Endothelial cells for treatment of anal fissure

Abstract

The present invention relates to the use of endothelial cells for the treatment of anal fissure (such as chronic anal fissure) and provides compositions and methods relating thereto. In some aspects of the present invention the endothelial cells are E4ORF1+ endothelial cells, such as E4ORF1+ human umbilical vein endothelial cells.

Description

[0001]International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 ENDOTHELIAL CELLS FOR TREATMENT OF ANAL FISSURE CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 651,344 filed on May 23, 2024, the content of which is hereby incorporated by reference in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 9, 2025, is named Angiocrine_037_WO1_SL.xml and is 1,841 bytes in size. INCORPORATION BY REFERENCE For the purposes of only those jurisdictions that permit incorporation by reference, all of the references cited in this disclosure and any manufacturers’ instructions or catalogues for any products cited or mentioned herein are hereby incorporated by reference in their entireties. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Numbers in square brackets following text herein refer to the numbered references in the Reference List section of this patent disclosure. BACKGROUND Anal fissure is a painful condition consisting of a tear in the anoderm that is typically linear and extends cephalad to the dentate line. The majority (around 90%) of anal fissures are in the posterior midline; however, anal fissures can also be located in the anterior midline, more frequently in women. An anal fissure in the lateral position may indicate other disease processes like inflammatory bowel disease [1]. The pain, especially associated with defecation, can be extreme and may last 15 minutes to 2 hours after a bowel movement [2]. Spasms of the internal anal sphincter can occur, which International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 may also be painful. Small amounts of anal bleeding can also occur. Current treatments for acute anal fissure consist of stool softeners, dietary fiber supplements, and topical ointments (e.g., containing analgesics and / or vasodilators). If healing is not achieved promptly, anal fissures can lead to a poor quality of life [1]. While many anal fissures heal by themselves in a few weeks, many do not. These are called chronic anal fissures. As many as 50% of patients who develop an acute anal fissure do not heal despite medical treatment [1] leading to a chronic anal fissure. Previously, it was thought that repeated infections of an acute anal fissure wound led to chronicity, with remnant epithelial cells in the wound being the culprit. However, more recently, ischemic pathophysiology has been highlighted as a potential cause of chronic anal fissures (CAF), where high resting anal sphincter pressure leads to reduced blood flow and poor healing or a lack of healing of the anal fissure wound [4-6]. Spontaneous healing of CAF may occur (reported to be occur in from 10% to as high as 35% of cases), but such healing is often plagued by relapse and may be associated with chronic fecal evacuation disorder [7]. Hence, CAF patients typically undergo medical management consisting of topical vasodilators (e.g., 2% diltiazem or 4mg / g glyceryl trinitrate ointment). The healing rate using topical vasodilators has been reported to be ~50% [3]. Botulinum toxin injection may also be employed, for example combined with topical vasodilator treatment or following failure of vasodilator treatment. Botulinum toxin doses of ≥ 50IU have been reported to be efficacious in 40-70% of cases. However, temporary anal incontinence and relapse remain as unmet needs [3]. Surgical intervention may be indicated for CAF patients who have failed medical therapy. Internal lateral anal sphincterotomy is considered to be the gold standard of surgical intervention. The procedure provides prompt symptomatic relief by reducing pathologically elevated pressures within the anal canal. Success rates following such surgeries have been reported to be around 90%. However, a significant downside to surgery is the risk of anal incontinence as a complication. Up to 50% of patients experience transient incontinence, varying from the inability to control gas to loss of formed stool resulting in soiling. Long-term incontinence rate has been reported between 5-14%. The rate for major incontinence (defined as involuntary loss of feces) has been reported to be approximately 2%. Some experts have advocated limiting the International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 sphincterotomy to the length of the anal fissure, which has been shown to reduce the risk of incontinence. However, this is associated with an increased risk of non-healing anal fissure or recurrence of anal fissures. Other complications of internal anal sphincterotomy include infection, bleeding, and fistula development [8]. Given the current medical landscape for chronic anal fissure, an unmet need exists for a medical intervention that is safe and effective, especially for those who have failed the currently available interventions of topical vasodilators and / or botulinum toxin injections, and / or who would like to avoid current surgical methods and the associated downsides. The present invention addresses this need. SUMMARY OF THE INVENTION The present invention provides methods for the treatment of anal fissures in mammalian subjects (preferably human subjects) in need thereof, the methods comprising administering to such subjects an effective amount of a composition comprising endothelial cells – which are preferably E4ORF1+ human umbilical vein endothelial cells. The methods of the present invention may provide several advantages over prior methods of treatment of anal fissures, including, but not limited to: increasing the probability of complete healing of an anal fissure, accelerating the healing of an anal fissure, decreasing pain on defecation, decreasing the duration of pain, decreasing bleeding, decreasing the duration of bleeding, decreasing spasm; decreasing the duration of spasm, improving anal incontinence, reducing the risk of significant temporary or permanent anal incontinence, improving quality-of-life, reducing the rate of relapse. Also, importantly the methods of the present invention can be performed on an out-patient basis in a procedure that takes only 5-10 minutes to perform and avoids the need for surgery, cutting into the sphincteric muscles and the sensitive perianal tissue. In preferred embodiments the anal fissures treated using the methods of the present invention are chronic anal fissures (CAF), such as chronic non-healing anal fissures and / or chronic recurring anal fissures. In some embodiments, the subjects treated using the methods of the present invention have chronic anal fissure. In some embodiments, the subjects treated using the methods of the International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 present invention have a non-healing chronic anal fissure. In some embodiments, the subjects treated using the methods of the present invention have a recurrent chronic anal fissure (CAF). In some embodiments, the subjects treated using the methods of the present invention have previously been unsuccessfully treated using another anal fissure medical treatment or surgery. In some embodiments, the subjects treated using the methods of the present invention have previously been unsuccessfully treated using a topical vasodilator. In some embodiments, the subjects treated using the methods of the present invention have previously been unsuccessfully treated using botulinum toxin. In some embodiments, the subjects treated using the methods of the present invention have previously been unsuccessfully treated using internal lateral anal sphincterotomy surgery. In some embodiments the endothelial cells are umbilical vein endothelial cells (UVECs). In some embodiments, the UVECs are human umbilical vein endothelial cells (HUVECs). In some embodiments the UVECs or HUVECs are E4ORF1+. In preferred embodiments the endothelial cells are E4ORF1+ HUVECs. In some embodiments the composition comprising the ECs (e.g. E4ORF1+ HUVECs) is administered to the subject locally (e.g., to the location of the anal fissure). In some embodiments the composition comprising the ECs (e.g., E4ORF1+ HUVECs) is administered to the subject by local injection. In some embodiments the composition comprising the ECs (e.g., E4ORF1+ HUVECs) is administered to the subject locally by percutaneous injection (e.g., to the location of the anal fissure). An important advantage of the methods of the present invention is that, because percutaneous delivery involves no intrusion into the anal sphincter, subjects can be treated in an outpatient setting and without damage to the anal sphincter. In the inventive method, the total EC dose administered to the subject may be dependent on (1) the length of the anal fissure (e.g., in cm or mm) or the area of the fissure wound (e.g. in cm2or mm2), (2) the number of injections applied along the sides of the fissure (for example, one, two, three, four, or more), (3) the specific number (dose) of ECs applied per centimeter of anal fissure length or per cm2of anal fissure area, (4) the number of anal fissures to be treated in the subject, and (5) the number of treatments given over a course of treatment (for example, one, two administered, for example, three weeks apart, three administered at intervals of, for example, three weeks, four administered at intervals of, for International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 example, two or four weeks, or more.). A course of treatment refers to repeating cycles of treatment, followed by a pause, rest, or other strategy, to maximize effectiveness. Each cycle in a course of treatment need not apply the same dose of ECs administered in a different cycle. The dose used in any one cycle may be determined on an ad hoc basis as indicated by the status of the subject’s anal fissure and / or the subject’s response to prior administration. In some embodiments, the ECs are administered to the subject at a dose (the number of ECs administered per treatment) of from about 5 x 106to about 200 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 150 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 120 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 100 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 50 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 40 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 30 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 15 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 7.5 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 7.5 x 106to about 60 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 7.5 x 106to about 30 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 7.5 x 106to about 15 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 15 x 106to about 30 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of about 7.5 x 106total ECs per anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose of about 15 x 106total ECs per anal fissure. In some International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 embodiments, the ECs are administered to the subject at a treatment dose of about 30 x 106total ECs per anal fissure. In some embodiments, the EC treatment dose (as described above) is administered to the subject along one side (one half) of the anal fissure. For example, in some embodiments, the ECs are administered to the subject at a dose of from about 5 x 106to about 200 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of from about 5 x 106to about 150 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of from about 5 x 106to about 120 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of from about 5 x 106to about 100 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of from about 5 x 106to about 50 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of from about 5 x 106to about 40 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of from about 5 x 106to about 30 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of from about 5 x 106to about 15 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of from about 5 x 106to about 7.5 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of from about 7.5 x 106to about 60 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of from about 7.5 x 106to about 30 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of from about 7.5 x 106to about 15 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of from about 15 x 106to about 30 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of about 7.5 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the ECs are administered to the subject at a dose of about 15 x 106total ECs along one side (one half) of the anal fissure. In some International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 embodiments, the ECs are administered to the subject at a dose of about 30 x 106total ECs along one side (one half) of the anal fissure. In some embodiments, the total EC treatment dose (as described above) is split such that, for example, half of the EC treatment dose is administered along one (first) side of the fissure and half of the EC treatment dose is administered along the other (second) side of the fissure. In some of such embodiments the treatment dose administered can be any of the treatment dose ranges or treatment doses listed above but split such that a portion (such as approximately half) of the EC treatment dose is administered to each of the two sides of the anal fissure. In some embodiments, the ECs are administered to the subject at a treatment dose (the number of ECs administered per treatment) of from about 5 x 106to about 200 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 150 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 120 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 100 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 50 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 40 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a dose of from about 5 x 106to about 30 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 5 x 106to about 15 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 7.5 x 106to about 50 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 7.5 x 106to about 40 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 7.5 x 106to about 30 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a treatment dose of from about 7.5 x 106to about 15 x 106ECs per cm of anal fissure length. In some embodiments, the ECs International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 are administered to the subject at a treatment dose of from about 15 x 106to about 30 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a treatment dose of about 7.5 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a treatment dose of about 15 x 106ECs per cm of anal fissure length. In some embodiments, the ECs are administered to the subject at a treatment dose of about 30 x 106ECs per cm of anal fissure length. In some embodiments the subject has more than one anal fissure (for example, two or three or up to ten separate anal fissures) to be treated. In some embodiments one or more anal fissure of the subject will be treated once, for example by administering a treatment dose (as described above) of a composition comprising ECs (e.g., E4ORF1+ HUVECs) to the subject once, in which case the day on which the composition is administered to the subject may be referred to as “Day 0.” In some embodiments one or more anal fissures of the subject will be treated once, for example by administering a composition comprising ECs (e.g., E4ORF1+ HUVECs) at a dose range or dose as described above, once. In some embodiments one or more anal fissures of the subject will be treated more than once, for example by administering a course of treatment in the form of a treatment dose (as described above) of a composition comprising ECs (e.g., E4ORF1+ HUVECs) administered to the subject multiple times (e.g., twice, or three times). In some embodiments one or more anal fissures of the subject will be treated more than once, for example by administering a treatment dose of a composition comprising ECs (e.g., E4ORF1+ HUVECs) at a treatment dose range or treatment dose as described above to the subject multiple times (e.g., twice or three times). In some such embodiments the time interval between treatments / administrations may be about one week, about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about seven weeks, about eight weeks, about nine weeks, about ten weeks, about twelve weeks, about fourteen weeks, about sixteen weeks, about eighteen weeks, about twenty weeks, or more. In some embodiments, the time interval between administrations is from about 2 weeks to about 12 weeks. In some embodiments, the time interval between administrations is from about 4 weeks to about 10 weeks. In some embodiments, the time interval between administrations is from 6 weeks to about 8 weeks. In some embodiments, the time interval International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 between administrations is from about 3 to about 4 weeks. For example, in some embodiments the composition comprising the ECs (e.g., E4ORF1+ HUVECs) is administered to the subject twice, once on Day 0 and again on Day 21. Similarly, in some embodiments the composition comprising the ECs (e.g., E4ORF1+ HUVECs) is administered to the subject twice, once on Day 0 again on Day 28. In some embodiments the time interval between administrations is determined on an ad hoc basis as indicated by the status of the subject’s anal fissure and / or the subject’s response to the prior administration. In some embodiments the treatment dose administered at any one treatment within a course of treatment may be different from that administered at any other treatment within that course; for example, the treatment dose delivered on Day 0 may be different from that delivered on Day 21. In some embodiments, the treatment methods of the present invention involve also performing internal lateral anal sphincterotomy surgery on the subject prior to administration of the ECs. In some embodiments, the treatment methods of the present invention involve also performing internal lateral anal sphincterotomy surgery on the subject concurrently with administration of the ECs. In some embodiments, the treatment methods of the present invention involve also performing internal lateral anal sphincterotomy surgery on the subject after administration of the ECs. In some embodiments, the treatment methods of the present invention involve also administering a vasodilator (e.g., diltiazem or glyceryl trinitrate) and / or botulinum toxin to the subject prior to administration of the ECs. In some embodiments, the treatment methods of the present invention involve also administering a vasodilator (e.g., diltiazem or glyceryl trinitrate) and / or botulinum toxin to the subject concurrently with administration of the ECs. In some embodiments, the treatment methods of the present invention involve also administering a vasodilator (e.g., diltiazem or glyceryl trinitrate) and / or botulinum toxin to the subject after administration of the ECs. In some embodiments, the treatment methods of the present invention can be performed in the absence of internal lateral anal sphincterotomy surgery, or in the absence of other surgical anal fissure interventions. Indeed, an important aspect of the present invention is that the methods described herein, while they can be used as an adjunct to surgical International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 interventions, can also be used as the primary treatment modality thus avoiding surgical procedures that can lead to various complications including incontinence. In some embodiments, the treatment methods of the present invention can be performed in the absence of other medical interventions, for example in the absence of vasodilator and / or botulinum toxin administration. In some embodiments the compositions comprising the ECs comprise ECs (e.g., E4ORF1+ HUVECs) in a saline solution (e.g., an isotonic saline or buffered saline). In some such embodiments the compositions comprise one or more additional components suitable for administration to living subjects selected from the group consisting of buffers, salts, polysaccharides, proteins, and preservatives. In some such embodiments the compositions comprise one or more of human serum albumin (HSA), Dextran40, and DMSO. In some such embodiments the compositions comprise up to about 1% DMSO. In some such embodiments the compositions comprise up to about 0.25% DMSO. In some such embodiments the compositions comprise about 6% Dextran 40. In some such embodiments the compositions comprise about 10% HSA. In some such embodiments the compositions comprise about 6% Dextran 40 and about 10% HSA. In some such embodiments the compositions comprise about 6% Dextran 40 and about 10% HSA and about 0.25% DMSO. In some such embodiments the compositions comprise about 6% Dextran 40 and about 10% HSA in an isotonic saline or buffered saline. In some such embodiments the compositions comprise about 6% Dextran 40 and about 10% HSA and about 0.25% DMSO in an isotonic saline or buffered saline. In some embodiments the compositions comprising the ECs comprise ECs (e.g., E4ORF1+ HUVECs) at a concentration of from about 0.5 x106cells / ml to 50 x106cells / ml. In some embodiments the compositions comprising the ECs comprise ECs (e.g., E4ORF1+ HUVECs) at a concentration of from about 1.0 x106cells / ml to about 25 x106cells / ml. In some embodiments the compositions comprising the ECs comprise ECs (e.g., E4ORF1+ HUVECs) at a concentration of about 1 x106cells / ml. In some embodiments the compositions comprising the ECs comprise ECs (e.g., E4ORF1+ HUVECs) at a concentration of about 5 x106cells / ml. International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 These and other embodiments of the invention are described further in other sections of this patent disclosure, and this Summary of the Invention is intended to be read in conjunction with all other sections of this patent disclosure. In addition, as will be apparent to those of skill in the art, certain modifications and combinations of the various embodiments described herein fall within the scope of the present invention. DETAILED DESCRIPTION The “Summary of the Invention,” “Examples,” and “Claims” sections of this patent disclosure describe many of the main embodiments of the present invention. This “Detailed Description” section provides certain additional description relating to the compositions and methods of the present invention and is intended to be read in conjunction with all other sections of this patent disclosure. Furthermore, and as will be apparent to those in the art, the different embodiments described throughout this patent disclosure can be combined in different ways, regardless of any subheadings. Such combinations of the specific embodiments described herein fall within the scope of the present invention. Definitions Certain definitions and abbreviations are provided below. Other terms or phrases may be defined elsewhere in this patent disclosure or may have meanings that are clear from the context in which they are used. Unless defined otherwise herein, or unless some other meaning is clear from their use in context herein, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. For example, The Dictionary of Cell and Molecular Biology (5th ed. J.M. Lackie ed., 2013), the Oxford Dictionary of Biochemistry and Molecular Biology (2d ed. R. Cammack et al. eds., 2008), and The Concise Dictionary of Biomedicine and Molecular Biology (2d ed. P-S. Juo, 2002) can provide one of skill with general definitions of some terms used herein. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably. International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone). Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10. Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and / or “consisting essentially of” are included. As used herein, the terms “about” and “approximately,” when referring to a stated numeric value, indicate a value within plus or minus 10% of the stated numeric value. In all embodiments where a numerical value is described using the terms “about” or “approximately” the analogous embodiment where the precise numerical value is used is also contemplated. For example, for an embodiment that involves a time of about 24 hours, the analogous embodiment involving a time of 24 hours (i.e., without the “about” qualification) is also contemplated. As used herein, cells are “positive” or “+” for a started marker when that marker is expressed to a detectable degree in the cells. For example, endothelial cells are positive for E4ORF1 (i.e., are E4ORF1+) if E4ORF1 is expressed to a detectable degree by the endothelial cells. In some situations, expression of a stated marker may be assessed in comparison to, e.g., an experimental negative control. As used herein, the abbreviation “E4ORF” refers to an open reading frame (ORF) present within the adenoviral early 4 (E4) region of an adenovirus genome, or a polypeptide / protein encoded by that ORF (whether the gene or the protein is referred to will be clear from the International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 context of use). As used herein, the abbreviation “E4ORF1” refers to open reading frame (ORF) 1 of the early 4 (E4) region of an adenovirus genome, or a polypeptide / protein encoded by that ORF (whether the gene or the protein is referred to will be clear from the context of use). As used herein, the abbreviation “E4ORF1+” when used with reference to a stated cell type refers to a cell that expresses E4ORF1 to a detectable degree. E4ORF1+ cells contain a recombinant E4ORF1 nucleic acid molecule and express the E4ORF1 protein. As used herein, the abbreviation “EC(s)” refers to an endothelial cell(s). As used herein, the abbreviation “UVEC” refers to umbilical vein endothelial cells. As used herein, the abbreviation “HUVEC” refers to human umbilical vein endothelial cells. As used herein, the terms “E-CELs” and “E-CEL UVECs” and “E-CEL UVEC cells” refer to E4ORF1+ HUVECs or compositions comprising E4ORF1+ HUVECs. Whether the cells per se or a composition comprising the cells is referred to will be clear from the context in which the terms are used. For example, reference to E-CELs in the context of administration to a subject necessarily denotes administration of a composition comprising the E-CELs. As used herein, the term “allogeneic” means deriving from, originating in, or being members of the same species, where the members are genetically related or genetically unrelated but genetically similar. For example, in embodiments involving administration of allogeneic ECs to a subject, the allogeneic ECs are obtained from a donor of the same species as the subject to whom the cells will be administered (i.e., the recipient). In some embodiments the allogeneic cells are obtained from a donor having a full or partial MHC / HLA match to the subject to whom the cells will be administered (i.e., the recipient) - i.e., the donor of the cells and the recipient of the cells are fully or partially MHC-matched or HLA-matched. In some embodiments, the ECs are: (a) obtained from a donor, (b) maintained and / or cultured and / or expanded ex vivo, and (c) subsequently administered into a subject of the same species as the donor. International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 As used herein, the term “autologous” means deriving from or originating in the same subject. For example, in embodiments involving administration of autologous ECs to a subject, the autologous cells are obtained from the subject to whom they will be administered (i.e., the donor and recipient of the ECs are the same individual). In some embodiments, ECs are: (a) obtained from a subject, (b) maintained and / or cultured and / or expanded ex vivo, and (c) subsequently administered to the same subject. As used herein the term “chronic” when used in reference to an anal fissure refers to an anal fissure that persists for ≥ 6 weeks. As used herein, the terms “treat,” “treating,” and “treatment” refer to achieving, to a detectable degree, an improvement in, or prevention of worsening of, one or more symptoms or sequelae of the condition being treated. For example, such terms, as used herein, include achieving, to a detectable degree (and optionally to a statistically significant degree and / or to a clinically significant degree), one or more of the following “treatment outcomes,” depending on the condition being treated: (a) healing of an anal fissure, (b) increased speed of healing of an anal fissure, (c) improvement (i.e., decrease) in, or prevention of worsening of, discharge from an anal fissure, (d) improvement in, or prevention of worsening of, an abscess in the vicinity of an anal fissure, (e) a reduction in the frequency or speed of relapse / recurrence of an anal fissure, (f) improvement (i.e., decrease) in, or prevention of worsening of, pain associated with an anal fissure, (g) healing of an anal fissure, or any one or more of the treatment outcomes or treatment endpoints described in Example 2, and the like. Any suitable methods or clinical tests for assessing these treatment outcomes / endpoints may be used and suitable methods for assessing such treatment outcomes will be well known to physicians practicing in the art of the present invention. In some embodiments, such treatment outcomes may be assessed by comparing a symptom or sequela in a single subject before and after treatment, or by comparing a symptom or sequela in a subject treated with a method as disclosed herein to that in a different individual who is not treated or is not treated or who is treated with a placebo, or by comparing a symptom or sequela in subject treated with a method as disclosed herein to a reference value for that symptom or sequela (e.g., a reference value for that symptom or sequela typically exhibited in a healthy person of the same sex, age and weight). For each of the embodiments described herein that refer to a method of treatment, an analogous International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 method directed to achieving any one or more of the treatment outcomes or endpoints listed above or listed in Example 2, or that results in any one or more of the treatment outcomes or endpoints listed above or in Example 2, is also provided. For example, in addition to providing methods useful for the treatment of an anal fissure in subjects in need thereof comprising administering to the subjects an effective amount of a composition comprising endothelial cells (ECs), the present invention also comprises an analogous method for the treatment of an anal fissure in subjects in need thereof comprising administering to the subjects an effective amount of a composition comprising endothelial cells (ECs) wherein the treatment results in healing of an anal fissure, or increased speed of healing of an anal fissure, or healing of an anal fissure, or improvement (i.e., decrease) in, or prevention of worsening of, discharge from an anal fissure, and / or improvement (i.e., decrease) in, or prevention of worsening of, pain associated with an anal fissure. As used herein the term “effective amount” refers to an amount of a stated cell type (e.g., E4ORF1+ HUVECs), or a composition comprising that stated cell type, that is sufficient to achieve the stated outcome (e.g., treatment of an anal fissure) to a detectable degree, or, in some cases to a specifically stated degree. In some embodiments the effective amount is specified (e.g., in terms of a number of E4ORF1+ HUVECs per anal fissure, or per centimeter of anal fissure length). In other embodiments an appropriate “effective amount” may be determined empirically, for example using standard techniques known in the art, such as dose escalation studies, and may be determined taking into account such factors as the planned route of administration, desired frequency of administration, etc. Furthermore, an “effective amount” may be determined using studies such as those described in the Examples section of this patent disclosure. The term “engineered” when used in relation to ECs refers to ECs cells that have been engineered by man to result in the recited phenotype (e.g., E4ORF1 expression), or to express a recited protein. The term “engineered cells” is not intended to encompass naturally occurring cells, but is, instead, intended to encompass, for example, cells that comprise a recombinant nucleic acid molecule, or cells that have otherwise been altered artificially, for example so that they express a protein that they would not otherwise express, or so that they express a protein at substantially higher levels than that observed in non-engineered ECs. International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 As used herein the abbreviation “HLA” refers to human leukocyte antigen. As used herein term “subject” refers to a mammalian individual. In some embodiments the subject is a human. When the subjects are human the terms “subject” and “patient” may be used interchangeably. As used herein, the term “anal fissure” is used consistently with its normal meaning in the art. Thus, the term “anal fissure” as used herein encompasses both acute and chronic anal fissures. The term “anal fissure” as used herein also encompasses anal fissures of any etiology, including anal fissures caused by, or associated with passing large or hard stools, constipation, long-lasting diarrhea, childbirth, Crohn's disease, inflammatory bowel disease(s), anal cancer, HIV, tuberculosis or syphilis. The term “anal fissure” also includes anal fissure of unknown etiology (i.e., idiopathic anal fissure). Methods of Treatment In some embodiments the present invention provides methods for the treatment of anal fissure in subjects in need thereof, the methods comprising administering to the subjects an effective amount of a composition comprising endothelial cells (ECs). In some embodiments the subjects treated using the methods of the present invention are mammalian subjects. In preferred embodiments the subjects treated using the methods of the present invention are human subjects. In some embodiments the ECs administered in the treatment methods of the present invention are autologous ECs. In some embodiments the ECs administered in the treatment methods of the present invention are allogeneic ECs. In some embodiments the ECs administered in the treatment methods of the present invention are umbilical vein endothelial cells (UVECs). In some embodiments the ECs administered in the treatment methods of the present invention are human umbilical vein endothelial cells (HUVECs). In some such embodiments the UVECs are E4ORF1+ UVECs. In preferred embodiments the UVECs are E4ORF1+ HUVECs. International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 In some embodiments, the treatment methods of the present invention involve administering ECs to the subject at a certain dose or at a certain dose range. Suitable doses and dose ranges are described in the Summary of the Invention section above. In some embodiments, the ECs are administered to the subject such that the total volume of the EC-containing composition administered in a single administration is from about 0.5 mls to about 10 mls, such as from about 0.5mls to about 1.0 ml. In some embodiments, the treatment methods of the present invention involve administering ECs to the subject with a certain administration schedule (referred to in the Summary of the Invention section above as “a course of treatment”). In some embodiments, the treatment methods of the present invention involve administering ECs to the subject once, i.e., in a single administration (for example a single administration of a dose of ECs as specified above). In some embodiments, the treatment methods of the present invention involve administering ECs to the subject multiple times, i.e., in multiple administrations (for example where each of the multiple administrations involves administration of a dose of ECs as specified above). For example, in some embodiments, the treatment methods of the present invention involve administering ECs to the subject twice, or three times, or four times, or more. In those embodiments that involve administering ECs to the subject multiple times, the time interval between administrations may be about one week, about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about seven weeks, about eight weeks, about nine weeks, about ten weeks, about twelve weeks, about fourteen weeks, about sixteen weeks, about eighteen weeks, about twenty weeks, or more. In some embodiments, the time interval between administrations is from about 2 weeks to about 12 weeks. In some embodiments, the time interval between administrations is from about 4 weeks to about 10 weeks. In some embodiments, the time interval between administrations is from 6 weeks to about 8 weeks. In some preferred embodiments, the time interval between administrations is about six weeks. In other preferred embodiments, the time interval between administrations is about twelve weeks. In some embodiments the time interval between administrations is determined on an ad hoc basis as indicated by the status of the subject’s anal fissure and / or the subject’s response to the prior administration. In some embodiments the dose of ECs administered at each treatment within a course of treatment is determined on an ad hoc basis as indicated by the status of the subject’s anal International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 fissure and / or the subject’s response to the prior administration. Other suitable administration schedules are described in the Summary of the Invention section above. In some embodiments, the treatment methods of the present invention involve administering ECs to the subject using any suitable route of administration. In preferred embodiments, the treatment methods of the present invention involve administering ECs to the subject locally at the site of the anal fissure. In some preferred embodiments, the treatment methods of the present invention involve administering ECs to the subject by local injection at the site of anal fissure, by percutaneous administration, e.g., using a needle. In some preferred embodiments, the treatment methods of the present invention involve administering ECs to the subject by local percuctaneous injection at the site of anal fissure using a 23- to 33-gauge needle. In some embodiments, the treatment methods of the present invention involve administering ECs to the subject under direct visualization (e.g., using imaging guidance). In some embodiments, the administration of the ECs involves depositing the ECs along the length of the anal fissure. In some embodiments, the administration of the ECs involves depositing the ECs in a single injection along the length of one side / half of the anal fissure. In some embodiments, the administration of the ECs involves depositing the ECs in two or more separate injections along the length of both sides of the anal fissure. In some embodiments, the administration of the ECs is performed by inserting a needle / syringe (e.g., through the skin) into the soft tissues adjacent to the anal fissure and injecting the EC- containing composition along the length of the anal fissure as the needle is withdrawn, thereby depositing the ECs along the length of the anal fissure. Such a method may be used to deposit / distribute the ECs relatively uniformly along the length of the anal fissure. This procedure can be performed on either one side or both sides of the anal fissure. In some embodiments the methods of treatment provided herein can be performed in conjunction with performing one or more additional medical treatments or surgical procedures on the subject. For example, in some embodiments the methods of treatment provided herein are performed in conjunction with performing internal lateral anal sphincterotomy surgery. Similarly, in some embodiments the methods of treatment provided herein are performed in conjunction with treating an anal fissure using a vasodilator and / or botulinum toxin. International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 In some embodiments the methods of treatment provided herein can be performed in conjunction with administering one or more additional agents (e.g., pharmaceutical agents, biologic agents, or cell therapy agents) to the subjects, i.e., in addition to the EC-containing compositions described herein. Additional agents that can be used in such methods include vasodilators, botulinum toxin, growth factors, cytokines, anti-inflammatory agents, immunosuppressive agents, anti-thymocyte globulin, mycophenylate mofetil, cyclosporin, methotrexate, cyclophosphamide, Rituxan, steroids, anti-bacterial agents, anti-viral agents, and anti-fungal agents, and other agents known in the art to be useful in the treatment of anal fissure or in the context of cell therapy or transplant therapy. In some such embodiments the one or more additional agents is administered in the same composition as the ECs (i.e., the additional agents can be added to the compositions of the present invention). In some embodiments, the one or more additional agents is administered separately from the compositions of the present invention. For example, in some embodiments, one or more additional agents can be administered to the subjects in a different composition to that in which the ECs are provided and administered to the subjects prior to, concurrently with, or after administration of the EC-containing compositions of the present invention. In some embodiments, the one or more additional agents is administered to the subject within about 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours 12 hours, 24 hours or more before or after administration of the EC-containing compositions of the present invention. In some embodiments, the treatment methods of the present invention lead to a detectable improvement in, or prevention of worsening of, one or more symptoms or sequelae of the anal fissure being treated. In some embodiments, the treatment methods of the present invention lead to one or more of (a) healing of a anal fissure, (b) increased speed of healing of a anal fissure, (c) improvement (i.e., decrease) in, or prevention of worsening of, discharge from a anal fissure, (d) improvement in, or prevention of worsening of, an abscess in the vicinity of the anal fissure, (e) a reduction in the frequency or speed of relapse of a anal fissure, and / or (f) improvement (i.e., decrease) in, or prevention of worsening of, pain associated with a anal fissure, (g) healing of a anal fissure, or (h) one or more of the outcomes / endpoints listed in Example 2. Various methods may be used to detect and / or assess the therapeutic effects of the methods of the present invention. Such methods include International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 visual inspection (including making measurements of the length or area of an anal fissure), imaging studies, biopsy studies, patient questionnaires, and the like. Endothelial Cells The methods and compositions of the present invention involve endothelial cells (ECs). In some embodiments the ECs are umbilical vein endothelial cells (UVECs). UVECs are isolated from umbilical cords. The UVECs disclosed herein can be isolated from the umbilical cord using any means known to those skilled in the art. By way of example only, UVECs can be isolated from umbilical vein tissue by a process wherein tissue is excised and mechanically broken into fragments. Enzymes may also be perfused through the major vessels of the umbilical cord to release the endothelial cells lining the interior of the vessels and any vessels downstream thereof. Once released, the cells can be suspended and cultured in a medium supportive of endothelial cell growth. In some embodiments the cell population so isolated can be further purified, for example, by immunoselection of endothelial cells using a marker or combination of markers that is selective for endothelial cells (for example, antibody directed to CD31). The endothelial cells may be expanded in culture. In some embodiments the endothelial cells are transfected with a gene conferring a growth advantage over non-transduced cells such that transduced cells grow more rapidly and / or more stably than the non-transduced cells from which they were derived. Additional methods for preparation of UVECs are provided in the Examples section of this patent disclosure. In some embodiments ECs (e.g., UVECs) are genetically modified. In preferred embodiments the ECs (e.g., UVECs) are genetically modified to express the adenovirus E4ORF1 protein, i.e., they are E4ORF1+ ECs (e.g., E4ORF1+ UVECs). In some embodiments, the ECs are engineered to express one or more ETS family transcription factors, (i.e., they are ETS+ ECs). In some embodiments, the ECs are engineered to express the ETS family transcription factor ETV2 (i.e., they are ETV2+ ECs). In some embodiments, the ECs are E4ORF1+ ETV2+ECs. International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 E4ORF1 Several of the embodiments of the present invention involve ECs that are E4ORF1+ - i.e., ECs that express an E4ORF1 polypeptide. The “E4ORF1” polypeptide is encoded by open reading frame (ORF) 1 of the early 4 (E4) region of the adenovirus genome. E4ORF1+ ECs for use in accordance with the present invention typically comprise a recombinant nucleic acid molecule that contains an E4ORF1 coding sequence operatively linked to a promoter suitable for expression of the E4ORF1 coding sequence in ECs. E4ORF1 amino acid sequences and nucleotide sequences are known in the art. Any such sequences may be used in accordance with the present invention. In some embodiments the E4ORF1 polypeptide may be from any suitable adenovirus type or strain, such as human adenovirus type 2, 3, 5, 7, 9, 11, 12, 14, 34, 35, 46, 50, or 52. In some preferred embodiments the polypeptide sequence used is from human adenovirus type 5. Amino acid sequences of such adenovirus polypeptides, and nucleic acid sequences that encode such polypeptides, are well known in the art and available in well-known publicly available databases, such as the Genbank database. For example, suitable sequences include the following: human adenovirus 9 (Genbank Accession No. CAI05991), human adenovirus 7 (Genbank Accession No. AAR89977), human adenovirus 46 (Genbank Accession No. AAX70946), human adenovirus 52 (Genbank Accession No. ABK35065), human adenovirus 34 (Genbank Accession No. AAW33508), human adenovirus 14 (Genbank Accession No. AAW33146), human adenovirus 50 (Genbank Accession No. AAW33554), human adenovirus 2 (Genbank Accession No. AP.sub.--000196), human adenovirus 12 (Genbank Accession No. AP.sub.--000141), human adenovirus 35 (Genbank Accession No. AP.sub.--000607), human adenovirus 7 (Genbank Accession No. AP.sub.--000570), human adenovirus 1 (Genbank Accession No. AP.sub.--000533), human adenovirus 11 (Genbank Accession No. AP.sub.--000474), human adenovirus 3 (Genbank Accession No. ABB 17792), and human adenovirus type 5 (Genbank accession number D12587). In some embodiments the E4ORF1 sequence used is that having NCBI accession number AZR66741.1. In some embodiments the E4ORF1 sequence used is that having NCBI accession number AP_000232.1. In some embodiments the E4ORF1 sequence used is has the amino acid sequence: International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 MAAAVEALFVVLEREGAILPRQEGFSGVYVFFSPINFVIPPMGAVMLSLRLRVCIP PGYFGRFLALTDVNQPDVFTESYIMTPDMTEELSVVLFNHGDQFFYGHAGMAVV RLMLIRVVFPVVRQASNV (SEQ ID NO.1). In some embodiments the E4ORF1 polypeptide may have amino acid sequence that is, or be encoded by a nucleic acid sequence that is, a variant, derivative, mutant, or fragment of any of the specific sequences provided herein or known in the art provided that such variants, derivatives, mutants, or fragments are, or encode, a polypeptide that has one or more of the functional properties of adenovirus E4ORF1 described herein. In some embodiments, the variants, derivatives, mutants, or fragments have about an 85% identity to the known sequence, or about an 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the known sequence. In some embodiments, a variant, derivative, mutant, or fragment of a known nucleotide sequence is used that varies in length by about 50 nucleotides, or about 45 nucleotides, or about 40 nucleotides, or about 35 nucleotides, or about 30 nucleotides, or about 28 nucleotides, 26 nucleotides, 24 nucleotides, 22 nucleotides, 20 nucleotides, 18 nucleotides, 16 nucleotides, 14 nucleotides, 12 nucleotides, 10 nucleotides, 9 nucleotides, 8 nucleotides, 7 nucleotides, 6 nucleotides, 5 nucleotides, 4 nucleotides, 3 nucleotides, 2 nucleotides, or 1 nucleotide relative to the known nucleotide sequence. In some embodiments, a variant, derivative, mutant, or fragment of a known amino sequence is used that varies in length about 50 amino acids, or about 45 amino acids, or about 40 amino acids, or about 35 amino acids, or about 30 amino acids, or about 28 amino acids, 26 amino acids, 24 amino acids, 22 amino acids, 20 amino acids, 18 amino acids, 16 amino acids, 14 amino acids, 12 amino acids, 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, or 1 amino acid relative to the known amino acid sequence. In some embodiments E4ORF1 sequences are used without other sequences from the adenovirus E4 region – for example not in the context of the entire E4 region or not together with other ORFs in the E4 region. However, in other embodiments E4ORF1 may be used in conjunction with one or more other ORFs from the E4 region, such as E4ORF2, E4ORF3, E4ORF4, E4ORF5 or E4ORF6 / 7 sequences. For example, although E4ORF1 sequences can be used in constructs (such as a viral vectors) that contain other sequences, genes, or coding regions (such as promoters, marker genes, antibiotic resistance genes, and International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 the like), in certain embodiments, the E4ORF1 sequences are used in constructs that do not contain the entire E4 region, or that do not contain other ORFs from the entire E4 region, such as E4ORF2, E4ORF3, E4ORF4, and / or E4ORF5. E4ORF1 encoding sequences can be present in constructs or vectors that contain various other sequences, genes, or coding regions, for example, promoters, enhancers, antibiotic resistance genes, reporter genes or expression tags (such as, for example nucleotides sequences encoding GFP), or any other nucleotide sequences or genes that might be desirable. E4ORF1-encoding nucleic acid molecules can be under the control of one or more promoters to allow for expression. Any promoter able to drive expression of the E4ORF1 nucleic acid sequences in endothelial cells can be used. Examples of suitable promoters include, but are not limited to, the CMV, SV40, RSV, HIV-Ltr, and MML promoters. The promoter can also be a promoter from the adenovirus genome, or a variant thereof. For example, in some embodiments the promoter may be a promoter that drives expression of E4ORF1 in nature in an adenovirus genome. However, in other embodiments the promoter is not one that drives expression of E4ORF1 in nature in an adenovirus genome. The E4ORF1-encoding sequences may comprise naturally occurring nucleotides, synthetic nucleotides, or a combination thereof. For example, in some embodiments the nucleic acid molecules of the invention can comprise RNA, such as synthetic modified RNA that is stable within cells and can be used to direct protein expression / production directly within cells. In other embodiments the E4ORF1-encoding sequences can comprise DNA. In embodiments where DNA is used, the DNA sequences may be operably linked to one or more suitable promoters and / or regulatory elements to allow (and / or facilitate, enhance, or regulate) expression within cells, and may be present in one or more suitable vectors or constructs. The E4ORF1-encoding sequences can be introduced into UVECs using any suitable system known in the art, including, but not limited to, transfection techniques and viral-mediated transduction techniques. Transfection methods that can be used in accordance with the present invention include, but are not limited to, liposome-mediated transfection, polybrene-mediated transfection, DEAE dextran-mediated transfection, electroporation, International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 calcium phosphate precipitation, microinjection, and micro-particle bombardment. Viral- mediated transduction methods that can be used include, but are not limited to, lentivirus- mediated transduction, adenovirus-mediated transduction, retrovirus-mediated transduction, adeno-associated virus-mediated transduction and herpesvirus-mediated transduction. In some embodiments the E4ORF1-encoding sequences are in a vector. In some embodiments the E4ORF1-encoding sequences are in a viral vector. In some embodiments the E4ORF1-encoding sequences are in a lentiviral vector. In some embodiments the E4ORF1-encoding sequences are in an adenoviral vector. In some embodiments the E4ORF1-encoding sequences are in adeno-associated virus vector. In some embodiments the E4ORF1-encoding sequences are in a retroviral vector. In some embodiments the E4ORF1-encoding sequences are in a Moloney murine leukemia virus (MMLV) vector (a type of retroviral vector). In some embodiments the presence of E4ORF1 coding sequences in the ECs can be confirmed and / or quantified using standard nucleic acid detection and / or quantification assays known in the art, such as PCR-based techniques (e.g., quantitative PCR) and sequencing-based techniques (e.g., quantitative next generation sequencing-based techniques). In some embodiments the presence of E4ORF1 polypeptides can be confirmed and / or quantified using standard protein detection and / or quantification assays known in the art, such as antibody-based techniques. In some embodiments the expression of functional E4ORF1 polypeptide (or an appropriate amount of functional E4ORF1 polypeptide can be confirmed and / or quantified using functional assays (e.g., in vitro or in vivo assays) for any of the functional properties of E4ORF1-expressing endothelial cells that are known in the art (such as any of those described in U.S. Patent No.8,465,732). The handling, manipulation, and expression of E4ORF1 sequences in ECs may be performed using conventional techniques of molecular biology and cell biology. Such techniques are well known in the art. For example, one may refer to the teachings of Sambrook, Fritsch and Maniatis eds., “Molecular Cloning A Laboratory Manual, 2nd Ed., Cold Springs Harbor Laboratory Press, 1989); the series Methods of Enzymology (Academic Press, Inc.), or any other standard texts for guidance on suitable techniques to use in handling, manipulating, and expressing nucleotide and / or amino acid sequences. International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 Additional aspects relevant to the handling and expression of E4ORF1 sequences in endothelial cells are described in U.S. Patent No. 8,465,732, the contents of which are hereby incorporated by reference. Compositions In some embodiments the compositions used in the methods of the present invention comprise an effective amount of ECs (e.g., E4ORF1+ HUVECs). Typically, such compositions comprise ECs in a saline solution (such as an isotonic saline, physiological saline, or buffered saline) suitable for administration to a subject. In some embodiments the compositions may comprise one or more additional excipients suitable for administration to human subjects and compatible with cell therapies, such as buffers, salts, preservatives, polysaccharides (e.g., dextrans), proteins (e.g., albumin) and the like. In some embodiments the compositions may comprise a low concentration of a cryopreservative. In some embodiments the compositions of the present invention comprise Dextran 40. In some embodiments the compositions of the present invention comprise human serum albumin (HSA). In some embodiments the compositions of the present invention comprise dimethyl sulfoxide (DMSO), for example at a concentration of up to 1%, or preferably at a concentration of up to 0.25%. In some embodiments the compositions of the present invention comprise ECs (such as E4ORF1+ HUVECs), Dextran40, HSA and DMSO in an isotonic saline. Other suitable compositions are described in the Summary of the Invention section above. In some embodiments the compositions of the present invention comprise both E4ORF1+ and E4ORF1-negative ECs. In some embodiments at least about 75% of the ECs in the compositions are E4ORF1+. In some embodiments at least about 80% of the ECs in the compositions are E4ORF1+. In some embodiments at least about 85% of the ECs in the compositions are E4ORF1+. In some embodiments at least about 90% of the ECs in the compositions are E4ORF1+. In some embodiments at least about 95% of the ECs in the compositions are E4ORF1+. In some embodiments at least about 98% of the ECs in the compositions are E4ORF1+. In some embodiments at least about 99% of the ECs in the compositions are E4ORF1+. International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 Typically, the ECs (e.g., E4ORF1+ HUVECs) for use in the methods of the present invention are supplied (e.g., to the medical facility where there will be administered to subjects) in frozen form – i.e., in a frozen composition containing the specified endothelial cells (e.g., E4ORF1+ HUVECs) and a cryopreservative. The frozen compositions are thawed prior to use, for example using standard methods known in the art. In some embodiments the frozen composition may be thawed and then administered to a subject without dilution. However, in other embodiments the frozen composition is thawed and then diluted in a suitable diluent to result in the generation of a composition containing the desired or specified concentration cells and the desired or specified concentrations of excipients, for example as described above. Kits In some embodiments the present invention provides a kit comprising a composition according to the present invention in sterile packaging and instructions for administering the therapeutic composition to a subject for treatment of an anal fissure. Such kits will typically include a label indicating the intended use of the contents of the kit. In this context, the term “label” includes any writing or recorded material supplied on or with the kit, or that otherwise accompanies the kit. The invention is further described with reference to the following non-limiting Examples. EXAMPLES The following examples are provided to demonstrate particular situations and settings in which this technology may be applied and are not intended to restrict the scope of the invention and the claims included in this disclosure. Example 1 Preparation of E4ORF1+ HUVECs HUVECs were isolated from the umbilical vein of an umbilical cord by enzymatic digestion with collagenase. Cells were then cultured in endothelial cell growth medium before transduction with a viral vector containing an E4ORF1 expression cassette (encoding SEQ International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 ID NO.1). The resulting E4ORF1+ HUVEC cells are referred to throughout these Examples and the Figures referenced therein as “E-CEL UVECs or “E-CEL UVEC cells.” For some studies E-CEL UVEC cells were transferred to bioreactors and expanded to yield the desired number of cells. Expanded E-CEL UVEC cells were cryopreserved. Prior to use, E-CEL UVEC cells were thawed, optionally further cultured / expanded, and prepared in a final composition (e.g., in a physiological saline) to be administered to subjects as described in subsequent Examples. Example 2 Human Clinical Trial of E4ORF1+ HUVECs for Treatment of Chronic Anal Fissure This Phase 1b clinical trial is conducted to evaluate the initial safety and efficacy of local (percutaneous) injections of a study drug that is a composition containing allogeneic (consented-maternal donor) human umbilical vein endothelial cells transduced to express adenovirus E4ORF1 (“E4ORF1+ HUVECs”) as an experimental treatment of patients with chronic anal fissure (CAF) who have failed medical therapy (i.e., topical vasodilators ± botulinum toxin injection). The study is a non-randomized, open-label, dose-escalating single arm study, meaning every study participant will receive some dose of the experimental study drug (no placebo). Inclusion criteria for patient participation in the clinical trial include: (1) a minimum age of 18 years, (2) a maximum age of 90 years, (3) any sex (treatment not gender based), (4) anterior or posterior chronic anal fissure (with chronicity defined as presence of anal fissure for ≥ 6 weeks), (5) inadequate response to medical treatment of anal fissure (e.g., 1 month of failed vasodilator treatment plus declined or failed botulinum toxin injection treatment), and (6) a recent history of pain on defecation at a level 4 or higher on the numerical rating scale (NRS). Exclusion criteria for clinical trial participation include: (1) lateral anal fissure, (2) the presence of peri-anal or rectovaginal fistula, rectal or anal stenosis, or peri-anal abscess or non-healing peri-anal post-surgical wounds that are not anal fissures (subjects with history of anorectal surgery with healed surgical wounds are not excluded), (3) active, untreated or medically unresponsive infection of an anal fissure or anal fistula (e.g., erythema and pus), International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 (4) active systemic infection (e.g., bacteremia, sepsis), (5) presence of inflammatory bowel diseases (e.g., Crohn’s, ulcerative colitis), (6) taking systemic chemotherapy or local pelvic radiation treatments, (7) renal impairment defined by serum creatinine ≥ 1.5 x upper limit of normality (ULN), (8) hepatic impairment defined by both of the following laboratory ranges: (a)total bilirubin ≥ 1.5 x ULN unless benign congenital hyperbilirubinemia; and (b) aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≥ 2.5 x ULN, (9) active alcohol or substance use that, in the opinion of the site investigator, will interfere with study follow-up, (10) active malignant tumor (tumors must be in remission for ≥ 6 months without maintenance chemotherapy and / or radiation), (11) ongoing or recent history (within 6 months) of abnormal, severe, progressive, or uncontrolled hepatic, hematological, gastrointestinal, endocrine, pulmonary, cardiac, neurological, psychiatric, or cerebral diseases, (12) congenital immunodeficiencies (13) history of major surgery or severe trauma within the previous 3 months, (14) Subjects who are actively being considered as candidates for solid organ transplantation or who may have a high likelihood of needing a solid organ transplant (e.g., progressive heart failure patients), (15) females who are pregnant or breastfeeding or planning to become pregnant or breastfeed during the study (180 days), (16) subjects who have known hypersensitivity or documented allergy to DMSO, and (17) subjects who do not wish to or cannot comply with study procedures. Consented, eligible participants will receive percutaneous injections of a composition containing E4ORF1+ HUVECs along both sides of the fissure. The treatments will be spaced 3 to 4 weeks apart. Initial safety and efficacy parameters will be observed over a 6- month period, followed by a long-term follow-up consisting of an annual questionnaire. The primary outcomes measured include: (1) safety of the E4ORF1+ HUVEC injections (incidence of acute injection reactions and serious adverse events attributed to the study drug), and (2) efficacy (assessed as proportion of subjects achieving ≥ 50% reduction in pain-on-defecation (using numerical rating scale (NRS)) and ≥ 50% reduction in fissure- wound area (based on clinical examination supplemented by photo-documentation) from baseline (Day 0)). Secondary outcome measures include: (1) safety of the of the E4ORF1+ HUVEC injections over a time frame of 180 days (incidence of adverse events, regardless of attribution, over 180 days following the first E4ORF1+ HUVEC injection), and (2) efficacy to include International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 fistula healing (assessed based on a reduction in size of the anal fissure, improvement in symptoms such as anal pain on defecation, time-to 50% fistula wound closure, etc.) over a time frame of 180 days. Treatment with the study drug will take place on Day 0, preferably in an outpatient setting. Day 0 is defined as the day-of-treatment. Screening and certain baseline tests can occur between Days -30 to 0. If multiple baseline measures are taken during this period, then the measure closest to or on Day 0 will be used as “baseline.” On the day-of-treatment (Day 0), the subject’s anal fissure is examined and photo-documented (e.g., using a ruler marked in millimeters visible in a digital photograph for documentation of the fissure length and / or area). If required, a per os (PO) anxiolytic may be administered to the subject prior to treatment. Similarly, if required, administration of an anesthetic (and if clinically indicated, short-term, intravenous analgesic or anxiolytic) will be provided. Appropriate preparation of the fissure is performed. The composition containing the E4ORF1+ HUVECs prepared as described in Example 1 is percutaneously injected into both sides of the anal fissure, with half of the total cell dose being injected into each side. The subject is then observed for at least 90 minutes for any post-treatment adverse events prior to leaving the clinical site. The subject then attends follow-up visits at certain intervals up to Day 180 after the initial treatment (Day 0) for evaluation of treatment outcomes (e.g., primary and secondary endpoints). Additional details of this clinical trial are provided in Table 1 below. Table 1 Phase 1B Clinical Trial of E-CEL UVEC Therapy for Chronic Anal Fissure International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 Example 3 Results of Human Clinical Trial of E4ORF1+ HUVECs for Treatment of Chronic Anal Fissure Three patients with chronic anal fissure meeting the inclusion and exclusion criteria specified in Example 2 were treated with E4ORF1+ HUVECs in accordance with the clinical trial summary shown in Example 2. On Study Day 0, both subjects were administered 30x106E4ORF1+ HUVECs in two injections of 15x106E4ORF1+ HUVECs along each side of the fissure. A second treatment was administered to the first two subjects using the same approach and dose between approximately Study Day 21 and Day 28. The third subject has not yet received a second dose. No subject exhibited a severe (Grade ≥ 3) injection site reaction (as per CTCAE v5.0 terms and grading) or serious adverse events attributed (related) to the E4ORF1+ HUVECs at any treatment. International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 The anal fissure for the first subject was deemed healed on Study Day 56. The second patient reported considerable reduction in symptoms and partial healing of the fissure by Day 28. This patient has yet to reach their Day 90 study visit. Prior to Study Day 28, the third patient showed ongoing healing with reduction in fissure size and residual symptoms following their first treatment. A second treatment is being scheduled for this patient. These data are consistent with a good safety profile and a positive E4ORF1+ HUVECs treatment effect. Reference List 1. Schlichtemeier, S. and A. Engel, Anal fissure. Aust Prescr, 2016.39(1): p.14-7. 2. Nelson, R.L., Anal fissure (chronic). BMJ Clin Evid, 2014.2014. 3. NICE, 2% Topical diltiazem hydrochloride for chronic anal fissure.2013, National Institute for Health and Care Excellence. 4. Shafik, A., A new concept of the anatomy of the anal sphincter mechanism and the physiology of defecation. XV. Chronic anal fissure: a new theory of pathogenesis. Am J Surg, 1982.144(2): p.262-8. 5. Schouten, W.R., J.W. Briel, and J.J. Auwerda, Relationship between anal pressure and anodermal blood flow. The vascular pathogenesis of anal fissures. Dis Colon Rectum, 1994.37(7): p.664-9. 6. Schouten, W.R., et al., Ischaemic nature of anal fissure. Br J Surg, 1996.83(1): p. 63-5. 7. Jain, M., et al., Fecal evacuation disorders in anal fissure, hemorrhoids, and solitary rectal ulcer syndrome. Indian J Gastroenterol, 2019.38(2): p.173-177. 8. Acar, T., et al., Treatment of chronic anal fissure: Is open lateral internal sphincterotomy (LIS) a safe and adequate option? Asian J Surg, 2019.42(5): p. 628-633. 9. Candotti, F., et al., Gene therapy for adenosine deaminase-deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans. Blood, 2012.120(18): p.3635-46. 10. Ding, B.S., et al., Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization. Cell, 2011.147(3): p.539-53. 11. Ding, B.S., et al., Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature, 2010.468(7321): p.310-5. 12. Poulos, M.G., et al., Endothelial transplantation rejuvenates aged hematopoietic stem cell function. J Clin Invest, 2017.127(11): p.4163-4178. 13. Poulos, M.G., et al., Vascular Platform to Define Hematopoietic Stem Cell Factors and Enhance Regenerative Hematopoiesis. Stem Cell Reports, 2015.5(5): p.881-894.

Claims

International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 CLAIMS We Claim:

1. A method of treating a chronic anal fissure in a human subject in need thereof, the method comprising administering to a human subject with a chronic anal fissure a composition comprising E4ORF1+ human umbilical vein endothelial cells (E4ORF1+ HUVECs), wherein the composition is administered to the subject by local administration at the site of the anal fissure, thereby treating the chronic anal fissure in the subject.

2. The method of claim 1, wherein the composition is administered to the subject by percutaneous injection at the site of the anal fissure.

3. The method of claim 1 or claim 2, wherein from about 5 x 106to about 200 x 106total E4ORF1+ HUVECs are administered at the site of the anal fissure.

4. The method of claim 3, wherein approximately half of the total E4ORF1+ HUVECs are administered on one (first) side of the fissure and half of the total E4ORF1+ HUVECs are administered on the other (second) side of the fissure.

5. The method of claim 1 or claim 2, wherein from about 5 x 106to about 50 x 106total E4ORF1+ HUVECs are administered at the site of the anal fissure.

6. The method of claim 5, wherein approximately half of the total E4ORF1+ HUVECs are administered on one (first) side of the fissure and half of the total E4ORF1+ HUVECs are administered on the other (second) side of the fissure.

7. The method of claim 1 or claim 2, wherein about 30 x 106total E4ORF1+ HUVECs are administered at the site of the anal fissure.

8. The method of claim 7, wherein approximately half of the total E4ORF1+ HUVECs are administered on one (first) side of the fissure and half of the total E4ORF1+ HUVECs are administered on the other (second) side of the fissure.International Patent Application Attorney Docket No.: Angiocrine.037.WO1 Electronically Filed: May 23, 2025 9. The method of any of the preceding claims, comprising repeating the administration of E4ORF1+ HUVECs to the subject multiple times, wherein the time interval between administrations is from about 2 weeks to about 12 weeks.

10. The method of claim 9, wherein the time interval between administrations is from about 3 weeks to about 4 weeks.

11. The method of any of the preceding claims, wherein the composition comprises E4ORF1+ HUVECs and one or more excipients suitable for administration to a human subject selected from the group consisting of diluents, buffers, salts, polysaccharides, proteins, and preservatives.

12. The method of claim 11, wherein the composition comprises human serum albumin (HSA).

13. The method of claim 11 or 12, wherein the composition comprises Dextran40.

14. The method of any one of claims 11-13, wherein the composition comprises up to about 0.25% DMSO.

15. The method of any one of claims 11-14, wherein the composition comprises HSA, Dextran40 and up to about 0.25% DMSO.

16. The method of any of the preceding claims, wherein the composition is administered percutaneously using a needle.

17. The method of any of the preceding claims, wherein the composition is administered percutaneously under imaging guidance.

18. The method of any of the preceding claims, wherein the subject has an anal fissure that is not responsive to treatment with a vasodilator or with botulinum toxin.

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