCCAGAATCCATCCGCCCAGGCCCTGGGACTGGGAAAGCACAACTACTGCCGGAATCCCGACGGCGATGCCAAGDocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO CCTTGGTGTCACGTGCTGAAGAATCGGAGACTGACCTGGGAGTA TTGTGATGTGCCAAGCTGCTCCACATGTGGCCTGAGGCAGTACTC CCAGCCCCAGTTTCGCCCTTGAVar3 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 138 CGGAGCCGTGTTCGTGAGCCCATCCCAGGAGATCCACGCAAGGT TTAGGAGAGGAGCACGCAGCTACCAGGTGATCTGTCGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGAGCTGGCTGAGACC CGTGCTGCGGTCCAACAGAGTGGAGTACTGCTGGTGTAATTCTG GCAGGGCACAGTGCCACAGCGTGCCTGTGAAGTCTTGCAGCGAG CCACGCTGTTTCAACGGCGGCACCTGTCAGCAGGCCCTGTATTTC TCCGACTTCGTGTGCCAGTGTCCTGAGGGCTTCGCCGGCAAGTGC TGTGAGATCGACACAGGCAACTCTGATTGCTACTTTGGCAATGG CTCCGCCTATAGGGGCACCCACTCTCTGACAGAGAGCGGCGCCT CCTGTCTGCCCTGGAACTCTATGATCCTGATCGGCAAGGTGTACA CAGCACAGAATCCTAGCGCCCAGGCCCTGGGACTGGGAAAGCAC AACTATTGCCGCAATCCAGACGGCGATGCCAAGCCCTGGTGTCA CGTGC TGAAGAATAGGCGCCTGACCTGGGAGTACTGCGATGTGC CCTCCI GCTCTAC A TGTGGCCTGCGGCAG TATTCCCAGCCCC AGT TTAGACCTTGAVar4 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 139 CGGAGCCGTGTTCGTGAGCCCATCCCAGGAGATCCACGCAAGGT TTAGGAGAGGAGCACGCAGCTACCAGGTGATCTGTCGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGTCTTGGCTGAGACC CG rGCTGCGGAGCAACAGAGIGGAGIACTGCTGGTGTAA TTCCG GCAGGGCACAG 1GCCACTCTGTGCCTGTGAAGTCT TGC AGCGAG CCACGCTGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTATTTC TCCGACTTCGTGTGCCAGTGTCCTGAGGGCTTCGCCGGCAAGTGC TGTGAGATCGACACCAGGGCCACATGCTACGAGGATCAGGGCATCAGCTATAGAGGAACCTGGAGCACAGCAGAGTCCGGAGCAGAGTGTACAAACTGGAATAGCTCCGCCCTGGCACAGAAGCCATACTCCGGCAGGCGCCCCGACGCAATCAGACTGGGCCTGGGCAAC CACAATTATTGCAGGAACCCTGACCGCGATTG CTACTTTGGCAATGGAAGCGCCTATAGGGGAACCCACTCCCTGA CAGAGAGCGGAGCCTCCTGrCTGCCCTGGAACTCCATGATCCTG ATCGGCAAGGTGTACACCGCCCAGAATCCTTCTGCCCAGGCCCT GGGACTGGGAAAGCACAACTATTGCAGGAATCCAGACGGCGAT G CC AAG CCCTGGTGTCACGTG CTG AAG A ATAGG CG CCTGACCTG GGAGTACTGCGATGTGCCCTCCTGCTCTACATGTGGCCTGCGGCA GTATAGCCAGCCCCAGTTTAGACCTTGAVarS ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 140 CGGAGCCGTGTTCGTGAGCCCATCCCAGGAGATCCACGCCCGGT TTCGGAGAGGCGCCAGAAGCTACCAGGTGATCTGTAGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGAGCTGGCTGAGGCC CGTGCTGCGGTCCAACAGAGTGGAGTACTGCTGGTGTAATTCTG GCCGGGCACAGTGCCACAGCGTGCCAGTGAAGTCTTGCAGCGAG CCCAGATGTTTCAACGGCGGCACCTGTCAGCAGGCCCTGTATTTC TCCGACTTCGTGTGCCAGTGTCCAGAGGGATTCGCCGGCAAGTG CTGTGAGATCGACACAGGCAACTCTGATTGCTACTTTGGCAATG GCTCCGCCTATCGGGGCACCCACTCTCTGACAGAGAGCGGCGCC TCCTGTCTGCCCTGGAACTCTATGATCCTGATCGGCAAGGTGTACACAGCACAGAATCCTAGCGCCCAGGCCCTGGGACTGGGAAAGCDocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO ACAACTATTGCAGGAATCCCGACGGCGATGCCAAGCCTTGGTGT CACGTGCTGAAGAATAGGCGCCTGACCTGGGAGTACTGCGACGT GCCTTCCTGCTCTACATGTGGCCTGAGGCAGTATTCCCAGCCTCA GTTTCGCGGCCAGAAGGATGTGCCAGGCGTGTACACCAAGGTGA CAAACTATCTGGACTGGATCAGGGATAATATGCGCCCATGAVari 1 ATGGACGCCATGAAGAGGGGACTG TGCTGCG TGCTGC TGCTG TG 141 CGGAGCCGTGTTCGTGTCCCCATCTCAGGAGATCCACGCAAGGT TTAGGAGAGGAGCACGCTCTTACCAGGATGAGAAGACCCAGATG ATCTATCAGCAGCACCAGAGCTGGCTGAGGCCAGTGCTGAGGTC CAACAGGGTGGAGAGCTGCTCCGAGCCCAGATGTTTCAATGGCG GCACATGTCAGCAGGCCCTGTACTTCTCCGACTTCGTGTGCCAGT GTCC IGAGGGCITFGCCGGCAAGTGC TGTGAGATCGACACCAGG GCCACATGCTACGAGGATCAGGGCATCAGCTATAGGGGAACCTG GAGCACAGCAGAGTCCGGAGCCGAGTGTACCAACTGGAATAGCT CCGCCCTGGCACAGAAGCCATACTCCGGCAGGCGCCCCGACGCA ATCAGGCTGGGCCTGGGCAACCACAATTATTGCCGGAACCCTGA CAGAGAITCCAAGCCAIGGTGITACGTGITCAAGGCCGGCAAGT ATTC TAGCGAG IT TTGC TCTACCCC AGCCTGTTCTGAGGGCAACA GCGATTGCTACTTCGGCAATGGCTCCGCCTATAGGGGCACCCAC TCTCTGACAGAGTCCGGCGCCTCTTGTCTGCCTTGGAACTCTATG ATCCTGATCGGCAAGGTGTACACAGCACAGAATCCAAGCGCCCA GGCCCTGGGACTGGGAAAGCACAACTATTGCCGCAATCCCGACG GCGATGCCAAGCCTTGGIGTCACGIGCTGAAGAATCGGAGACTG ACCTGGGAGTACTGCGATGTGCCTTCTTGCAGCACATGTGGCCTG CGGCAGTATAGCCAGCCCCAGTTTAGACCTTGAVar12 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 142 CGGAGCCGTGTTCGTGTCCCCATCTCAGGAGATCCACGCCCGGTT TCGGAGAGGCGCCAGATCTTACCAGGTGATCTGCAGGGATGAGA AGACCCAGATGATCTATTGCTGGTGTAACTCCGGCCGCGCACAG TGTCACTCTGTGCCAGTGAAGAGCTGCTCCGAGCCCCGGTGTITC AATGGCGGCACATGCCAGCAGGCCCTGTACTTCTCCGACTTCGT GTGCCAGTGTCCTGAGGGCTTTGCCGGCAAGTGCTGTGAGATCG ACACCCGGGCCACATGTTACGAGGATCAGGGCATCAGCTATAGA GGAACCTGGAGCACAGCAGAGTCCGGAGCAGAGTGCACCAACT GGAATAGCTCCGCCCTGGCACAGAAGCCATACTCCGGCAGGCGC CCCGACGCAATCAGGCTGGGCCTGGGCAACCACAATTA TTGCAG GAACCCTGACCGCGATTCCAAGCCATGGTGTTACGTGTTCAAGG CCGGCAAGTATTCTAGCGAGTTTТGCTCTACCCCAGCCTGTTCTGAGGGCAACAGCGATTGTTACTTCGGCAATGGCAGCGCCTATAGA GGAACCCACTCCCTGACAGAGTCCGGAGCATCTTGCCTGCCTTG GAACTCTATGATCCTGATCGGCAAGGTGTACACAGCACAGAATC CAAGCGCCCAGGCCCTGGGACTGGGAAAGCACAACTATTGCAGG AATCCCGACGGCGATGCCAAGCCTTGGTGTCACGTGCTGAAGAA TCGGAGACTGACCTGGGAGTACTGCGATGTGCCTTCTTGCAGCA CATGTGGCCTGAGGCAGTATAGCCAGCCCCAGTTTCGCCCTTGAVar13 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 143CGGAGCCGTGTTCGTGTCCCCATCTCAGGAGATCCACGCAAGGTDocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO TTAGGAGAGGAGCACGCTCTTACCAGGTGATCTGCCGGTGTAAC TCCGGCAGAGCCCAGTGCCACTCTGTGCCAGTGAAGAGCTGCTC CGAGCCCCGCTGTTTCAATGGCGGCACCTGTCAGCAGGCCCTGT ATTTCTCCGATTTCGTGTGCCAGTGTCCTGAGGGCTTTGCCGGCA AGTGCTGTGAGATCGACACCAGGGCCACATGCTACGAGGATCAG GGCATCAGCTATAGGGGAACCTGGAGCACAGCAGAGTCCGGAG CCGAGTGTACAAACTGGAATAGCTCCGCCCTGGCACAGAAGCCA TACTCCGGCAGGCGCCCCGACGCAATCAGGCTGGGCCTGGGCAA CCACAATTATTGCAGGAACCCTGACCGCGATTCCAAGCCATGGT GTTACGTGTTCAAGGCCGGCAAGTATTCTAGCGAGTTTТGCTCTA CCCCAGCCTGTTCTGAGGGCAACAGCGATTGCTACTTCGGCAAT GGAAGCGCCTATAGGGGAACCCACTCCCTGACAGAGTCCGGAGC CTCTTGTCTGCCTTGGAACTCTATGATCCTGATCGGCAAGGTGTA CACAGCACAGAATCCAAGCGCCCAGGCCCTGGGACTGGGAAAG CACAACTATTGCAGAAATCCCGACGGCGATGCCAAGCCTTGGTG TCACGTGCTGAAGAATCGGAGACTGACCTGGGAGTACTGCGACG TGCCTTCTTGCAGCACATGTGGCCTGCGGCAGTATAGCCAGCCCC AGTTTAGACCTTGAVar14 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 144 CGGAGCCGTGTTCGTGTCCCCATCTCAGGAGATCCACGCCCGGTT TCGGAGAGGCGCCAGATCTTACCAGGTGATCTGCTGGTGTAACT CCGGCAGGGCACAGTGCCACTCTGTGCCAGTGAAGAGCTGCTCC GAGCCCCGCTGTTTCAATGGCGGCACCTGTCAGCAGGCCCTGTA TTTCTCCGATTTCGTGTGCCAGTGTCCTGAGGGCTTTGCCGGCAA GTGCTGTGAGATCGACACCCGGGCCACATGCTACGAGGATCAGG GCATCAGCTATAGAGGAACCTGGAGCACAGCAGAGTCCGGAGC AGAGTGTACAAACTGGAATAGCTCCGCCCTGGCACAGAAGCCAT ACTCCGGCAGGCGCCCCGACGCAATCAGACTGGGCCTGGGCAAC CACAATTATTGCAGGAACCCTGACCGCGATTCCAAGCCATGGTG TTACGTGTTCAAGGCCGGCAAGTATTCTAGCGAGTTTTGCTCTAC CCCAGCCTGTTCTGAGGGCAACAGCGATTGCTACTTCGGCAATG GAAGCGCCTATAGGGGAACCCACTCCCTGACAGAGTCCGGAGCC TCTTGTCTGCCTTGGAACTCTATGATCCTGATCGGCAAGGTGTAC ACAGCACAGAATCCAAGCGCCCAGGCCCTGGGACTGGGAAAGC ACAACTATTGCAGAAATCCCGACGGCGATGCCAAGCCTTGGTGT CACGTGCTGAAGAATCGGAGACTGACCTGGGAGTACTGCGACGT GCCTTCTTGCAGCACATGTGGCCTGAGGCAGTATAGCCAGCCCC AGTTTCGCCCTTGA Var15 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 145 CGGAGCCGTGTTCGTGTCCCCTTCTCAGGAGATCCACGCCCGGTT TCGGAGAGGCGCCAGATCCTACCAGAGCTGCTCCGAGCCACGCT CTTCAACGGCGGCACCTGTCAGCAGGCCCTGTATTTCTCTGATT TCGTGTGCCAGTGTCCTGAGGGCTTTGCCGGCAAGTGCTGTGAG ATCGACACCCGGGCCACATGCTACGAGGATCAGGGCATCAGCTA TAGAGGAACCTGGAGCACAGCAGAGTCCGGAGCAGAGTGTACA AACTGGAATAGCTCCGCCCTGGCACAGAAGCCATACTCCGGCAGGCGCCCCGACGCAATCAGGCTGGGCCTGGGCAACCACAA ITATTDocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO GCAGGAATCCTGACCGCGATTCTAAGCCATGGTGTTACGTGTTC AAGGCCGGCAAGTATTCTAGCGAGTTTTGCAGCACCCCCGCCTG TTCTGAGGGCAACAGCGATTGCTACTTCGGCAATGGCTCCGCCT ATCGGGGCACCCACTCTCTGACAGAGTCCGGCGCCTCTTGTCTGC CATGGAACAGCATGATCCTGATCGGCAAGGTGTACACAGCCCAG AATCCATCCGCCCAGGCCCTGGGACTGGGAAAGCACAACTATTG CAGGAATCCCGACGGCGATGCCAAGCCTTGGTGTCACGTGCTGA AGAACCGGAGACTGACCTGGGAGTACTGCGACGTGCCTTCTTGC AGCACATGTGGCCTGAGGCAGTATAGCCAGCCCCAGTTTCGCCC TTGAVar21 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 146 CGGAGCCGTGTTCGTGTCCCCATCTCAGGAGATCCACGCAAGGT TTAGGAGAGGAGCACGCAGCTACCAGGTGATCTGTAGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGTCTTGGCTGAGGCC TGTGCTGAGGAGCAACAGGGTGGAGTACTGCTGGTGTAATTCCG GCCGGGCACAGTGCCACTCTGTGCCAGTGAAGAGCTGCTCCGAG CCCAGATGTTTCAACGGCGGCACCTGTCAGCAGGCCCTGTACTTC AGCGACTTCGTGTGCCAGACCAGGGCCACATGTTACGAGGATCA GGGCATCTCCTATAGGGGAACCTGGAGCACAGCAGAGTCCGGAG CCGAGTGCACAAACTGGAATAGCTCCGCCCTGGCACAGAAGCCA TACTCCGGCAGGCGCCCCGACGCAATCAGGCTGGGCCTGGGCAA CCACAATTATTGCCGGAATCCTGACAGAGATTCTAAGCCATGGT GTTACGTGTTCAAGGCCGGCAAGTATTCTAGCGAGTTTТGCTCCA CCCCAGCCTGTTCTGAGGGCAACAGCGATTGCTACTTCGGCAAT GGAAGCGCCTATAGGGGAACCCACTCCCTGACAGAGTCCGGAGC CTCTTGTCTGCCTTGGAACTCCATGATCCTGATCGGCAAGGTGTA CACAGCCCAGAATCCATCTGCCCAGGCCCTGGGACTGGGAAAGC ACAACTATTGCAGAAATCCCGACGGCGATGCCAAGCCTTGGTGT CACGTGCTGAAGAACCGGAGACTGACCTGGGAGTACTGCGACGT GCCTTCTTGCAGCACATGTGGCCTGCGGCAGTATAGCCAGCCCC AGTTTAGACCTTGAVar22 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 147 CGGAGCCGTGTTCGTGTCCCCATCTCAGGAGATCCACGCAAGGT TTAGGAGAGGAGCACGCTCTTACCAGGTGATCTGTCGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGAGCTGGCTGAGACC TGTGCTGAGGTCCAACCGCGTGGAGTACTGCTGGTGTAATTCCG GCTGGGCACAGTGCCACTCTGTGCCAGTGAAGAGCCAGTGTCCC GAGGGCTTCGCCGGCAAGTGCTGTGAGATCGACACCAGGGCCAC ATGCTACGAGGATCAGGGCATCAGCTATAGGGGAACCTGGTCCA CAGCAGAGTCTGGAGCCGAGTGTACAAACTGGAATAGCTCCGCC CTGGCACAGA AGCCTTACTCCGGCAGGCGCCCAGACGCAATCAG ACTGGGACTGGGCAACCACAATTATTGCCGGAACCCCGACAGAG ATTCCAAGCCTTGGTGTTACGTGTTCAAGGCCGGCAAGTATTCTA GCGAGTTTТGCTCTACCCCAGCCTGTAGCGAGGGCAACTCCGATT GCTACTTCGGCAATGGCTCCGCCTATAGGGGCACCCACTCTCTGA CAGAGTCTGGCGCCAGCTGTCTGCCTTGGAACTCTATGATCCTGATCGGCAAGGTGTACACAGCACAGAATCCAAGCGCCCAGGCCCTGDocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO GGACTGGGAAAGCACAACTATTGCCGCAATCCAGACGGCGATGC CAAGCCCTGGTGTCACGTGCTGAAGAATCGGAGACTGACCTGGG AGTACTGCGACGTGCCTAGCTGCTCCACATGTGGCCTGCGGCAG TATAGCCAGCCCCAGTTTAGACCTTGAVar23 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 148 CGGAGCCG TGTTCGT GTCCCC TIC TCAGGAGA ICC ACGCAAGGTT TAGGAGA GGAGCACGCAGCTACCAGGTGATCTGTAGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGTCTTGGCTGAGGCC CGTGCTGAGGAGCAACAGGGTGGAGTACTGCTGGTGTAATTCCG GCCGGGCACAGTGCCACTCTGTGCCTGTGAAGAGCTGTTCCGAG CCAAGACCTGAGGGATTCGCCGGAAAGTGCTGTGAGATCGACAC CAGGGCCACATGCTACGAGGATCAGGGCA1CTCTTATAGGGGAA CCTGGAGCACAGCAGAGTCCGGAGCCGAGTGTACAAACTGGAAT AGCTCCGCCCTGGCACAGAAGCCTTACTCCGGCAGGCGCCCAGA CGCAATCAGGCTGGGACTGGGAAACCACAATTATTGCCGGAACC CCGACAGAGATAGCAAGCCTTGGTGTTACGTGTTCAAGGCCGGC AAGrATrCTAGCGAGTnTGCTCCACCCCAGCCTGTICTGAGGGC AACAGCGATTGCTACITCGGCAATGGAAGCGCCTATAGGGGAAC CCACTCCCTGACAGAGTCCGGAGCCTCTTGTCTGCCTTGGAACTC CATGATCCTGATCGGCAAGGTGTACACAGCCCAGAATCCATCTG CCCAGGCCCTGGGACTGGGAAAGCACAACTATTGCAGAAATCCA GACGGCGATGCCAAGCCCTGGTGTCACGTGCTGAAGAATCGGAG ACTGACCTGGGAGTACTGCGACGTGCCCTCITGCAGCACATGIG GCCTGCGGCAGTATTCCCAGCCACAGTTTAGACCCTGAVar24 ATGGACGCCATGAAGAGAGGACTGTGCTGCGTGCTGCTGCTGTG 149 CGGAGCCGTGTTCGTGTCCCCATCTCAGGAGATCCACGCAAGGT TTAGGAGAGGAGCACGCTCTTACCAGGTGATCTGTCGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGAGCTGGCTGAGACC TGTGCTGAGGTCCAACCGCGTGGAGTACTGCTGGTGTAATAGCG GCCGGGCACAGTGCCACTCTGTGCCAGTGAAGAGCGACACCAGG GCCACATGTTACGAGGATCAGGGCATCTCCTATAGGGGAACCTG GTCCACAGCAGAGTCTGGAGCCGAGTGCACAAACTGGAATAGCT CCGCCCTGGCACAGAAGCCATACTCCGGCAGGCGCCCCGACGCA ATCAGGCTGGGCCTGGGCAACCACAATTATTGCCGGAACCCTGA CAGAGATTCCAAGCCATGGTGTTACGTGTTCAAGGCCGGCAAGT A TIC TAGCGAG IT ITGC TC TACCCCCGCC TGTAGCGAGGGCAACT CX'GATTGCTACTTCGGCAATGGCTCCGCCTATAGGGGCACCCACT CTCTGACAGAGTCTGGCGCCAGCTGTCTGCCATGGAACTCTATG ATCCTGATCGGCAAGGTGTACACAGCCCAGAATCCAAGCGCCCA GGCCCTGGGACTGGGAAAGCACAACTATTGCCGCAATCCCGACG GCGATGCCAAGCCn’GGTGTCACGTGCTGAAGAATCGGAGACTG ACCTGGGAGTACTGCGACGTGCCTAGCTGCTCCACATGTGGCCT GCGGCAGTATAGCCAGCCCCAGTTTAGACCTTGAVar31 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 150 CGGAGCCGTGTrCGTGTCTCCTAGCCAGGAGATCCACGCCCGGTTTCGGAGAGGCGCCAGAAGCTACCAGGTGATCTGTCGGGATGAGAAGACCCAGATGATCTATCAGCAGCACCAGAGCTGGCTGAGACCDocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO AGTGCTGCGGTCCAACAGAGTGGAGTACTGCTGGTGTAATTCTG GCAGGGCACAGTGCCACAGCGTGCCAGTGAAGTCCTGCTCTGAG CCTCGCTGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTATTTC AGCGACTTCGTGTGCCAGTGTCCCGAGGGCTTTGCCGGCAAGTG CTGTGAGATCGACACCCGGGCCACATGCTACGAGGATCAGGGCA TCTCCTATAGAGGAACCTGGTCCACAGCAGAGTCTGGAGCAGAG TGCACCAACTACTGTAGGAATCCAGACCGCGATTCCAAGCCCTG GTGTTACGTGTTCAAGGCCGGCAAGTATAGCTCCGAGTTTTGCTC TACACCTGCCTGTAGCGAGGGCAACTCCGACTGCTACTTCGGCA ATGGCTCCGCCTATAGGGGCACCCAC TC TCTGACAGAGTC TGGC GCC AGCTG TCTGCCATGGAACTC TATGATCCTGATCGGC AAGGT GTACACCGCCCAGAATCCAAGCGCCCAGGCCCTGGGACTGGGAA AGCACAACTATTGCCGCAATCCTGACGGCGATGCCAAGCCATGG TGTCACGTGCTGAAGAATAGGCGCCTGACCTGGGAGTACTGCGA TGTGCCCAGCTGCTCCACATGTGGCCTGAGGCAGTATTCCCAGCC CC AGT 1TCGCCCTIGAVar32 ATGGACGCCATGAAGAGGGGAC TGTGC TGCGTGCTGCTGC TGTG 151 CGGAGCCGTGITCGTGTCTCCTAGCCAGGAGATCCACGC AAGGT TTAGGAGAGGAGCACGCTCTTACCAGGTGATCTGTCGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGTCCTGGCTGAGACC AGTGCTGCGGTCTAACAGAGTGGAGTACTGCTGGTGTAATAGCG GCAGGGCACAGTGCCACTCCGTGCCTGTGAAGTCCTGCTCTGAG CCACGCTGTTTCAACGGCGGCACCTGTCAGCAGGCCCTGTACTrC TCCGACTTCGTGTGCCAGTGTCCAGAGGGCTTTGCCGGCAAGTG CTGTGAGATCGACACCAGGGCCACATGCTATGAGGATCAGGGCA TCAGCTCCACACCCGCCTGCTCCGAGGGCAACTCTGATrGTTACT TCGGCAATGGCTCTGCCTATCGCGGCACCCACAGCCTGACAGAG TCTGGCGCCAGCTGTCTGCCCTGGAACAGCATGATCCTGATCGG CAAGGTGTACACAGCACAGAATCCn’CCGCCCAGGCCCTGGGAC TGGGAAAGCACAACTATTGCAGGAATCCAGACGGCGATGCCAA GCCCTGGTGTCACGTGCTGAAGAATAGGCGCCTGACCTGGGAGT ACTGCGACGTGCCCAGCTGCTCCACATGTGGCCTGCGGCAGTAT AGCCAGCCCCAGTTTAGACCTTGAVar33 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 152 CGGAGCCGTGTTCGTGTCTCCAAGCCAGGAGATCCACGCAAGGT TI AGGAGAGGAGCACGCAGCTACCAGGIGATC TGTAGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGAGCTGGCTGAGGCC CGTGCTGCGGTCCAACAGAGTGGAGTACTGCTGGTGTAATTCTG GCCGGGCACAGTGCCACAGCGTGCCTGTGAAGTCCTGCTCTGAG CCAAGATGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTATTT CTCCGACTFCGTGTGCCAGTGTCCTGAGGGCTTrGCCGGCAAGTGCTG TGAGA TCGACACCAGGGCCACATGC TACGAGGATCAGGGCA TCTCCTATAGGGGAACCTGGTCCACAGCAGAGTCTGGAGCCGAG TGTACCAATAAGGCCGGCAAGTACAGCTCCGAGTTCTGCTCTAC ACCAGCCTGTAGCGAGGGCAACTCCGATTGCTACTTTGGCAATG GCTCCGCCTATCGGGGCACCCACTCTCTGACAGAGTCTGGCGCCAGCTGTCTGCCCTGGAACTC'IATGATCCTGA TCGGC AAGG TGTACDocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO ACCGCACAGAATCCTAGCGCCCAGGCCCTGGGACTGGGAAAGCA CAACTATTGCAGGAATCCAGACGGCGATGCCAAGCCCTGGTGTC ACGTGCTGAAGAACAGGCGCCTGACCTGGGAGTACTGCGACGTG CCCAGCTGCTCCACATGTGGCCTGCGGCAGTATAGCCAGCCCCA GTTCAGACCTTGAVar34 ATGGACGCCATGAAGAGGGGACTG TGCTGCG TGCTGC TGCTG TG 153 CGGAGCCGTGTTCGTGTCTCCTAGCCAGGAGATCCACGCAAGGT TTAGGAGAGGAGCACGCTCTTACCAGGTGATCTGTCGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGTCCTGGCTGAGACC AGTGCTGCGGTCTAACAGAGTGGAGTACTGCTGGTGTAATAGCG GCAGGGCACAGTGCCACTCCGTGCCTGTGAAGTCCTGCTCTGAG CC ACGCTGTTTC AACGGCGGCACATG TCAGC AGGCCCTGTATTTC TCCGACTTCGTGTGCCAGTGTCCCGAGGGCTTTGCCGGCAAGTGC TGTGAGATCGATAAGCCTTGGTGCTACGTGTTCAAGGCCGGCAA GTATAGCTCCGAGTTTTGCTCCGAGGGCAACTCTGACTGTTACTT CGGCAATGGCTCTGCCTATAGGGGCACCCACAGCCTGACAGAGT CTGGCGCCAGCTGTCTGCCATGGAACAGCATGATCCTGA TCGGC AAGGTGTACACCGCCCAGAATCCATCCGCCCAGGCCCTGGGACT GGGAAAGCACAACTATTGCCGCAATCCAGACGGCGATGCCAAGC CCTGGTGTCACGTGCTGAAGAATAGGCGCCTGACCTGGGAGTAC TGCGATGTGCCCAGCTGCTCCACATGTGGCCTGCGGCAGTATAG CCAGCCCCAGTTTAGACCTTGAVar35 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 154 CGGAGCCGTGITCGTGTCTCCI AGCCAGGAGATCCACGCAAGGT T TAGGAGAGGAGC ACGCTCTTACC AGGTGA'rCTGTCGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGTCCTGGCTGAGACC AGTGCTGCGGTCTAACAGAGTGGAGTACTGCTGGTGTAATAGCG GCAGGGCACAGTGCCACTCCGTGCCTGTGAAGTCCTGCTCTGAG CCACGCTGTTTCAACGGCGGCACCTGTCAGCAGGCCCTGTATTTC TCCGACTTCGTGTGCCAGIGTCCAGAGGGCTrTGCCGGCAAGTG CTGTGAGATCGATACAAGGGCCACCTGCACAAACTGGAATAGCT CCGCCCTGGCACAGAAGCCATGGTGCTCCGAGGGCAACTCTGAC TGTTACTTCGGCAATGGCTCTGCCTATCGCGGCACCCACAGCCTG ACAGAGTCTGGCGCCAGCTGTCTGCCCTGGAACAGCATGATCCT GATCGGCAAGGTGTACACAGCACAGAATCCTTCCGCCCAGGCCC TGGGAC I GGGAAAGCAC AACTA I TGCAGGAATCC AGACGGCGAT GCXAAGCCCTGGTGTCACGTGCTGAAGAATAGGCGCCTGACCTG GGAGTACTGCGATGTGCCCAGCTGCTCCACATGTGGCCTGCGGC AGTATAGCCAGCCCCAGTTTAGACCTTGAVar36 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 155 CGGAGCCGTGTTCGTGAGCCCATCCCAGGAGATCCACGCAAGGT TTAGGAGAGGAGCACGCAGCTACCAGGTGATCTGTCGGGATGAG AAGACCCAGATGATCrATCAGCAGCACCAGTCTrGGCTGAGACC CGTGCTGCGGAGCAACAGAGTGGAGTACTGCTGGTGTAATTCCG GCAGGGCACAGTGCCACTCTGTGCCTGTGAAGTCTTGCAGCGAG CCACGCTGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTATTTCTCCGACTTCGTGTGCCAGTGTCCTGAGGGCTTCGCCGGCAAGTGCDocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO TGTGAGATCGACTCTGAGGGCAACAGCGATTGCTACTTTGGCAA TGGCAGCGCCTATAGGGGAACCCACTCCCTGACAGAGAGCGGAG CCTCCTGTCTGCCCTGGAACTCCATGATCCTGATCGGCAAGGTGT ACACCGCCCAGAATCCTTCTGCCCAGGCCCTGGGACTGGGAAAG CACAACTATIGCCGCAATCCAGACGGCGATGCCAAGCCCTGGTG TCACGTGCTGAAGAATAGGCGCCTGACCTGGGAGTACTGCGATG TGCCCTCCTGCTCTACATGTGGCCTGCGGCAGTATAGCCAGCCCC AGTTTAGACCTTGAVar41 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 156 CGGAGCCGTGTTCGTGTCCCCATCTCAGGAGATCCACGCAAGGT TTAGGAGAGGAGCACGCTCTTACCAGGTGATCTGTAGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGTCTTGGCTGAGGCC TGTGCTGAGGAGCAACAGGGTGGAGTACTGCTGGTGTAATTCCG GCCGGGCACAGTGCCACTCTGTGCCAGTGAAGAGCTGCTCCGAG CCCAGATGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTATTTC TCCGACTTCGTGTGCCAGTGTCCAGAGGGCTTTGCCGGCAAGTG CTG TGAGA TCGACACC AGGGCCACATGC TACGAGGATCAGGGCA TCAGCTATAGGGGAACCTGGAGCACAGCAGAGTCCGGAGCCGA GTGTACCAACTGGAATAGCTCCGCCCTGGCACAGAAGCCTTACT CCGGCAGGCGCCCAGACGCAATCAGGCTGGGACTGGGAAACCA CAATTATTGCCGGAATCCCGACAGAGATTCCAAGCCTTGGTGTT ACGTGTTCAAGGCCGGCAAGTATTCTAGCGAGTTTTGCTCTACAC CTGCC TGTTCTGAGGGCAACAGCGA ITGC TACT TCGGCAATGGA AGCGCCTATAGGGGAACCCACTCCCTGACAGAGTCCGGAGCCTC TTGCCTGCCATACTGTAGAAACCCCGACGGCGATGCCAAGCCTT GGTGCCACGTGCTGAAGAATCGGAGACTGACCTGGGAGTACTGT GATGTGCCATCTTGCAGCACATGTGGCCTGCGGCAGTATAGCCA GCCCCAGTTTAGACCTTGAVar42 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 157 CGGAGCCGTGTrCGTGICCCCTrCTCAGGAGATCCACGCCCGGTr TCGGAGAGGCGCCAGAAGCTACCAGGTGATCTGTCGGGATGAGA AGACCCAGATGATCTATCAGCAGCACCAGTCCTGGCTGAGACCA GTGCTGCGGTCTAACAGAGTGGAGTACTGCTGGTGTAATTCTGG CAGGGCACAGTGCCACAGCGTGCCAGTGAAGAGCTGCTCCGAGC CTCGCTGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTATTTCT CCGACT TCGTGTGCCAGTGTCCCGAGGGC ITTGCCGGCAAGTGCT GTGAGATCGACACCCGGGCCACATGCTACGAGGATCAGGGCATC TCCTATAGAGGAACCTGGTCCACAGCAGAGTCTGGAGCAGAGTG TACCAACTGGAATAGCTCCGCCCTGGCCCAGAAGCCATACTCTG GCAGGCGCCCCGATGCAATCAGACTGGGACTGGGCAACCACAAT TAn'GCAGGAATCCTGACCGCGATrCTAAGCCATGGTGTTACGTG TTCAAGGCCGGCAAGrATTCTAGCGAGTITTGCAGCACCCCAGC CTGTAGCGAGGGCAACTCCGACTGCTACTTCGGCAATGGCAGCG CCGATGTGCCCTCTTGCAGCACATGTGGCCTGAGGCAGTATTCCC AGCCCCAGTTTCGCCCTTGAVar43 ATGGACGCCATGAAGAGAGGACTGTGCTGCGTGCTGCTGCTGTG 158CGGAGCCGTGTTCGTGTCCCCTTCTCAGGAGATCCACGCAAGGTTDocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO TAGGAGAGGAGCACGCTCTTACCAGGTGATCTGTAGGGATGAGA AGACCCAGATGATCTATCAGCAGCACCAGTCTTGGCTGAGGCCA GTGCTGAGGAGCAACAGGGTGGAGTACTGCTGGTGTAATTCCGG CCGGGCACAGTGCCACTCTGTGCCAGTGAAGAGCTGCTCCGAGCCrAGATGTITCAACGGCGGCACATGTCAGCAGGCCCrGTATTTCT CCGACTTCGTGTGCCAGTGTCCCGAGGGCTTTGCCGGCAAGTGCT GTGAGATCGACACCAGGGCCACATGCTACGAGGATCAGGGCATC AGCTATAGGGGAACCTGGAGCACAGCAGAGTCCGGAGCCGAGT GTACCAACTGGAATAGCTCCGCCCTGGCACAGAAGCCATACTCC GGCAGGCGCCCCGATGCAATCAGGCTGGGCCTGGGCAACCACAA T TAI TGCCGGAACCCTGACAGAGATTCCAAGCC ATGGTGTTACG TGTTCAAGGC CGGC AAGTATTCTAGCGAG 1111 GCTCTACACCTG CCTGTTCTGAGGGCAACAGCGACTGCTACTTCGGCAATGGAAGC GCCTATAGGGGAACCCACTCCCTGACAGAGTCCGGAGCCTCTTG CCTGCCACTGAAGAATCGGAGACTGACCTGGGAGTACTGTGATG TGCCCTC ITGCAGCACATGTGGCCTGCGGCAG TATAGCCAGCCC CAGTTTAGACCTTGAVar44 ATGGACGCC Al GAAGAGGGGACTG TGCTGCGTGCTGC TGCTG TG 159 CGGAGCCGTGTTCGTGTCCCCTTCTCAGGAGATCCACGCAAGGTT TAGGAGAGGAGCACGCTCTTACCAGGTGATCTGTAGGGATGAGA AGACCCAGATGATCTATCAGCAGCACCAGTCTTGGCTGAGGCCA GTGCTGAGGAGCAACAGGGTGGAGTACTGCTGGTGTAATAGCGG CCGGGCACAGTGCCACTCCGTGCCAGTGAAGAGCTGCTCCGAGC CCAGATGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTATTTC AGCGACTTCGTGTGCCAGTGTCCCGAGGGCTTTGCCGGCAAGTG CTGTGAGATCGACACCAGGGCCACATGCTACGAGGATCAGGGCA TCTCCTATAGGGGAACCTGGTCTACAGCAGAGAGCGGAGCCGAG TGTACCAACTGGAATAGCTCCGCCCTGGCACAGAAGCCATACTC CGGCAGGCGCCCCGACGCAATCAGGCTGGGCCTGGGCAACCACA ATTATTGCCGGAACCCTGACAGAGATTCTAAGCCATGGTGTTAC GTGTTCAAGGCCGGCAAGTATTCTAGCGAGTTTTGCTCCACCCCC GCCTGTTCTGAGAAGCCTTGGTGCCACGTGCTGAAGAATCGGAG ACTGACATGGGAGTACTGCGATTGTGGCCTGCGGCAGTATTCCC AGCCCCAGTICAGACCTTGAVar45 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 160 CGGAGCCGrGTTCGIGTCCCCATCTCAGGAGAICCACGCCCGGIT TCGGAGAGGCGCCAGAAGCTACCAGGTGATCTGCCGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGTCTTGGCTGAGACC CGTGCTGCGGAGCAACAGAGTGGAGTACTGCTGGTGTAATAGCG GCAGGGCACAGTGCCACTCCGTGCCAGTGAAGAGCTGCTCCGAG CCTCGCTGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTATTTC TCCGACTTCGTGTGCCAGTGTCCTGAGGGCTTTGCCGGCAAGTGC TGTGAGATCGACACCCGGGCCACATGCTACGAGGATCAGGGCAT CTCTTATAGAGGAACCTGGTCTACAGCAGAGAGCGGAGCAGAGT GTACCAACTGGAATAGCTCCGCCCTGGCACAGAAGCCATACTCC GGCAGGCGCCCCGACGCAATCAGGCTGGGCCTGGGCAACCACAAITA ITGCAGGAATCCTGACCGCGATAGCAAGCCA IGGTGTIACDocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO GTGTTCAAGGCCGGCAAGTATTCTAGCGAG 1111 GC 1 CCACCCCC GCCTGTTCCGAGGGCAACTCTGATTGCTACTTCGGCAATGGCTCC GCCTATAGGGGCACATGTGGCCTGCGCCAGTACTCTCAGCCCCA GTTTCGCCCTTGAVar46 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 161 CGGAGCCG TGTTCGT GTCCCCATCTC AGGAGATCCACGCCCGGTT TCGGAGAGGCGCCAGATCTTACCAGGTGATCTGTCGGGATGAGA AGACCCAGATGATCTATCAGCAGCACCAGTCTTGGCTGAGACCC GTGCTGCGGAGCAACAGAGTGGAGTACTGCTGGTGTAATAGCGG CAGGGCACAGTGCCACTCCGTGCCAGTGAAGAGCTGCTCCGAGC CTCGCTGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTATTTCA GCGACTTCGTGTGCCAGTGTCCTGAGGGCTITGCCGGCAAGTGCT GTGAGATCGACACCCGGGCCACATGCTACGAGGATCAGGGCATC TCCTATAGAGGAACCTGGTCTACAGCAGAGAGCGGAGCAGAGTG TACCAACTGGAATAGCTCCGCCCTGGCACAGAAGCCATACTCCG GCAGGCGCCCCGATGCAATCAGACTGGGACTGGGCAACCACAAT TATTGCAGGAATCCTGACCGCGATTCTAAGCC A TGGTGT TACGTG TTCAAGGCCGGCAAGTATTC TAGCGAGTTTTGC TCCACCCCCGCC TGCTCTGAGACATGTGGCCTGAGGCAGTACTCCCAGCCCCAGTT CCGCCCTTGAVar51 ATGGATGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 162 CGGAGCCGTGTTCGTGTCCCCTTCTCAGGAGATCCACGCAAGGTT TAGGAGAGGAGCACGCAGCTACCAGGTGATCTGCAGGGACGAG AAGACCCAGATGATCTATCAGCAGCACCAGTCITGGCTGAGACC AGrGCTGCGGAGCAACAGAGTGGAGTACTGCTGGTGrAA'ITCCG GCAGGGCCCAGTGTCACTCTGTGCCAGTGAAGAGCTGCTCCGAG CCCCGCTGTTTCAACGGCGGCACATGCCAGCAGGCCCTGTATTTC TCCGATTTCGTGTGCCAGTGTCCAGAGGGCTTTGCCGGCAAGTGC TGTGAGATCGATACCCGGGCCACATGTTACGAGGACCAGGGCAT CTCCTATAGAGGAACCTGGAGCACAGCAGAGTCCGGAGCAGAGT GCACCAACTGGAATAGCTCCGCCCTGGCCCAGAAGCCATACTCT GGCAGGCGCCCCGATGCAATCAGACTGGGACTGGGCAACCACA ATTATTGTCGGAATCCTGATAGAGACTCTAAGCCATGGTGCTAC GTGTTCAAGGCCGGCAAGTATTCTAGCGAGTTTTGCAGCACCCCT GCCTGTTCTGAGGGCAACAGCGACTGTTACTTCGGCAATGGAAG CGCCTATAGGGGAACCCACTCCC TGACAGAGTCCGGAGCCTCT T GCXTGCCATGGAACTCCATGATCCTGATCGGCAAGGTGTACACA GCACAGAATCCATCTGCCCAGGCCCTGGGACTGGGAAAGCACAA CTATTGTAGAAATCCCGATGGCGACGCCAAGCCTTGGTGCCACG TGCTG A AG A ACCGG AG A CTG ACCTGGG AGTA CTGCG ATGTGCCT TCI rGCAGCACAlGTGGCCTGCGGCAGTArAGCCAGCCACAGTr CAGAATCAAGGGCGGCCTGTTTGCAGACATCGCAAGCCACCCAT GGCAGGCAGCCATCTTCGCAAAGCACAGGCGCTCCCCTGGAGAG AGGTTTCTGTGCGGCGGCATCCTGATCTCCTCTTGTTGGATCCTG TCCGCCGCACACTGCTTCCAGGAGCGGTTCCCCCCTCACCACCTG ACCGTGATCCTGGGAAGGACATACAGGGTGGTGCCAGGAGAGGAGGAGCAGAAGTTCGAGGTGGAGAAGTACATCGTGCACAAGGADocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO GTTTGACGATGACACCTATGATAATGACATCGCCCTGCTGCAGCT GAAGAGCGACAGCTCCAGGTGTGCCCAGGAGTCTAGCGTGGTGC GCACAGTGTGCCTGCCTTGAVar51-Kl ATGGATGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 163 CGGAGCCGTGTTCGTGAGCCCATCCCAGGAGATCCACGCAAGGTTrAGGAGAGGAGCACGCAGCTACCAGGTGATCTGCAGGGACGA GAAGACCCAGATGATCTATCAGCAGCACCAGAGCTGGCTGAGGC CTGTGCTGAGGTCCAACAGGGTGGAGTACTGCTGGTGTAATTCT GGCCGGGCACAGTGTCACAGCGTGCCTGTGAAGTCTTGCAGCGA GCCAAGATGTTTCAACGGCGGCACATGCCAGCAGGCCCTGTATT TCAGCGATTTCGTGTGCCAGTGTCCTGAGGGCTTCGCCGGCAAGT GCTGTGAGATCGATGGCAACTCCGACTGTrAClTTGGCAATGGCT CCGCCTATAGGGGCACCCACTCTCTGACAGAGAGCGGCGCCTCC TGCCTGCCTTGGAACTCTATGATCCTGATCGGCAAGGTGTACACC GCACAGAATCCAAGCGCCCAGGCCCTGGGACTGGGAAAGCACA ACTATTGTCGCAATCCAGATGGAGACGCAAAGCCATGGTGCCACGlGCTGAAGAATAGGCGCCIGACCTGGGAGTACrGCGATGTGCC dec TGCTCT ACATG TGGCCTGCGGCAGTATTCTCAGCCTC AGT T CAGAATCAAGGGCGGCCTGTTTGCAGACATCGCATCCCACCCAT GGCAGGCAGCCATCTTCGCAAAGCACCGGAGATCTCCAGGAGAG CGGTTTCTGTGCGGAGGAATCCTGATCAGCTCCTGTTGGATCCTG TCCGCCGCCCACTGCTTCCAGGAGAGATTTCCCCCTCACCACCTG ACCGIGATCCIGGGCCGGACATACAGAGTGGTGCCAGGCGAGGA GGAGCAGAAGTTCGAGGTGGAGAAGTACATCGTGCACAAGGAG TTTGACGATGACACCTATGATAACGACATCGCCCTGCTGCAGCT GAAGTCCGACTCTAGCAGGTGTGCCCAGGAGTCCTCTGTGGTGC GCACAGTGTGCCTGCCCTGAVar52 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 164 CGGAGCCGTGTTCGTGTCCCCATCTCAGGAGATCCACGCCCGGTT TCGGAGAGGCGCCAGAAGC I AC CAGGTGATCTGC CGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGTCTTGGCTGAGACC TGTGCTGAGGAGCAACCGCGTGGAGTACTGCTGGTGTAATTCCG GCAGGGCACAGTGCCACTCTGTGCCAGTGAAGAGCTGCTCCGAG CCTCGCTGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTACTTC TCCGACTTCGTGTGCCAGTGTCCTGAGGGCTTTGCCGGCAAGTGC TGTGAGATCGACACCCGGGCCACATGCTACGAGGATCAGGGCAT CTCTTATAGAGGAACCTGGAGCACAGCAGAGTCCGGAGCAGAGT GTACCAACTGGAATAGCTCCGCCCTGGCACAGAAGCCATACTCC GGCAGGCGCCCTGACGCAATCAGACTGGGACTGGGCAACCACA ATTATTGCCGGAATCCAGACAGAGATAGCAAGCCCTGGTGTTAC G TGTTCA AGGCCGGC A AGTA ITCT AGCGAGl f TTGCTCCA CCCCC GCCTG rTCrGAGGGCAACAGCGATTGCTACTTCGGCAAIGGCAG CGCCTATAGGGGAACCCACTCCCTGACAGAGTCCGGAGCCTCTT GTCTGCCTTGGAACTCCATGATCCTGATCGGCAAGGTGTACACA GCCCAGAATCCATCTGCCCAGGCCCTGGGACTGGGAAAGCACAA CTATTGCCGCAATCCTGACGGCGATGCCAAGCCATGGTGTCACGTGC TGAAGAACCGGAGACTGACCTGGGAG TACTGCGACG TGCCADocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO TCTTGCAGCACATGTGGCCTGAGGCAGTATAGCCAGCCACAGTT TCGCTGTGGCCAGAAGGATGTGCCCGGCGTGTACACCAAGGTGA CAAACTATCTGGACTGGATCAGGGATAATATGCGCCCCTGAVar52-Kl ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 165 CGGAGCCGTGTTCGTGAGCCCATCCCAGGAGATCCACGCCCGGT TTCGGAGAGGCGCCAGAAGCTACCAGGTGATCTGCAGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGAGCTGGCTGAGGCC CGTGCTGCGGTCCAACAGAGTGGAGTACTGCTGGTGTAATTCTG GCCGGGCACAGTGCCACAGCGTGCCAGTGAAGTCTTGCAGCGAG CCCAGATGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTATTTC TCCGACTTCGTGTGCCAGTGTCCAGAGGGATTCGCCGGCAAGTG CTGrGAGATCGACGGCAACTCrGATrGCTACTTTGGCAAl GGCTC CGCCTATCGGGGCACCCACTCTCTGACAGAGAGCGGCGCCTCCT GTCTGCCCTGGAACTCTATGATCCTGATCGGCAAGGTGTACACC GCACAGAATCCTAGCGCCCAGGCCCTGGGACTGGGAAAGCACA ACTATTGCAGGAATCCCGACGGCGATGCCAAGCCTTGGTGTCACGlGCTGAAGAATAGGCGCCIGACCTGGGAGTACrGCGACGTGCC ITCCTGCTCTACATGTGGCCTGAGGCAGTAlTCCCAGCCTCAGTf TCGCTGTGGCCAGAAGGATGTGCCAGGCGTGTACACCAAGGTGA CAAACTATCTGGACTGGATCAGGGATAATATGCGCCCATGAVar53 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 166 CGGAGCCGTGTTCGTGTCCCCATCTCAGGAGATCCACGCAAGGT TTAGGAGAGGAGCACGCAGCTACCAGGTGATCTGCAGGGATGA GAAGACCCAGATGATCTATCAGCAGCACCAGTCTTGGCTGAGAC CTGTGCTGAGGAGCAACCGCGTGGAGTACrGCTGGTGIAATTCC GGCAGGGCACAGTGCCACTCTGTGCCAGTGAAGAGCTGCTCCGA GCCCCGCTGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTATTT CTCCGACTTCGTGTGCCAGTGTCCAGAGGGCTTTGCCGGCAAGT GCTGTGAGATCGACACCCGGGCCACATGCTACGAGGATCAGGGC ATC rCTIATAGAGGAACCTGGAGCAC AGCAG AG TCCGGAGCAGA GTGTACCAACTGGAATAGCTCCGCCCTGGCACAGAAGCCATACT CCGGCAGGCGCCCCGACGCAATCAGGCTGGGCCTGGGCAACCAC AATTATTGCCGGAATCCTGACAGAGATAGCAAGCCATGGTGTTA CGTGTTCAAGGCCGGCAAGTATTCTAGCGAGTTTTGCTCCACCCC TGCCTGTTCTGAGGGCAACAGCGATTGCTACTTCGGCAATGGCA GCGCCTATAGGGGAACCCACTCCCTGACAGAGTCCGGAGCCTC T TGTCTGCCTTGGAACTCCATGATCCTGATCGGCAAGGTGTACACA GCCCAGAATCCATCTGCCCAGGCCCTGGGACTGGGAAAGCACAA CTATTGCCGCAATCCCGACGGCGATGCCAAGCCTTGGTGTCACG TGCTGAAGAACCGGAGACTGACCTGGGAGTACTGCGATGTGCCC TCriGTAGCACACCHGAVar54 ATGGACGCCATGAAGAGGGGACTGTGCTGCGTGCTGCTGCTGTG 167 CGGAGCCGTGTFCGTG'ICCCCATCTCAGGAGATCCACGCCCGGIT TCGGAGAGGCGCCAGAAGCTACCAGGTGATCTGCAGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGTCTrGGCTGAGGCCTGTGCTGAGGAGCAACAGGGTGGAGTACTGCTGGTGTAATTCCGGCCGGGCACAGTGCCACTCTGTGCCAGTGAAGAGCTGCTCCGAGDocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NO CCTAGATGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTATTTC TCCGACTTCGTGTGCCAGTGTCCAGAGGGCTTTGCCGGCAAGTG CTGTGAGATCGACACCAGGGCCACATGCTACGAGGATCAGGGCA TCTCTTATAGGGGAACCTGGAGCACAGCAGAGTCCGGAGCCGAG TGTACCAACTGGAATAGCTCCGCCCTGGCACAGAAGCCATACTC CGGCAGGCGCCCTGACGCAATCAGGCTGGGACTGGGAAACCAC AATTATTGCCGGAATCCAGACAGAGATAGCAAGCCCTGGTGTTA CGTGTTCAAGGCCGGCAAGTATTCTAGCGAGTTTTGCTCCACCCC CGCCTGTTCTGAGGGCAACAGCGATTGCTACTTCGGCAATGGAA GCGCCTATAGGGGAACCCACTCCCTGACAGAGTCCGGAGCCTCT TGTCTGCCITGGAACl'CCATGATCCTGAl'CGGCAAGGIGTACACA GCCCAGAATCCATCTGCCCAGGCCCTGGGACTGGGAAAGCACAA CTATTGCAGAAATCCTGACGGCGATGCCAAGCCATGGTGTCACG TGCTGAAGAACCGGAGACTGACCTGGGAGTACTGCGACGTGCCA TCTTGTAGCACAGGCCAGAAGGATGTGCCCGGCGTGTACACCAA GGTGACAAACTATCTGGACTGGATCAGGGATAATATGCGCCCTT GAVar54-Kl ATGGACGCC Al GAAGAGGGGACTG TGCTGCGTGCTGC TGCTG TG 168 CGGAGCCGTGTTCGTGAGCCCATCCCAGGAGATCCACGCCCGGT TTCGGAGAGGCGCCAGAAGCTACCAGGTGATCTGCCGGGATGAG AAGACCCAGATGATCTATCAGCAGCACCAGAGCTGGCTGAGACC CGTGCTGCGGTCCAACAGAGTGGAGTACTGCTGGTGTAATTCTG GCAGGGCACAGTGCCACAGCG IGCCAG TGAAGTC I TGCAGCGAG CCCCGCTGTTTCAACGGCGGCACATGTCAGCAGGCCCTGTATTTC TCCGACTTCGTGTGCCAGTGTCCTGAGGGCTTCGCCGGCAAGTGC TGTGAGATCGACGGCAACTCTGATTGCTACTTTGGCAATGGCTCC GCCTATAGGGGCACCCACTCTCTGACAGAGAGCGGCGCCTCCTG TCTGCCCTGGAACTCTATGATCCTGATCGGCAAGGTGTACACCGC ACAGAA ICC I’AGCGCCCAGGCCCTGGGAC TGGGAAAGCACAACT ATTGCCGCAATCCCGACGGCGATGCCAAGCCTTGGTGTCACGTG CTGAAGAATAGGCGCCTGACCTGGGAGTACTGCGACGTGCCTTC CTGTTCTACAGGCCAGAAGGATGTGCCAGGCGTGTACACCAAGG TGACAAACTATCTGGACTGGATCAGGGATAATATGCGCCCCTGAVar3 (NO) atggatgcaatgaagagagggctctgctgtgtgctgctgctgtgtggagcagtcttcgtttcgcccagccag 169 gaaatccatgcccgattcagaagaggagccagatcttaccaagtgatctgcagagatgaaaaaacgcagat gatataccagcaacatcagtcatggctgcgccctgtgctcagaagcaaccgggtggaatattgctggtgca acagtggcagggcacagtgccactcagtgcctgtcaaaagttgcagcgagccaaggtgtttcaacggggg cacctgccagcaggccctgtacttctcagatttcgtgtgccagtgccccgaaggatttgctgggaagtgctgt gaaacagataccggaaacagtgactgetactttgggaatgggtcagcctaccgtggcacgcacagcctcac cgagtcgggtgcctcctgcctcccgtggaattccatgatcctgataggcaaggtttacacagcacagaaccc cagtgcccaggcactgggcctgggcaaacataattactgccggaatectgatggggatgccaagccctgg tgccacgtgctgaagaaccgcaggctgacgtgggagtactgtgatgtgccctcctgctccacctgcggcct gagacagtacagccagcctcagtttcgcccgtgaVarlOl atggacgccatgaagaggggactgtgctgcgtgctgctgctgtgcggagccgtgttcgtgtccccatctca 170 ggagatccacgcccggtttcggagaggcgccagatcttaccaggtgatctgtcgggatgagaagacccag atgatctatcagcagcaccagtcttggctgagacccgtgctgcggagcaacagagtggagtactgctggtgtaatagcggcagggcacagtgccactccgtgccagtgaagagctgctccgagcctcgctgtttcaacggcDocket No: 100-2369WO01Construct Nucleic Acid Sequence SEQ ID NOggcacatgtcagcaggccctgtatttcagcgacttcgtgtgccagtgtcctgagggctttgccggcaagtgc tgtgagatcgacacccgggccacatgctacgaggatcagggcatctcctatagaggaacctggtctacagc agagagcggagcagagtgtaccaactggaatagctccgccctggcacagaagccatactccggcaggc gccccgatgcaatcagactgggactgggcaaccacaatattgcaggaatcctgaccgcgattctaagcca tggtgttacgtgttcaaggccggcaagtattctagcgagttttgctccacacccgcctgctctgagtgtggcct gaggcagtactcccagccccagttccgcccttgaVari 03 atggacgccatgaagaggggactgtgctgcgtgctgctgctgtgcggagccgtgttcgtgagcccttccca 171 ggagatccacgcccggtttcggagaggcgccagatcctaccaggtgatctgtagggatgagaagaccca gatgatctatcagcagcaccagagctggctgaggccagtgctgaggtccaacagggtggagtactgctgg tgtaatccggccgggcacagtgccactctgtgccagtgaagtcttgcagcgagcccagatgtttcaacggc ggcacatgtcagcaggccctgtacttctctgacttcgtgtgccagtgtccagagggattcgccggcaagtgc tgtgagatcgattgcggcctgaggcagtatagccagccccagtttcgcccttgaVari 15 atggacgccatgaagaggggactgtgctgcgtgctgvtgctgtgcggagccgtgttcgtgagvccatcxca 172 ggagatccacgcacggtttcggagaggcgccagaagctaccagtgctccgagcctagatgtttcaacggc ggcacctgtcagcaggccctgtatttcagcgatttcgtgtgccagtgtccagagggcttcgccggcaagtgc tgtgagatcgacggcaactccgaitgctacttggcaatggctctgcctataggggaacccacagcctgaca gagtccggagcctcttgtctgccttggaactccatgatcctgatcggcaaggtgtacacagcccagaatcca tctgcccaggccctgggactgggaaagcacaactattgccggaatccagacggcgatgccaagccctggt gtcacgtgctgaagaataggcgcctgacctgggagtactgcgacgtgccctcttgcagcacatgtggcctg aggcagtattctcagccccagtttcgcccttgaVari 24 atggacgccatgaagaggggactgtgctgcgtgctgctgctgtgcggagccgtgttcgtgagcccatccca 173 ggagatccacgcaaggtttaggagaggagcacgctcctaccaggtgatctgtagggatgagaagaccca gaigatctatcagcagcaccagagctggctgaggcccgtgctgcggtccaacagagtggagtactgctgg tgtaatctggccgggcacagtgccacagcgtgcctgtgaagtccggcaactctgactgttacttcggcaat ggctccgcctatagaggcacccactctctgacagagagcggcgcctcctgcctgccttggaactctatgatc ctgatcggcaaggtgtacacagcacagaatccaagcgcccaggccctgggactgggaaagcacaactatt gccggaatccagacggcgatgccaagccctggtgtcacgtgctgaagaacaggcgcctgacctgggagt actgtgatgtgccctcttgcagcacatgtggcctgcggcagtattctcagccccagtttagacctga

[0170] Brain microvascular endothelial cells were procured from Cell Systems (an AnaBios company; Cat#ACBR1376) and cultured in T75 flasks as per the manufacturer's protocol. One day prior to transfection, cells were seeded at 200,000 cells / well in 12-well plates in duplicate wells for each transgene. The next day, the media was changed prior to the transfection using Lipofectamine LTX Plus reagents (Thermo Fisher, Cat #15338100). All the PLAT variant plasmids were diluted to 100 ng / pl and 1 pg plasmid was used per well to transfect the cells. As a negative control, an empty GFP plasmid was used at the same concentration. Next day, the media was changed, and cells were allowed to grow for 48 hours post transfection before harvesting. Total RNA was isolated from duplicate wells together using RNAeasy Plus micro kit (Qiagen, Cat#74034), converted to cDNA for the maximum amount. 8 pl cDNA was used forDocket No: 100-2369WO01MMP9 amplification using specific TaqMan assay and 2 pl from 1: 10,000 dilution of the cDNA was used for amplifying 18S rRNA as the endogenous control. The relative CT values of MMP9 were normalized by the corresponding endogenous control (18S) and fold change for each transgene was calculated with respect to Variant 0. Based on previous results, Variant 2 was considered the negative control. Results are shown in FIG. 2 and in Table 9 below:Table 9: Activities of exemplary tPA variantsVariant Fold change in MMP9 expression Statistically Significant as as compared to VAR0 compared to VAR2 (Y / N) 0 1.11 Y1 0.56 N2 0.62 N3 1.66 Y4 1.98 Y5 1.13 Y11 1.17 Y12 0.87 N13 1.72 Y14 2.00 Y15 3.02 Y21 1.97 Y22 1.97 Y23 2.00 Y24 0.25 N31 0.51 N32 0.61 N33 0.17 Y (decrease) 34 0.42 N35 0.59 N36 2.20 YDocket No: 100-2369WO01Variant Fold change in MMP9 expression Statistically Significant as as compared to VAR0 compared to VAR2 (Y / N) 41 1.69 Y42 1.83 Y43 1.93 Y44 1.20 Y45 1.54 Y46 3.19 Y51 2.29 Y51-K1 3.29 Y52 2.13 Y52-K1 2.78 Y53 2.30 Y54 1.18 Y54-K1 1.92 Y101 1.45 Y103 2.25 Y115 0.81 N124 4.41 Y

[0171] As shown in FIG. 2, transfection of the tPA variants had varying effects in average MMP9 transcription over the negative control. Variants 0. 101, 103, 11, 124, 13, 14, 15, 21, 22, 23, 3, 36, 4, 41, 42, 43, 44, 45, 46, 5, 51, 51-K1, 52, 52-K1, 53, 54, and 54-K1 each produced a statistically significant increase in MMP9 transcription as compared to Variant 2. Variant 33 results in statistically significant decrease in MMP9 transcription. Variants 1, 12, 2, 24, 31, 32, 34, 35, and 115 did not produce a significant difference in MMP9 transcription as compared to negative control.

[0172] It is known that tPA can be processed, completely or partially, by cleavage between the tail and the catalytic domain to form two polypeptide chains, namely, chain A and chain B (see Table 1). Without wishing to be bound by theory, it is contemplated that Variants 51 andDocket No: 100-2369WO0151 -KI may also be cleaved similarly, resulting in truncated constructs with deletion of the catalytic domain, where the chain B fragment is not retained in the processed protein. Amino acid sequences of processed Variants 51 and 51-K1 are set forth in SEQ ID NOs: 175 and 176, respectively. It is contemplated that the observed activities of Variants 51 and 51 -KI may be ascribed to the untruncated protein, the truncated protein, or both. However, given that the truncated Variant 51 is expected to have an identical sequence as Variant 1 except for lacking the proline residue at the C -terminus, its higher activity than Variant 1 suggests that the cleavage only occurred partially and that in the untruncated protein, the fragment of catalytic domain of SEQ ID NO: 110 may have conferred certain benefit, for example, in facilitating LRP-1 binding. Similarly, given that the truncated Variant 51-K1 is expected to have a similar sequence as Variant 3, except for lacking a threonine residue between the EGF-like and Kringle 2 domains and a proline residue at the C-terminus, its higher activity than Variant 3 suggests that the cleavage only occurred partially and that in the untruncated protein, the fragment of catalytic domain of SEQ ID NO: 110 may have conferred certain benefit, for example, in facilitating LRP-1 binding.

[0173] Variants 31-35, which included fragments of a Kringle 1 domain, did not induce increase in MMP9 transcription. However, the constructs containing either an intact Kringle 1 domain (Variants 11, 13-15, 21-23, 41-46, 51-54, and 101) or a deletion of Kringle 1 domain (Variants 3, 4, 5, and 36) effectively increased MMP9 transcription.

[0174] Without wishing to be bound by any particular theory, it is believed that the Finger and EGF-like domains are important, given that at least one of the domains appears to be needed for the ability of tPA to induce MMP9 transcription (compare the active Variants 15 and 124 with the inactive Variant 2). When a tPA variant includes a Finger domain, a complete EGF-like domain appears to be dispensable. For example, Variants 21-23, which included fragments of the EGF-like domain, still induced MMP9 transcription, even in the absence of the critical Tyr67 (numbered according to SEQ ID NO: 27) in Variants 22 and 23. When a tPA variant includes an EGF-like domain, a complete Finger domain is dispensable. For example, Variant 12, which removed certain amino acids but maintained the two disulfide bonds within the Finger domain (one formed between the cysteine residues at positions 3 and 33 of SEQ ID NO: 2, the other formed between the cysteine residues at positions 31 and 40 of SEQ ID NO: 2), showed reduced activity. In contrast, Variants 11, 13, and 14, which removed certain amino acids and disruptedDocket No: 100-2369WO01at least one of the disulfide bonds (by removal of one or more of the cysteine residues), were capable of inducing MMP9 transcription. Without wishing to be bound by theory, it is contemplated that the disulfide bond disruption may have changed the stereo conformation of the molecule, compared to Variant 12, and restored activity of the tPA variant.

[0175] Overall, the results of the present example provide novel tPA variants which exhibit increased activity as determined by MMP9 transcription. In some aspects, the present results are unexpected as variants which may have been expected to be inactive were instead active and vise versa.INCORPORATION BY REFERENCE

[0176] The entire disclosure of each of the patent documents and scientific articles referred to herein, including but not limited to U. S. Patent No. 10,946,076 and International Application Nos. PCT / US2022 / 016043 and PCI7US2024 / 047456, is incorporated by reference for all purposes.EQUIVALENTS

[0177] 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

Docket No: 100-2369WO01WHAT IS CLAIMED IS:

1. A catalytically inactive tissue plasminogen activator (tPA) variant, the tPA variant comprising:(i) a Finger domain,(ii) an optional EGF-like domain,(iii) a deletion of a Kringle 1 domain,(iv) an optional Kringle 2 domain, and(v) an optional tail region,provided that,the tPA variant comprises at least one of an EGF-like domain and a Kringle 2 domain, and the tPA variant does not comprise the amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 46, or SEQ ID NO: 49.

2. The tPA variant of claim 1, comprising (ii) a deletion of the EGF-like domain, (iv) a Kringle 2 domain, and (v) a tail region.

3. The tPA variant of claim 1, comprising (ii) an EGF-like domain and (iv) a deletion of a Kringle 2 domain.

4. The tPA variant of any one of claims 1-3, further comprising (vi ) a catalytically inactive fragment of a catalytic domain.

5. The tPA variant of claim 4, comprising (ii) an EGF-like domain, (iv) a Kringle 2 domain, and (v) a tail region.

6. The tPA variant of claim 4 or 5, wherein the catalytically inactive fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 110.

7. The tPA variant of claim 4 or 5, wherein the catalytically inactive fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 115.8 The tPA variant of claim 4, comprising (ii) an EGF-like domain, (iv) a Kringle 2 domain, and (v) a deletion of the tail region.Docket No: 100-2369WO019. The tPA variant of claim 8, wherein the catalytically inactive fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 47.

10. The tPA variant of any one of claims 1-9, wherein the tPA variant comprises an ammo acid sequence at least 80% (e.g., at least 85%, at least 90%. at least 95%. or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 91, 92, 113, 114, 118, 119, 125, 126, 129, 130, 133, 134, and 176.

11. A catalytically inactive tissue plasminogen activator (tPA) molecule, the tPA variant comprising:(i) a fragment of a Finger domain or a deletion of a Finger domain,(ii) an EGF-like domain,(iii) a Kringle 1 domain, and(iv) a Kringle 2 domain.

12. The tPA variant of claim 11. comprising (i) a fragment of a Finger domain comprising an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NO: 50, 54, 57, and 60.

13. The tPA variant of claim 11. comprising (i) a deletion of the Finger domain.

14. The tPA variant of any one of claims 11-13, further comprising a tail region.

15. The tPA variant of any one of claims 11-14, comprising a deletion of a catalytic domain.

16. The tPA variant of any one of claims 11-15. wherein the tPA variant comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 51, 53. 55, 56, 58. 59, 61. 62, 63, and 64.

17. A catalytically inactive tissue plasminogen activator (tPA) molecule, the tPA variant comprising:(i) a Finger domain,(ii) a fragment of an EGF-like domain or a deletion of an EGF-like domain,(iii) an optional Kringle 1 domain, and(iv) a Kringle 2 domain.

18. The tPA variant of claim 17, comprising (ii) a fragment of an EGF-like domain and (iii) a Kringle 1 domain.Docket No: 100-2369WO0119. The tPA variant of claim 18, wherein the fragment of the EGF-like domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NO: 65, 68, and 71.

20. The tPA variant of claim 17, comprising (ii) a deletion of an EGF-like domain and (iii) a deletion of a Kringle 1 domain21. I’he tPA variant of any one of claims 17-20. further comprising a tail region.

22. The tPA variant of any one of claims 17-21, further comprising a deletion of a catalytic domain.

23. The tPA variant of any one of claims 17-22. wherein the tPA variant comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%. at least 95%. or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 66, 67, 69, 70. 72, 73, 133, and 134.

24. A catalytically inactive tissue plasminogen activator (tPA) molecule, the tPA variant comprising:(i) a Finger domain,(li) an EGF-like domain,(iii) a Kringle 1 domain, and(iv) a fragment of a Kringle 2 domain or a deletion of a Kringle 2 domain.

25. The tPA vanant of claim 24, comprising (iv) a deletion of a Kringle 2 domain.

26. The tPA variant of claim 24, comprising (iv) a fragment of a Kringle 2 domain.

27. The tPA variant of claim 26, wherein the fragment of the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g, at least 85%>. at least 90%, at least 95%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 93, 96, 99, 102, and 105.

28. The tPA variant of any one of claims 24-27. further comprising a tail region.

29. The tPA variant of any one of claims 24-28, comprising a deletion of a catalytic domain.

30. The tPA variant of any one of claims 24-29. wherein the tPA variant comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%. or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 108, 109, 127, and 128.Docket No: 100-2369WO0131. A catalytically inactive tissue plasminogen activator (tPA) molecule, the tPA variant comprising:(i) a Finger domain,(ii) an EGF-like domain,(iii) a Kringle 1 domain,(iv) a Kringle 2 domain.(v) an optional tail region, and(vi) a catalytically inactive fragment of a catalytic domain.

32. The tPA variant of claim 31, comprising (v) a tail region, wherein the fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 110.

33. The tPA variant of claim 31, comprising (v) a tail region, wherein the fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 115.

34. The tPA variant of claim 31, comprising (v) a deletion of a tail region, wherein the fragment of the catalytic domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 47.

35. The tPA variant of any one of claims 31-34, wherein the tPA variant comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%. or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 111, 112, 116, 117, 123, and 124.

36. A catalytically inactive tissue plasminogen activator (tPA) molecule, the tPA variant comprising:(i) a Finger domain,(ii) an EGF-like domain,(iii) a Kringle 1 domain,(iv) a Kringle 2 domain.(v) a deletion of a tail region, and(vi) an optional catalytically inactive fragment of a catalytic domain.

37. lire tPA variant of claim 36, comprising a deletion of the catalytic domain.

38. The tPA variant of claim 36. comprising a fragment of the catalytic domain that comprises an amino acid sequence at least 80*9, (e.g, at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 47.Docket No: 100-2369WO0139. The tPA variant of any one of claims 36-38, wherein the tPA variant comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%. at least 95%. or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 120, 121, 123, and 124.

40. The tPA variant of any one of claims 1-39, wherein the Finger domain comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%. at least 95%. or 100%) identical to SEQ ID NO: 2.

41. The tPA vanant of any one of claims 1 -40, wherein the EGF-like domain comprises an amino acid sequence at least 80% (e.g., at least 85%. at least 90%. at least 95%, or 100%) identical to SEQ ID NO: 3.

42. The tPA variant of any one of claims 1 -41, wherein the Kringle 1 domain comprises an amino acid sequence at least 80% (e.g., at least 85%. at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 4.

43. The tPA variant of any one of claims 1 -42, wherein the Kringle 2 domain comprises an amino acid sequence at least 80% (e.g., at least 85%. at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 5.

44. The tPA variant of any one of claims 1-10, 14-16, 21-23, and 28-35, and 40-43, wherein the tail comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 8.

45. A catalytically inactive tissue plasminogen activator (tPA) molecule, the tPA variant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 56, 59, 62, 64, 67, 70, 73, 75. 78, 81, 84, 87, 90, 92, 95, 98, 101, 104, 107, 109, 112, 114, 117, 119, 121, 124, 126. 128, 130. 132, 134, 175, and 176.

46. A vector comprising a nucleic acid sequence encoding the tPA variant of any one of claims 1-45.

47. The vector of claim 46, wherein the nucleic acid sequence encodes a signal peptide at an N-terminus of the tPA variant.Docket No: 100-2369WO0148. The vector of claim 47, 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: 11.

49. The vector of any one of claims 46-48, further comprising a promoter operably linked to the nucleic acid sequence encoding the tPA variant.

50. The vector of claim 49, wherein the promoter is active in trabecular meshwork cells.

51. The vector of any one of claims 46-50, further comprising a 5’UTR located upstream of the nucleic acid sequence encoding the tPA vari nt52. The vector of claim 51, wherein the 5’ UTR comprises a nucleic acid sequence at least 70% e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identical to SEQ ID NO: 52.

53. The vector of any one of claims 46-52, wherein the vector is a viral vector.

54. The vector of claim 53, wherein the viral vector is an adeno-associated virus (AAV) vector.

55. The vector of claim 54, wherein the AAV vector is a self-complementary AAV vector.

56. The vector of claim 54 or 55, wherein the AAV vector comprises capsid proteins VP1, VP2, and VP3 comprising amino acid sequences at least 80% (e.g., 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 NOs: 12, 13, and 14, respectively.

57. Use of the tPA variant of any one of claims 1-45 or the vector of any one of claims 46-56 in the preparation of a medicament.

58. A method of increasing outflow facility 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 1-45 or the vector of any one of claims 46-56.Docket No: 100-2369WO0159. 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 1-45 or the vector of any one of claims 46-56.

60. The method of claim 58 or 59, wherein the subject has open angle glaucoma.

61. The method of claim 60, wherein the subject has juvenile open angle glaucoma.

62. The method of any one of claims 58-61, 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.

63. The tPA variant of any one of claims 1-45, wherein the tPA variant upregulates the expression of matrix metalloproteinases (MMPs).

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