Endothelial cells for the treatment of gastrointestinal fistulas

Abstract

The present invention relates to the use of endothelial cells for the treatment of gastrointestinal fistulas, and provides related compositions and methods. In some embodiments of the present invention, endothelial cells are E4ORF1 + Endothelial cells, for example, E4ORF1 + These are human umbilical vein endothelial cells.

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 499,189, filed on 28 April 2023, the entirety of which is incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML format, the entire contents of which are incorporated herein by reference. The XML copy, created on April 29, 2024, is named Angio_035_WO1_Sequence_Listing and has a size of 2,070 bytes.

[0003] Inclusion by quotation For the purposes of jurisdictions that permit inclusion by quotation only, all documents cited herein shall be incorporated in their entirety by quotation. Furthermore, manufacturer's instructions or catalogs of products cited or referred to herein shall be incorporated by quotation. Any documents incorporated herein by quotation or any teachings therein may be used in the practice of the present invention. [Background technology]

[0004] background A gastrointestinal (GI) fistula is an abnormal connection between the epithelialized surface of the digestive system and another part of the body (often the skin), commonly taking the form of a tunnel called a fistula tract. An anal fistula is a specific type of GI fistula in which there is an abnormal connection between the epithelialized surface of the anal canal and the skin or perianal area around the anus. Many GI fistulas occur as complications of abdominal surgery. However, GI fistulas can also occur spontaneously, sometimes as a result of inflammation or infection in the abdominal cavity. Anal fistulas most often result from anal abscess infection, but Crohn's disease and radiation proctitis are also major causes of anal fistulas. If left untreated, GI fistulas can lead to serious complications such as bacterial infection and life-threatening sepsis. Symptoms of a GI fistula include abscess, pain, discharge of pus, blood, and / or intestinal contents (sometimes large amounts), and fecal incontinence, which dramatically reduce the patient's quality of life (QOL).

[0005] The primary goals of GI fistula treatment are fistula closure and healing of the fistula wound. The current standard treatment is fistula incision, a surgical procedure that involves incising the entire length of the fistula and opening it to heal as a flat scar. Other current treatments include fistula excision, fistula curettage, glue endovascular embolization, advance flap surgery, and plug placement. However, many current fistula treatments carry a high risk of persistent or recurrent fistulas and a high risk of treatment-related fecal incontinence. In particular, surgical fistula incision and / or fistula excision for anal fistula repair often results in damage to sphincter tissue during surgery, leading to fecal incontinence.

[0006] Therefore, there is a need in the art for novel and improved methods for treating GI fistulas, including anal fistulas. This invention addresses this need using cell therapy. [Overview of the project]

[0007] The present invention is based in part on certain studies and findings described in the Examples section of this specification. In particular, topical administration of genetically modified human umbilical vein endothelial cells (HUVECs) to gastrointestinal fistulas has been found to be safe and associated with improved fistula repair in a variety of different clinical protocols and in at least two types of gastrointestinal fistulas, including anal fistulas. Based on these studies and findings, the present invention provides a variety of novel and useful methods and compositions for the treatment of GI fistulas, as further described in the Detailed Description, Examples, and Claims of this specification, as summarized below.

[0008] Accordingly, in some embodiments, the present invention provides a method for treating gastrointestinal ("GI") fistulas in mammalian subjects requiring such treatment, the method comprising administering an effective amount of a composition comprising endothelial cells (ECs) to the subject.

[0009] In some embodiments, the GI fistula treated using the method of the present invention is any fistula comprising any portion of the GI duct. For example, in some embodiments, the GI fistula treated using the method of the present invention is any fistula comprising any portion of the small intestine or the large intestine. Similarly, in some embodiments, the GI fistula treated using the method of the present invention is any fistula comprising any portion of the oral cavity, esophagus, stomach, duodenum, jejunum, ileum, colon, rectum, or anus.

[0010] In some embodiments, the GI fistula treated using the method of the present invention is an anal fistula, anorectal fistula, anorovaginal fistula, appendiceal fistula, biliary fistula, cecal fistula, colonic fistula, colorectal fistula, cocovesical fistula, duodenal fistula, duodenocolonic fistula, enterovesical fistula, esophageal fistula, gallbladder fistula, gastrostomy, gastrocolic fistula, gastrojejunal fistula, ileal fistula, jejunal fistula, oral fistula, pancreatic fistula, rectal fistula, rectovaginal fistula, or salivary gland fistula. In some preferred embodiments, the GI fistula is an anal fistula.

[0011] In some embodiments, the GI fistula treated using the method of the present invention is an intestinal fistula. In some embodiments, the GI fistula treated using the method of the present invention is a non-healing intestinal fistula. In some embodiments, the GI fistula treated using the method of the present invention is an intestinal fistula. In some embodiments, the gastrointestinal fistula treated using the method of the present invention is a non-healing intestinal fistula.

[0012] In some embodiments, the GI fistula treated using the method of the present invention is a blind fistula. In some embodiments, the GI fistula treated using the method of the present invention is a complete fistula. In some embodiments, the GI fistula treated using the method of the present invention is an incomplete fistula. In some embodiments, the GI fistula treated using the method of the present invention is a simple fistula. In some embodiments, the GI fistula treated using the method of the present invention is a complex fistula. In some embodiments, the GI fistula treated using the method of the present invention is a primary fistula. In some embodiments, the GI fistula treated using the method of the present invention is a recurrent fistula.

[0013] 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 + It is HUVEC.

[0014] In some embodiments, EC (e.g., E4ORF1) + The composition containing HUVEC is administered topically to the subject (e.g., at the site of a GI fistula). In some embodiments, EC (e.g., E4ORF1 + Compositions containing HUVEC are administered to the subject by local injection. In some embodiments, EC (e.g., E4ORF1 + The composition containing HUVEC is administered to the subject by transdermal injection (for example, at the site of a GI fistula).

[0015] In the method of the present invention, the total dose of EC administered to a subject can vary depending on (1) the length of the fistula, (2) the number of injections applied along the side of the fistula (e.g., 1, 2, or 3), (3) whether the internal (mucosal) opening is treated (and at what dose), (4) the specific number (dose) of EC applied per 1 cm of the fistula, and (5) the number of fistulas treated in the subject.

[0016] In some embodiments, EC is administered to a subject having a fistula with a length of from about 1 cm to about 8 cm.

[0017] In some embodiments, EC is administered to the subject at a total amount of EC of from about 5×10 6 to about 200×10 6 per fistula. In some embodiments, EC is administered to the subject at a total amount of EC of from about 5×10 6 to about 150×10 6 per fistula. In some embodiments, EC is administered to the subject at a total amount of EC of from about 5×10 6 to about 120×10 6 per fistula. In some embodiments, EC is administered to the subject at a total amount of EC of from about 5×10 6 to about 100×10 6 per fistula. In some embodiments, EC is administered to the subject at a total amount of EC of from about 5×10 6 to about 50×10 6 per fistula. In some embodiments, EC is administered to the subject at a total amount of EC of from about 5×10 6 to about 40×10 6 per fistula. In some embodiments, EC is administered to the subject at a total amount of EC of from about 5×10 6 to about 30×10 6 per fistula. In some embodiments, EC is administered to the subject at a total amount of EC of from about 5×10 6 to about 15×10 6 per fistula. In some embodiments, EC is administered to the subject at a total amount of EC of from about 5×10 6 to about 7.5×10 6 per fistula. In some embodiments, EC is administered to the subject at a total amount of EC of from about 7.5×10 6From approximately 60 x 10 6 The total amount of EC is administered to the subject. In some embodiments, the EC is approximately 7.5 × 10 per fistula. 6 From approximately 30 x 10 6 The total amount of EC is administered to the subject. In some embodiments, the EC is approximately 7.5 × 10 per fistula. 6 From approximately 15 x 10 6 The total amount of EC is administered to the subject. In some embodiments, the EC is approximately 15 × 10 per fistula. 6 ~Approx. 30×10 6 The total amount of EC is administered to the subject. In some embodiments, the EC is approximately 7.5 × 10 per fistula. 6 The total amount of EC is administered to the subject. In some embodiments, the EC is approximately 15 × 10 per fistula. 6 The total amount of EC is administered to the subject. In some embodiments, the EC is approximately 30 × 10 per fistula. 6 ~Approx. 30×10 6 The total amount of EC is administered to the target.

[0018] In some embodiments, EC is administered to the subject along one side (one half) of the fistula. For example, in some embodiments, EC is administered along one side (one half) of the fistula, approximately 5 × 10 6 From approximately 200 x 10 6 The total EC dose is administered to the subject. In some embodiments, the EC is administered along one side (one half) of the fistula, approximately 5 × 10 6 From approximately 150 x 10 6 The total amount of EC is administered to the subject. In some embodiments, EC is administered along one side (one half) of the fistula, approximately 5 × 10 6 From approximately 120 x 10 6 The total EC dose is administered to the subject. In some embodiments, the EC is administered along one side (one half) of the fistula, approximately 5 × 10 6 From approximately 100 x 10 6 The total amount of EC is administered to the subject. In some embodiments, EC is administered along one side (one half) of the fistula, approximately 5 × 10 6 From approximately 50 x 10 6 The total amount of EC is administered to the subject. In some embodiments, EC is administered along one side (one half) of the fistula, approximately 5 × 10 6From approximately 40 x 10 6 The total amount of EC is administered to the subject. In some embodiments, EC is administered along one side (one half) of the fistula, approximately 5 × 10 6 From approximately 30 x 10 6 The total amount of EC is administered to the subject. In some embodiments, EC is administered along one side (one half) of the fistula, approximately 5 × 10 6 From approximately 15 x 10 6 The total amount of EC is administered to the subject. In some embodiments, EC is administered along one side (one half) of the fistula, approximately 5 × 10 6 From approximately 7.5 x 10 6 The total EC dose is administered to the subject. In some embodiments, the EC is administered along one side (one half) of the fistula, approximately 7.5 × 10 6 From approximately 60 x 10 6 The total EC dose is administered to the subject. In some embodiments, the EC is administered along one side (one half) of the fistula, approximately 7.5 × 10 6 From approximately 30 x 10 6 The total amount of EC is administered to the subject. In some embodiments, EC is administered along one side (one half) of the fistula in an amount of approximately 7.5 × 10 6 From approximately 15 x 10 6 The total amount of EC is administered to the subject. In some embodiments, EC is administered along one side (one half) of the fistula, approximately 15 × 10 6 From approximately 30 x 10 6 The total amount of EC is administered to the subject. In some embodiments, EC is administered along one side (one half) of the fistula in an amount of approximately 7.5 × 10 6 The total amount of EC is administered to the subject. In some embodiments, EC is administered along one side (one half) of the fistula, approximately 15 × 10 6 The total amount of EC is administered to the subject. In some embodiments, EC is administered along one side (one half) of the fistula, approximately 30 × 10 6 The total amount of EC is administered to the target.

[0019] In some embodiments, EC is administered to the subject along two sides of the fistula, for example, at the 12 o'clock and 6 o'clock positions on the circumference of the fistula. The term "12 o'clock" as used herein refers to the position on the anterior midline, which may also be called the cephalic position, and all other positions referred to using the term "o'clock" are positioned relative to the 12 o'clock position. In some such embodiments, the total dose administered may be any of the above dose ranges or doses, but a portion of the total EC dose (e.g., about half) is divided so that it is administered to each of the two sides of the fistula tract. In some embodiments, EC is administered at approximately 5 × 10⁻⁶ 6 From approximately 200 x 10 6 The total EC dose is administered to the subject along two sides of the fistula (for example, at the 12 o'clock and 6 o'clock positions on the circumference of the fistula). In some embodiments, the EC dose is approximately 5 × 10⁻¹⁰. 6 From approximately 150 x 10 6 The total EC dose is administered to the subject along two sides of the fistula (for example, at the 12 o'clock and 6 o'clock positions on the circumference of the fistula). In some embodiments, the EC dose is approximately 5 × 10⁻¹⁰. 6 From approximately 120 x 10 6 The total EC dose is administered to the subject along two sides of the fistula (for example, at the 12 o'clock and 6 o'clock positions on the circumference of the fistula). In some embodiments, the EC is approximately 5 × 10⁻¹⁰. 6 From approximately 100 x 10 6 The total EC dose is administered to the subject along two sides of the fistula (for example, at the 12 o'clock and 6 o'clock positions on the circumference of the fistula). In some embodiments, the EC dose is approximately 5 × 10⁻¹⁰. 6 From approximately 50 x 10 6 The total EC dose is administered to the subject along two sides of the fistula (for example, at the 12 o'clock and 6 o'clock positions on the circumference of the fistula). In some embodiments, the EC is approximately 5 × 10⁻¹⁰. 6 From approximately 40 x 10 6 The total EC dose is administered to the subject along two sides of the fistula (for example, at the 12 o'clock and 6 o'clock positions on the circumference of the fistula). In some embodiments, the EC is approximately 5 × 10⁻¹⁰. 6 From approximately 30 x 10 6With the total EC dosage, it is administered to the subject along two sides of the fistula (e.g., at the 12 o'clock and 6 o'clock positions of the circumference of the fistula). In some embodiments, EC is about 5×10 6 to about 15×10 6 With the total EC dosage, it is administered to the subject along two sides of the fistula (e.g., at the 12 o'clock and 6 o'clock positions of the circumference of the fistula). In some embodiments, EC is about 7.5×10 6 to about 60×10 6 With the total EC dosage, it is administered to the subject along two sides of the fistula (e.g., at the 12 o'clock and 6 o'clock positions of the circumference of the fistula). In some embodiments, EC is about 7.5×10 6 to about 30×10 6 With the total EC dosage, it is administered to the subject along two sides of the fistula (e.g., at the 12 o'clock and 6 o'clock positions of the circumference of the fistula). In some embodiments, EC is about 7.5×10 6 to about 15×10 6 With the total EC dosage, it is administered to the subject along two sides of the fistula (e.g., at the 12 o'clock and 6 o'clock positions of the circumference of the fistula). In some embodiments, EC is about 5×10 6 to about 30×10 6 With the total EC dosage, it is administered to the subject along two sides of the fistula (e.g., at the 12 o'clock and 6 o'clock positions of the circumference of the fistula). In some embodiments, EC is administered to the subject with a total EC dosage of about 7.5×10 6 along two sides of the fistula. In some embodiments, EC is administered to the subject with a total EC dosage of about 15×10 6 along two sides of the fistula. In some embodiments, EC is administered to the subject with a total EC dosage of about 30×10 6 along two sides of the fistula.

[0020] In some embodiments, EC is administered to the subject along three sides of the fistula (e.g., at the 12 o'clock, 4 o'clock, and 8 o'clock positions of the circumference of the fistula), or along four sides of the fistula (e.g., at the 12 o'clock, 3 o'clock, 6 o'clock, and 9 o'clock positions of the circumference of the fistula). In some such embodiments, the total dose administered can be any of the above dose ranges or doses, but a portion (about one-third) of the total EC dose is administered to each of the three sides of the fistula, or a portion (about one-fourth) of the total EC dose is divided so as to be administered to each of the four sides of the fistula tube.

[0021] In some embodiments, EC is administered to the subject at an EC dose of about 5×10 6 to about 200×10 6 per centimeter of the length of the fistula. In some embodiments, EC is administered to the subject at an EC dose of about 5×10 6 to about 150×10 6 per centimeter of the length of the fistula. In some embodiments, EC is administered to the subject at an EC dose of about 5×10 6 to about 120×10 6 per centimeter of the length of the fistula. In some embodiments, EC is administered to the subject at an EC dose of about 5×10 6 to about 100×10 6 per centimeter of the length of the fistula. In some embodiments, EC is administered to the subject at an EC dose of about 5×10 6 to about 50×10 6 per centimeter of the length of the fistula. In some embodiments, EC is administered to the subject at an EC dose of about 5×10 6 to about 40×10 6 per centimeter of the length of the fistula. In some embodiments, EC is administered to the subject at an EC dose of about 5×10 6 to about 30×10 6 per centimeter of the length of the fistula. In some embodiments, EC is administered to the subject at an EC dose of about 5×10 6 to about 15×10 6 per centimeter of the length of the fistula. In some embodiments, EC is administered to the subject at an EC dose of about 7.5×10 6 to about 50×10 6The EC dose is administered to the subject. In some embodiments, the EC dose is approximately 7.5 × 10 per centimeter of fistula length. 6 From approximately 40 x 10 6 The EC dose is administered to the subject. In some embodiments, the EC dose is approximately 7.5 × 10 per centimeter of fistula length. 6 From approximately 30 x 10 6 The EC dose is administered to the subject. In some embodiments, the EC dose is approximately 7.5 × 10 per centimeter of fistula length. 6 From approximately 15 x 10 6 The EC dose is administered to the subject. In some embodiments, the EC dose is approximately 15 × 10 per centimeter of fistula length. 6 From approximately 30 x 10 6 The EC dose is administered to the subject. In some embodiments, the EC dose is approximately 7.5 × 10⁶ per 1 cm of fistula length. 6 The subject is administered an EC dose of approximately 15 × 10¹⁴ per 1 cm of fistula length. In some embodiments, EC is administered at a dose of approximately 15 × 10¹⁴ per 1 cm of fistula length. 6 The subject is administered an EC dose of approximately 30 × 10¹¹ units per 1 cm of fistula length. In some embodiments, EC is administered at a dose of approximately 30 × 10¹¹ units per 1 cm of fistula length. 6 The individual is administered the appropriate EC dose to the target patient.

[0022] In some embodiments, EC is also administered to the subject at the internal opening of the fistula (in other words, EC is administered to both the fistula and the internal opening of the fistula, as described above and elsewhere in this specification). In some embodiments, EC is administered to the subject at the sutured internal opening of the fistula. In some embodiments, the dose of EC delivered to the internal opening is approximately 5 × 10⁻⁶. 6 Approximately 200 x 10 from EC 6 It is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 5 × 10⁻⁶. 6 Approximately 150 x 10 from EC 6 It is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 5 × 10⁻⁶. 6 Approximately 120 x 10 from EC 6 It is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 5 × 10⁻⁶. 6 Approximately 100 x 10 from EC 6It is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 5 × 10⁻⁶. 6 Approximately 50 x 10 from EC 6 It is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 5 × 10⁻⁶. 6 Approximately 40 x 10 from EC 6 It is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 5 × 10⁻⁶. 6 Approximately 30 x 10 from EC 6 It is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 5 × 10⁻⁶. 6 Approximately 15 x 10 from EC 6 This is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 7.5 × 10⁻⁶. 6 From EC, approximately 60 x 10 6 This is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 7.5 × 10⁻⁶. 6 Approximately 30 x 10 from EC 6 This is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 7.5 × 10⁻⁶. 6 Approximately 15 x 10 from EC 6 It is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 15 × 10⁻⁶. 6 Approximately 30 x 10 from EC 6 This is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 7.5 × 10⁻⁶. 6 It is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 15 × 10⁻⁶. 6 It is EC. In some embodiments, the dose of EC delivered to the internal opening is approximately 30 × 10 6This is EC. In some embodiments, the dose range or dose of EC delivered to the internal orifice (as described above) is divided into two or more parts, each part administered to a different point on the circumference of the internal orifice. For example, the total cellular dose can be administered to three parts of the circumference of the internal orifice; for example, at the 12 o'clock (cranial), 4 o'clock, and 8 o'clock positions on the circumference of the internal orifice. In some embodiments, the EC delivered to the internal orifice is administered, for example, by injection and / or under direct visualization (for example, with image guidance), approximately 3 mm submucosa below the sutured internal orifice.

[0023] In some embodiments, the subject has one or more fistulas to be treated (e.g., two or three, or up to ten separate fistulas).

[0024] In some embodiments, one or more fistulas of the target are, for example, EC (e.g., E4ORF1 + A single dose of a composition containing HUVEC can be administered to the target. In some embodiments, one or more fistulas in the target are treated with, for example, EC (e.g., E4ORF1 + Treatment can be administered once by administering a composition containing HUVEC in the above-described dose range or dose.

[0025] In some embodiments, one or more fistulas of the target are, for example, EC (e.g., E4ORF1 + The target can be treated two or more times by administering a composition containing HUVEC multiple times. In some embodiments, one or more fistulas in the target can be treated with, for example, EC (e.g., E4ORF1 +A subject may be treated more than once by administering a composition containing HUVEC) to the subject multiple times in the dose range or doses described above. In some such embodiments, the time interval between treatments / administrations may be about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 14 weeks, about 16 weeks, about 18 weeks, about 20 weeks, or longer. In some embodiments, the administration interval is about 2 weeks to about 12 weeks. In some embodiments, the administration interval is about 4 weeks to about 10 weeks. In some embodiments, the administration interval is 6 weeks to about 8 weeks. In some preferred embodiments, the administration interval is about 6 weeks. In other preferred embodiments, the administration interval is about 12 weeks. In some embodiments, the time interval between administrations is determined on an ad hoc basis, as indicated by the state of the subject's GI fistula and / or the subject's response to the prior administration.

[0026] In some embodiments, the therapeutic method of the present invention also involves curettage of the GI fistula before administration of EC. In some such embodiments, the administration of EC includes distributing EC along the length of the curettage of the GI fistula.

[0027] In some embodiments, the therapeutic method of the present invention also includes performing a fistula incision before administering EC, in which case EC can be administered into the fistula opened during the fistula incision.

[0028] In some embodiments, the therapeutic methods of the present invention may be performed without fistulotomy or other surgical GI fistula interventions such as fistulotomy. Indeed, an important aspect of the present invention is that the methods described herein can be used as primary therapeutic methods as well as adjuncts to surgical interventions, thus avoiding surgical procedures that may damage the sphincter and cause incontinence.

[0029] In some embodiments, a composition containing EC is a saline solution (e.g., isotonic saline or buffered saline) containing EC (e.g., E4ORF1 +The composition includes HUVEC). In some such embodiments, the composition comprises one or more further components suitable for administration to a living subject, selected from the group consisting of buffers, salts, polysaccharides, proteins, and preservatives. In some such embodiments, the composition comprises one or more of human serum albumin (HSA), dextran 40, and DMSO.

[0030] In some such embodiments, the composition contains up to about 1% DMSO. In some such embodiments, the composition contains up to about 0.25% DMSO. In such embodiments, the composition contains about 6% dextran 40. In some such embodiments, the composition contains about 10% HSA. In such embodiments, the composition contains about 6% dextran 40 and about 10% HSA. In some such embodiments, the composition contains about 6% dextran 40 and about 10% HSA and about 0.25% DMSO. In such embodiments, the composition contains about 6% dextran 40 and about 10% HSA in isotonic or buffered saline. In some such embodiments, the composition contains about 6% dextran 40 and about 10% HSA and about 0.25% DMSO in isotonic or buffered saline.

[0031] In some embodiments, compositions containing EC include EC (e.g., E4ORF1 + HUVEC) approximately 0.5 × 10 6 From cells / ml to 50 x 10 6 It is contained at a concentration of cells / ml. In some embodiments, the composition containing EC is EC (e.g., E4ORF1 + HUVEC) approximately 1.0 × 10 6 cells / ml ~ approx. 25×10 6 It contains cells / ml at a concentration. In some embodiments, the composition containing EC is about 1 × 10 6 EC (e.g., E4ORF1) at a concentration of cells / ml + Includes HUVEC). In some embodiments, the composition containing EC is about 5 × 10 6 EC (e.g., E4ORF1) at a concentration of cells / ml + (Including HUVEC)

[0032] These and other aspects of the present invention are described further in other sections of this specification, and the summary of the invention is intended to be read in conjunction with all other sections described herein. In addition, as will be apparent to those skilled in the art, certain modifications and combinations of the various aspects described herein are included within the scope of the invention. [Modes for carrying out the invention]

[0033] Detailed explanation The “Summary of the Invention,” “Examples,” and “Claims” sections of this specification describe many of the main aspects of the present invention. This “Detailed Description” section provides specific additional descriptions 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 specification. Furthermore, as will be apparent to those skilled in the art, the different aspects described throughout this specification can be combined in various different ways, regardless of the subheadings. Such combinations of the specific aspects described herein are within the scope of the present invention.

[0034] definition The following are arbitrary definitions and abbreviations. Other terms or phrases may be defined elsewhere in this patent disclosure or may have meanings that are obvious from the context in which they are used. Unless otherwise defined herein or unless other meanings become obvious from their use in the context herein, all technical and scientific terms and abbreviations used herein have the same meanings as commonly understood by those skilled in the art in which this invention relates. For example, The Dictionary of Cell and Molecular Biology (5th ed. JM Lackie ed., 2013), the Oxford Dictionary of Biochemistry and Molecular Biology (2nd ed. R. Cammack et al. eds., 2008), and The Concise Dictionary of Biomedicine and Molecular Biology (2nd ed. PS. Juo, 2002) can provide those skilled in the art with general definitions of some of the terms used herein.

[0035] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. Not only the term “a” (or “an”), but also the terms “one or more” and “at least one” may be used interchangeably.

[0036] Furthermore, “and / or” can be understood as a specific disclosure of whether each of the two identified functions or components is accompanied by or not accompanied by the other function or component. Thus, the term “and / or” as used in phrases such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Similarly, the term “and / or” as used in phrases 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).

[0037] Units, prefixes, and symbols are expressed in the form recognized by the International System of Units (SI). A numerical range includes the numerical values ​​that define that range, and each individual value provided herein can serve as the endpoint of a range that includes other individual values ​​provided herein. For example, the set of values ​​such as 1, 2, 3, 8, 9, and 10 is also a disclosure of the numerical range from 1 to 10.

[0038] When an aspect is described with the word “includes,” it also includes other similar aspects described with the words “consist of” and / or “essentially become from.”

[0039] As used herein, the terms “about” and “approximately” refer to a given number within plus or minus 10% of that number. In all embodiments in which a number is described using the terms “about” or “approximately,” similar embodiments in which an exact number is used are also intended. For example, in an embodiment involving a time of approximately 24 hours, a similar embodiment involving a time of 24 hours (in other words, without the “about” modifier) ​​is also intended.

[0040] As used herein, a cell is “positive” or “+” for a marker when the described marker is expressed to a detectable level within the cell. For example, if E4ORF1 is expressed to a detectable level by endothelial cells, the endothelial cells are E4ORF1 positive (in other words, E4ORF1+). In some situations, the expression of a described marker may be evaluated in comparison to, for example, a negative control in the experiment.

[0041] As used herein, the abbreviation "E4ORF" refers to the open reading frame (ORF) located within the adenovirus (early) 4 (E4) region of the adenovirus genome, or the polypeptide / protein encoded by that ORF (whether a gene or protein is being referred to may be clear from the context of use). As used herein, the abbreviation "E4ORF1" refers to open reading frame (ORF) 1 in the early (early) 4 (E4) region of the adenovirus genome, or the polypeptide / protein encoded by that ORF (whether a gene or protein is being referred to may be clear from the context of use).

[0042] As used herein, the abbreviation "E4ORF1+" means, when used in relation to the described cell types, cells that express E4ORF1 to a detectable degree. E4ORF1+ cells contain recombinant E4ORF1 nucleic acid molecules and express the E4ORF1 protein.

[0043] In this specification, the abbreviation "EC" refers to endothelial cells.

[0044] The abbreviation "UVEC" used herein refers to umbilical vein endothelial cells.

[0045] The abbreviation "HUVEC" used herein refers to human umbilical vein endothelial cells.

[0046] In this specification, the terms "E-CEL" and "E-CEL UVEC" refer to E4ORF1 + HUVEC or E4ORF1 + This refers to a composition containing HUVEC. Whether it refers to the cells themselves or a composition containing cells may be clear from the context in which the term is used. For example, a reference to E-CEL in the context of administration to a subject necessarily means the administration of a composition containing E-CEL.

[0047] As used herein, the term “allogeneic” means derived from, originating from, or belonging to a species that is genetically related, genetically disrelated but genetically similar. For example, in embodiments involving the administration of allogeneic ECs to a subject, the allogeneic ECs are obtained from a donor of the same species as the subject to which the cells are administered (in other words, the recipient). In some embodiments, allogeneic cells are obtained from a donor whose MHC / HLA matches completely or partially with that of the subject to which the cells are administered (in other words, the recipient)—that is, the cell donor and the cell recipient have completely or partially matching MHC or HLA. In some embodiments, ECs are (a) obtained from a donor, (b) maintained and / or cultured and / or grown ex vivo, and (c) subsequently administered to a subject of the same species as the donor.

[0048] As used herein, the term “autologous” means derived from or originating from the same subject. For example, in embodiments involving the administration of autologous ECs to a subject, the autologous cells are obtained from the subject to which they are administered (in other words, the EC donor and recipient are the same individual). In some embodiments, ECs are (a) obtained from a subject, (b) maintained and / or cultured and / or grown ex vivo, and (c) subsequently administered to the same subject.

[0049] As used herein, the terms “to treat,” “to treat,” and “treatment” mean achieving a detectable improvement or prevention of worsening of one or more symptoms or sequelae of the condition being treated. For example, as used herein, such terms include achieving, to a detectable degree (if necessary, to a statistically significant degree and / or clinically significant degree), one or more of the following “treatment outcomes,” depending on the condition being treated: (a) healing of the GI fistula; (b) an increase in the rate of healing of the GI fistula; (c) an improvement (in other words, a decrease) or prevention of worsening of drainage from the GI fistula; (d) an improvement or prevention of worsening of an abscess near the fistula; (e) a decrease in the frequency or rate of recurrence of the GI fistula; (f) an improvement (i.e., a decrease) or prevention of worsening of pain associated with the GI fistula; (g) EC (e.g., E4ORF1 +These include the healing of GI fistula within six weeks of administration of a composition containing HUVEC, etc. Any suitable method or clinical trial can be used to evaluate these treatment outcomes, and suitable methods for evaluating such treatment outcomes are well known to physicians in the art of the present invention. In some embodiments, such treatment outcomes may be evaluated by comparing symptoms or sequelae in a single subject before and after treatment, or by comparing symptoms or sequelae in a subject treated in the manner disclosed herein with symptoms in another individual that is untreated or treated with a placebo, or by comparing symptoms or sequelae in a subject treated in the manner disclosed herein with a baseline value of the symptom or sequelae (e.g., a baseline value of the symptom or sequelae typically shown in a healthy person of the same sex, age, and weight). For each embodiment described herein that refers to a treatment method, similar methods are also provided that aim to achieve one or more of the above treatment outcomes, or result in one or more of the above treatment outcomes. For example, in addition to providing a method useful for treating GI fistulas in subjects requiring it, comprising administering an effective amount of a composition containing endothelial cells (EC) to a subject, the present invention also provides a similar method for treating GI fistulas in subjects requiring it, comprising administering an effective amount of a composition containing endothelial cells (EC) to a subject, wherein the treatment is for healing the GI fistula, or increasing the rate of healing of the GI fistula, or for EC (e.g., E4ORF1 + To result in healing of the GI fistula within 6 weeks of administration of EC, or improvement (i.e., reduction) or prevention of exacerbation of drainage from the GI fistula, and / or improvement (i.e., reduction) or prevention of exacerbation of pain associated with the GI fistula.

[0050] As used herein, the term "effective dose" means a sufficient amount of the described cell type (e.g., E4ORF1) to achieve the described result (e.g., treatment of GI fistula) to a detectable extent, and in some cases to a specifically stated extent. +This means the amount of a composition containing HUVEC, or the described cell type. In some embodiments, the effective amount is (e.g., per fistula or per 1 cm of fistula length) E4ORF1 + The effective dose is determined by the number of HUVECs. In other embodiments, the appropriate “effective dose” may be determined empirically using standard techniques known in the art, such as dose escalation studies, or by taking into account factors such as the intended route of administration and the desired frequency of administration. Furthermore, the “effective dose” may be determined using tests such as those described in the Examples section of this patent specification.

[0051] When used in relation to EC, the term “engineered” means EC cells that have been manipulated by humans to produce the mentioned phenotype (e.g., E4ORF1 expression) or to express the mentioned protein. The term “genetically modified cell” is not intended to include naturally occurring cells, but rather to include cells that contain recombinant nucleic acid molecules, or cells that have been artificially modified to express proteins that would not otherwise be expressed, or to express proteins at substantially higher levels than observed in non-recombinant EC cells.

[0052] The abbreviation "HLA" as used herein means human leukocyte antigen.

[0053] As used herein, the term "subject" refers to an individual mammal. In certain embodiments, the subject is a human being. When the subject is a human being, the terms "subject" and "patient" may be used interchangeably.

[0054] The term "GI fistula" as used herein shall be used in accordance with its common meaning in the art. Therefore, as used herein, the term "GI fistula" encompasses both enterocutaneous fistulas and entero-enteral fistulas related to any part of the digestive tract (GI) (e.g., oral cavity, esophagus, stomach, duodenum, jejunum, ileum, colon, rectum, or anus), including, for example: anorectal fistula, anovaginal fistula, appendiceal fistula, biliary fistula, cecal fistula, colonic fistula, colorectal fistula, colovesical fistula, duodenal fistula, and duodeno-colic fistula. Anal fistulas are a specific type of GI fistula in which there is an abnormal connection between the epithelialized surface of the anal canal and the perianal skin or perianal area. Such fistulas may also be called perianal fistulas or fistula-in-ano.

[0055] As used herein, the term “GI fistula” also encompasses GI fistulas of any etiology. This includes GI fistulas caused by or associated with injury, surgery, or disease (including infectious and inflammatory diseases). For example, the term “GI fistula” includes GI fistulas caused by or associated with inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. The term “GI fistula” also includes GI fistulas of unknown etiology (i.e., idiopathic GI fistulas).

[0056] Treatment method In some embodiments, the present invention provides a method for treating GI fistulas in subjects requiring such treatment, the method comprising administering an effective amount of a composition comprising endothelial cells (ECs) to the subject.

[0057] In some embodiments, the GI fistula treated using the method of the present invention is an enterocutaneous fistula. In some embodiments, the GI fistula treated using the method of the present invention is a non-healing enterocutaneous fistula. In some embodiments, the GI fistula treated using the method of the present invention is an enterojejunostomy. In some embodiments, the GI fistula treated using the method of the present invention is a non-healing enterojejunostomy.

[0058] In some embodiments, the GI fistula treated using the method of the present invention is a fistula of the small intestine. In some embodiments, the GI fistula treated using the method of the present invention is a fistula of the large intestine.

[0059] In some embodiments, the GI fistula treated using the method of the present invention is an anal fistula, anorectal fistula, anorovaginal fistula, appendiceal fistula, biliary fistula, cecal fistula, colon fistula, colonorectal fistula, covesicostomy, duodenal fistula, duodenocolonic fistula, enterovestomy, esophageal fistula, gallbladder fistula, gastrostomy, gastrocolic fistula, gastrojejunocolonic fistula, ileal fistula, jejunal fistula, oral fistula, pancreatic fistula, rectal fistula, rectovaginal fistula, or salivary gland fistula.

[0060] In some preferred embodiments, the GI fistula treated using the method of the present invention is an intestinal cutaneous fistula. In some preferred embodiments, the GI fistula treated using the method of the present invention is an anal fistula.

[0061] In some embodiments, the GI fistula treated using the method of the present invention is a blind fistula. In some embodiments, the GI fistula treated using the method of the present invention is a complete fistula. In some embodiments, the GI fistula treated using the method of the present invention is an incomplete fistula. In some embodiments, the GI fistula treated using the method of the present invention is a simple fistula. In some embodiments, the GI fistula treated using the method of the present invention is a complex fistula. In some embodiments, the GI fistula treated using the method of the present invention is a primary fistula. In some embodiments, the GI fistula treated using the method of the present invention is a recurrent fistula.

[0062] In some embodiments, the subjects treated using the method of the present invention are mammals. In preferred embodiments, the subjects treated using the method of the present invention are humans.

[0063] In some embodiments, the EC administered in the therapeutic method of the present invention is autologous EC. In some embodiments, the EC administered in the therapeutic method of the present invention is an allogeneic EC.

[0064] In some embodiments, the EC administered in the therapeutic method of the present invention is umbilical vein endothelial cells (UVEC). In some embodiments, the EC administered in the therapeutic method of the present invention is human umbilical vein endothelial cells (HUVEC). In some such embodiments, the UVEC is E4ORF1+ UVEC. In preferred embodiments, the UVEC is E4ORF1 + It is HUVEC.

[0065] In some embodiments, the therapeutic method of the present invention involves administering a certain dose or a certain dose range to an EC. Appropriate doses and dose ranges are described in the summary section of the invention above.

[0066] In some embodiments, EC is administered to a subject such that the total volume of the EC-containing composition administered in a single dose is approximately 0.5 ml to approximately 20 ml, for example, approximately 5 ml to 15 ml.

[0067] In some embodiments, the therapeutic method of the present invention includes administering EC to a subject in a specific dosing schedule. In some embodiments, the therapeutic method of the present invention includes administering EC once, i.e., a single dose (e.g., a single dose of EC in the dose specified above). In some embodiments, the therapeutic method of the present invention includes administering EC multiple times, i.e., multiple doses (e.g., each of the multiple doses includes an administration of EC in the dose specified above). For example, in some embodiments, the therapeutic method of the present invention includes administering EC to a subject two, three, four, or more times. In embodiments in which EC is administered to a subject multiple times, the dosing interval can be about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 14 weeks, about 16 weeks, about 18 weeks, about 20 weeks, or longer. In some embodiments, the dosing interval is about 2 weeks to about 12 weeks. In some embodiments, the dosing interval is about 4 weeks to about 10 weeks. In some embodiments, the dosing interval is 6 to about 8 weeks. In some preferred embodiments, the dosing interval is about 6 weeks. In other preferred embodiments, the dosing interval is about 12 weeks. In some embodiments, the interval between dosing is determined on an ad hoc basis, as indicated by the condition of the subject's GI fistula and / or the subject's response to prior administration. Other suitable dosing schedules are described in the summary section of the invention above.

[0068] In some embodiments, the therapeutic method of the present invention includes administering EC to a subject using any suitable route of administration. In a preferred embodiment, the therapeutic method of the present invention includes locally administering EC to a subject at the site of the GI fistula. In some preferred embodiments, the therapeutic method of the present invention includes administering EC to a subject by local injection at the site of the GI fistula, for example, using a hypodermic needle. In some preferred embodiments, the therapeutic method of the present invention includes administering EC to a subject by local injection at the site of the GI fistula, for example, using a 25-gauge needle. In some embodiments, the therapeutic method of the present invention includes administering EC to a subject under direct visualization (for example, using image guidance).

[0069] In some embodiments, EC administration involves positioning the EC along the length of the GI fistula. In some embodiments, EC administration involves positioning the EC with a single injection along the length of one side / half of the GI fistula. In some embodiments, EC administration involves positioning the EC with two or more separate injections along the length of two or more sides of the GI fistula. In some embodiments, EC is administered to the subject along two sides of the fistula (e.g., the 12 o'clock and 6 o'clock directions of the circumference of the fistula). In some embodiments, EC is administered to the subject along three sides of the fistula (e.g., the 12 o'clock, 4 o'clock, and 8 o'clock directions of the circumference of the fistula). In some embodiments, EC is administered to the subject along four sides of the fistula (e.g., the 12 o'clock, 3 o'clock, 6 o'clock, and 9 o'clock directions of the circumference of the fistula). In some embodiments, EC administration involves placing EC in the soft tissue outside the GI fistula (along one or more of its sides). In some embodiments, EC administration involves placing EC in the soft tissue outside the GI fistula (along one or more of its sides) along the length of the GI fistula. In some embodiments, EC administration involves inserting a needle / syringe (e.g., through the skin) into the soft tissue adjacent to the fistula and up to the level of the internal opening of the fistula, and injecting the EC-containing composition along the length of the fistula as the needle is withdrawn toward the external opening of the fistula, thereby placing the EC outside the fistula along its length. Such methods can be used to place / distribute EC relatively uniformly along the length of the fistula. This procedure can be performed on either one or both sides of the fistula.

[0070] In some embodiments, the subject is treated by curettage of the GI fistula before administration of EC. In some such embodiments, administration of EC includes distributing the EC along the length of the curettage of the GI fistula. In some such embodiments, administration of EC includes distributing half of the EC along one side of the curettage of the GI fistula and the other half of the EC along the opposite side of the curettage of the GI fistula.

[0071] In some embodiments, the treatment methods provided herein can be performed in combination with one or more additional medical procedures (such as surgical procedures) on the subject. For example, in some embodiments, the treatment methods provided herein can be performed in combination with fistulotomy (a surgical procedure that involves making an incision along the entire length of the fistula and opening the fistula so that it can potentially heal as a flat scar, and is a standard treatment for anal fistulas). Similarly, in some embodiments, the treatment methods provided herein can be performed in combination with fistulomectomy (a non-surgical intervention). And in some embodiments, the treatment methods provided herein can be performed in combination with fistulomectomy (a surgical intervention that completely removes the fistula). In some embodiments, the treatment methods provided herein can be performed in combination with surgical closure of the opening of the GI fistula, for example, using sutures. In some embodiments, the treatment methods provided herein are carried out in combination with surgical closure of the internal opening of the GI fistula.

[0072] In a configuration in which the internal opening of a GI fistula is surgically closed, additional EC (i.e., in addition to the EC administered as described above) may also be administered submucosa around the surgically closed internal opening of the GI fistula.

[0073] In some embodiments, the therapeutic methods provided herein may be carried out in combination with the administration of one or more additional agents (e.g., pharmaceutical agents, biological agents, or cell therapies) to the target in addition to the EC-containing compositions described herein. Additional agents that can be used in such methods include growth factors, cytokines, anti-inflammatory agents, immunosuppressants, antithymocyte globulins, mycophenylate mofetil, cyclosporine, methotrexate, cyclophosphamide, rituxan, steroids, antibacterial agents, antiviral agents, and antifungal agents, as well as other agents known in the art to be useful in the treatment of GI fistula or in cell therapy or transplantation therapy.

[0074] In some such embodiments, one or more additional agents are administered in the same composition as the EC (i.e., the additional agents can be added to the composition of the present invention). In some embodiments, one or more additional agents are administered separately from the composition of the present invention. For example, in some embodiments, one or more additional agents may be administered to a subject in a composition different from the composition in which the EC is provided, and may be administered to the subject before, simultaneously with, or after administration of the EC-containing composition of the present invention. In some embodiments, one or more additional agents may be administered to a subject about 5 minutes or 5 minutes before or after administration of the EC-containing composition of the present invention, 10 minutes or 10 minutes before or after, 15 minutes or 15 minutes before or after, 30 minutes before or after, 1 hour before or after, 2 hours before or after, 3 hours before or after, 4 hours before or after, 5 hours before or after, 6 hours before or after, 12 hours before or after, 24 hours before or after, or more before or after administration of the EC-containing composition of the present invention.

[0075] In some embodiments, the therapeutic methods of the present invention result in a detectable improvement or prevention of exacerbation of one or more symptoms or sequelae of the GI fistula being treated. In some embodiments, the therapeutic methods of the present invention result in one or more of the following: (a) healing of the GI fistula, (b) an increase in the rate of healing of the GI fistula, (c) improvement (i.e., reduction) or prevention of exacerbation of discharge from the GI fistula, (d) improvement or prevention of exacerbation of abscesses near the GI fistula, (e) a decrease in the frequency or rate of recurrence of the GI fistula, and / or (f) improvement (i.e., reduction) or prevention of exacerbation of pain associated with the GI fistula, and (g) healing of the GI fistula within six weeks after administration of a composition containing EC (e.g., E4ORF1+HUVEC). Various methods can be used to detect and / or evaluate the therapeutic effect of the methods of the present invention. Such methods include visual inspection, imaging, biopsy, and patient questionnaires.

[0076] 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 the umbilical cord. The UVECs described herein can be isolated from the umbilical cord using means known to those skilled in the art.

[0077] As an example, UVECs can be isolated from umbilical vein tissue, a method involving tissue excision and mechanical fragmentation. Alternatively, endothelial cells lining the vascular surface and downstream vessels can be released by perfusing the major vessels of the umbilical cord with an enzyme. The released cells can be suspended and cultured in a medium that promotes endothelial cell proliferation. In one embodiment, the cell population thus isolated can be further purified by immunoselection, for example, using a marker selective to endothelial cells (e.g., an antibody against CD31) or a combination of markers. The endothelial cells can be grown in culture. In one embodiment, the endothelial cells are transformed with a gene that confers a proliferative advantage over untransformed cells, such that the transformed cells proliferate more rapidly and / or more stably than the untransformed cells from which they originate.

[0078] Further methods for preparing UVECs are described in the Examples section of this specification.

[0079] In some embodiments, EC (e.g., UVEC) is genetically modified.

[0080] In a preferred embodiment, the EC (e.g., UVEC) is genetically modified to express the adenovirus E4ORF1 protein, i.e., E4ORF1 + EC (for example, E4ORF1) + It is UVEC.

[0081] In some embodiments, the EC is engineered to express one or more ETS family transcription factors (i.e., the EC is ETS+EC). In some embodiments, the EC is engineered to express the ETS family transcription factor ETV2 (i.e., the EC is ETV2+EC). In some embodiments, the EC is E4ORF1 + ETV2 + It is an e-commerce site.

[0082] E4ORF1 Some aspects of the present invention include E4ORF1+ ECs, i.e., ECs expressing the E4ORF1 polypeptide. The “E4ORF1” polypeptide is encoded by open reading frame (ORF) 1 in the early 4 (E4) region of the adenovirus genome. E4ORF1+ ECs for use according to the present invention generally include recombinant nucleic acid molecules containing an E4ORF1 coding sequence operably linked to a promoter suitable for the expression of the E4ORF1 coding sequence in the EC.

[0083] The E4ORF1 amino acid and nucleotide sequences are known in the art. Any such sequence can be used in accordance with the present invention. In some embodiments, the E4ORF1 polypeptide may be derived from any suitable adenovirus type or strain, such as human adenovirus types 2, 3, 5, 7, 9, 11, 12, 14, 34, 35, 46, 50, or 52. In some preferred embodiments, the polypeptide sequence used is derived from human adenovirus type 5. The amino acid sequences of such adenovirus polypeptides and the nucleic acid sequences encoding such polypeptides are well known in the art and can be obtained from well-known public databases such as the Genbank database. For example, suitable sequences include: human adenovirus type 9 (Genbank accession number CAI05991), human adenovirus type 7 (Genbank accession number AAR89977), human adenovirus type 46 (Genbank accession number AAX70946), human adenovirus type 52 (Genbank accession number ABK35065), human adenovirus type 34 (Genbank accession number AAW33508), human adenovirus type 14 (Genbank accession number AAW33146), human adenovirus type 50 (Genbank accession number AAW33554), human adenovirus Human adenovirus type 2 (Genbank accession number AP.sub.jp.--000196), human adenovirus type 12 (Genbank accession number AP.sub.--000141), human adenovirus type 35 (Genbank accession number AP.sub.--000607), human adenovirus type 7 (Genbank accession number AP.sub.--000570), human adenovirus type 1 (Genbank accession number AP.sub.--000533), human adenovirus type 11 (Genbank accession number AP.sub.--000474), human adenovirus type 3 (Genbank accession number ABB 17792), human adenovirus type 5 (Genbank accession number D12587). In one embodiment, the E4ORF1 sequence used has NCBI accession number AZR66741.1. In one embodiment, the E4ORF1 sequence used has the NCBI accession number AP_000232.1.In one embodiment, the E4ORF1 sequence used has the amino acid sequence: MAAAVEALFVVLEREGAILPRQEGFSGVYVFFSPINFVIPPMGAVMLSLRLRVCIPPGYFGRFLALTDVNQPDVFTESYIMTPDMTEELSVLFNHGDQFFYGHAGMAVVRLMLIRVFPVVRQASNV (Sequence ID 1).

[0084] In some embodiments, the E4ORF1 polypeptide may be encoded by a nucleic acid sequence having or encoding an amino acid sequence, which is a modification, derivative, variant, or fragment of any particular sequence provided herein or known in the art, wherein such modification, derivative, variant, or fragment is a polypeptide having or encoding one or more of the functional properties of adenovirus E4ORF1. In some embodiments, the modification, derivative, variant, or fragment has about 85% identity to a known sequence, or about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a known sequence. In some embodiments, a variation, derivative, variant, or fragment of a known nucleotide sequence is used in various lengths relative to the known nucleotide sequence, such as approximately 50 nucleotides, or approximately 45 nucleotides, or approximately 40 nucleotides, or approximately 35 nucleotides, or approximately 30 nucleotides, or approximately 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. In some embodiments, modifications, derivatives, variants, or fragments of known amino acid sequences are used in various lengths relative to the known amino acid sequence, such as approximately 50 amino acids, 45 amino acids, 40 amino acids, 35 amino acids, 30 amino acids, 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.

[0085] In some embodiments, the E4ORF1 sequence is used without other sequences from the adenovirus E4 region, for example, without using the entire E4 region, and not with other ORFs within the E4 region. However, in other embodiments, E4ORF1 may be used in combination with one or more other ORFs from the E4 region, such as E4ORF2, E4ORF3, E4ORF4, E4ORF5, or E4ORF6 / 7 sequences. For example, the E4ORF1 sequence can be used in constructs (such as viral vectors) that include other sequences, genes, or coding regions (such as promoters, marker genes, or antibiotic resistance genes). In certain embodiments, the E4ORF1 sequence is used in constructs that do not include the entire E4 region, or that do not include other ORFs from the entire E4 region, such as E4ORF2, E4ORF3, E4ORF4, and / or E4ORF5.

[0086] The E4ORF1 coding sequence may be present in constructs or vectors containing various other sequences, genes, or coding regions, such as promoters, enhancers, antibiotic resistance genes, reporter genes, or expression tags (e.g., nucleotide sequences encoding GFP), or any other desired nucleotide sequence or gene.

[0087] The nucleic acid molecule encoding E4ORF1 can be placed under the control of one or more promoters to enable expression. Any promoter capable of expressing the E4ORF1 nucleic acid sequence in endothelial cells can be used. Examples of preferred promoters include, but are not limited to, the CMV, SV40, RSV, HIV-Ltr, and MML promoters. The promoter may also be an adenovirus genome-derived promoter or a variant thereof. For example, in some embodiments, the promoter may be a promoter that naturally expresses E4ORF1 in the adenovirus genome. However, in other embodiments, the promoter may not naturally express E4ORF1 in the adenovirus genome.

[0088] The sequence encoding E4ORF1 may include native nucleotides, synthetic nucleotides, or combinations thereof. For example, in some embodiments, the nucleic acid molecule of the present invention may include RNA, such as synthetically modified RNA, which is stable in cells and can be used to directly result in protein expression / production in cells. In other embodiments, the sequence encoding E4ORF1 may include DNA. In embodiments in which DNA is used, the DNA sequence may be operably ligated to one or more suitable promoters and / or regulatory elements that enable (and / or promote, enhance or regulate) expression in cells, and may be present in one or more suitable vectors or constructs.

[0089] The sequence encoding E4ORF1 can be introduced into UVEC using any suitable system known in the art, including but not limited to transfection techniques and virus-mediated introduction techniques. Transfection methods that can be used according to the present invention include, but are not limited to, liposome-mediated transfection, polybrene-mediated transfection, DEAE-dextran-mediated transfection, electroporation, calcium phosphate precipitation, microinjection, and microparticle gun methods. Virus-mediated introduction methods that can be used include, but are not limited to, lentivirus-mediated introduction, adenovirus-mediated introduction, retrovirus-mediated introduction, adeno-associated virus-mediated introduction, and herpesvirus-mediated introduction.

[0090] In some embodiments, the sequence encoding E4ORF1 is located in the vector. In some embodiments, the sequence encoding E4ORF1 is located in the viral vector. In some embodiments, the sequence encoding E4ORF1 is located in the lentiviral vector. In some embodiments, the sequence encoding E4ORF1 is located in the adenovirus vector. In some embodiments, the sequence encoding E4ORF1 is located in the adeno-associated virus vector. In some embodiments, the sequence encoding E4ORF1 is located in the retroviral vector. In some embodiments, the sequence encoding E4ORF1 is located in the Moloney's mouse leukemia virus (MMLV) vector (a type of retroviral vector).

[0091] In some embodiments, the presence of the E4ORF1 coding sequence in EC 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 the E4ORF1 polypeptide 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 characteristics of E4ORF1-expressing endothelial cells known in the art (such as those described in U.S. Patent No. 8,465,732).

[0092] The handling, manipulation, and expression of the E4ORF1 sequence in endothelial cells (EC) can be performed using conventional molecular and cell biological techniques. Such techniques are well known in the art. See, for example, Sambrook, Fritsch, and Maniatis eds., “Molecular Cloning: A Laboratory Manual,” 2nd Ed., Cold Springs Harbor Laboratory Press, 1989; the teachings in the Methods of Enzymology series (Academic Press, Inc.), and standard texts for guidance on appropriate techniques for handling, manipulating, and expressing nucleotide and / or amino acid sequences. Further aspects relating to the handling and expression of the E4ORF1 sequence in endothelial cells are described in U.S. Patent No. 8,465,732, which is incorporated herein by reference.

[0093] composition In some embodiments, the composition used in the method of the present invention contains an effective amount of EC (e.g., E4ORF1 + The composition contains EC in a saline solution (such as isotonic saline, saline, or buffered saline) suitable for administration to a subject. In some embodiments, the composition may contain one or more additional excipients suitable for administration to a human subject and suitable for cell therapy, such as buffers, salts, preservatives, polysaccharides (e.g., dextran), and proteins (e.g., albumin). In some embodiments, the composition may contain a low concentration of cryopreservative. In some embodiments, the composition of the present invention contains dextran 40. In some embodiments, the composition of the present invention contains human serum albumin (HSA). In some embodiments, the composition of the present invention contains dimethyl sulfoxide (DMSO) at a concentration of up to 1%, preferably up to 0.25%. In some embodiments, the composition of the present invention contains EC in an isotonic saline solution (E4ORF1 +The composition includes HUVEC, dextran 40, HSA, and DMSO. Other suitable compositions are described in the summary section of the invention above.

[0094] In some embodiments, the composition of the present invention contains both E4ORF1+ and E4ORF1- (negative) EC. In some embodiments, at least about 75% of the EC in the composition is E4ORF1+. In some embodiments, at least about 80% of the EC in the composition is E4ORF1+. In some embodiments, at least about 85% of the EC in the composition is E4ORF1+. In some embodiments, at least about 90% of the EC in the composition is E4ORF1+. In some embodiments, at least about 95% of the EC in the composition is E4ORF1+. In some embodiments, at least about 98% of the EC in the composition is E4ORF1+. In some embodiments, at least about 99% of the EC in the composition is E4ORF1+.

[0095] Generally, EC (e.g., E4ORF1) is used in the method of the present invention. + HUVEC) is in a frozen form, i.e., a specific endothelial cell (e.g., E4ORF1) + The frozen composition, containing HUVEC and a cryopreservative, is supplied (for example, to a medical facility where it will be administered to a subject). The frozen composition is thawed before use, for example, using a standard method known in the art. In some embodiments, the frozen composition may be administered to a subject without dilution after thawing. However, in other embodiments, the frozen composition is thawed and then diluted with a suitable diluent to produce a composition containing a desired or specified concentration of cells and a desired or specified concentration of excipients, for example, as described above.

[0096] kit In some embodiments, the present invention provides a kit comprising sterile-packaged compositions according to the present invention and instructions for administering the therapeutic compositions to a subject for the treatment of GI fistulas. Such kits typically include a label indicating the intended use of the kit's contents. In this specification, the term “label” includes any written or recorded material attached to or accompanying the kit.

[0097] The present invention will be further described by reference to the following non-limiting embodiments. [Examples]

[0098] Examples The following examples are provided to illustrate specific situations and settings to which the present invention may be applied and are not intended to limit the scope of the present invention and claims contained herein.

[0099] Example 1 E4ORF1 + Preparation of HUVEC HUVEC was isolated from the umbilical vein by enzymatic digestion with collagenase. The cells were then cultured in endothelial cell proliferation medium before being introduced with a viral vector containing an E4ORF1 expression cassette (encoding SEQ ID NO: 1). The resulting E4ORF1 + HUVEC cells are referred to as "E-CEL UVEC" or "E-CEL UVEC cells" throughout these examples and the drawings referenced therein.

[0100] In several studies, E-CEL UVEC cells were transferred to a bioreactor and grown to obtain the desired cell count. The grown E-CEL UVEC cells were cryopreserved. Before use, the E-CEL UVEC cells were thawed and, if necessary, further cultured / expanded to prepare the final composition for administration to the subject, as described in the subsequent examples.

[0101] Example 2 E4ORF1 + Human clinical trial of HUVEC treatment for anal fistula An anal fistula is an abnormal connection between the epithelial surface of the anal canal and the perianal skin or perianal area. Its etiology is crypt adenitis in 90% of cases and, in most cases, develops from anal abscess infection that is spontaneously discharged. Fistulas are classified into simple and complex fistulas based on anatomical and clinical features [6]. Complex fistulas have a higher risk of disease persistence or recurrence and a higher risk of treatment-related fecal incontinence. Thus, treatment of complex anal fistulas is more difficult, while simple fistulas can be effectively treated with fistula incision, the standard treatment for simple fistulas [7, 8]. Treatment of more complex fistulas is difficult due to the higher risk of treatment-related fecal incontinence.

[0102] Sphincter-preserving surgeries such as fistula incision or fistula excision have a high success rate, but can result in fecal incontinence in patients with complex anal fistulas. Therefore, clinically, these sphincter-preserving surgeries are a last resort for both patients and physicians and are contraindicated for complex anal fistulas [9-11]. Sphincter-preserving methods (such as adhesive injection, forward flap, and plug placement) have also been developed, but have a low success rate [12, 13]. Another current treatment option involves passing reactive sutures or rubber through the fistula tract and tightening them at regular intervals. This can prevent extensive destruction of the anal sphincter associated with fistula incision or fistula excision. However, this is not a true sphincter-preserving surgery and may not be tolerable for all patients. In summary, prior to the present invention, there was no optimal treatment for patients with complex anal fistulas. Improving the treatment outcomes for these patients remains an unmet medical need.

[0103] The present invention provides an opportunity to treat anal fistulas minimally invasively, on an outpatient basis, and, importantly, while structurally preserving the sphincter muscle.

[0104] Part A of the clinical trial was designed to test the safety and efficacy of E-CEL UVEC cells as an adjunct to fistula incision for simple fistula repair. In Part A of the trial, six subjects with simple fistulas were treated with fistula incision and E-CEL UVEC cell infusion. The top-level results of Part A of the clinical trial were as follows:

[0105] [Table 1]

[0106] All subjects received the dose assigned to their respective cohort (see Table 2). Each dose level was tolerable as determined by the Safety Monitoring Committee. No adverse events (including reactions to E-CEL UVEC cells) were observed. Preliminary efficacy evaluations (including physical examination and photographic documentation) showed that administration of E-CEL UVEC cells in conjunction with fistula incision promoted healing at the treatment site.

[0107] Fistula incision is the standard treatment for simple perianal fistulas, but it is an invasive surgical procedure that carries the risk of damaging the anal sphincter. Sphincter damage can lead to fecal incontinence, and if the fistula recurs, the risk of further anal sphincter damage increases. Based on the confirmation of the safety of injecting E-CEL UVEC cells into the fistula in Part A of this clinical trial, and the early signs of clinical utility (by physical examination and photographic documentation) of complete healing of the fistula wound by week 6, Part B1 was added to the clinical trial. This is to investigate the potential of E-CEL UVEC cell therapy after curettage of simple fistulas, without fistula incision and therefore without the risk of inducing sphincter damage and associated fecal incontinence. Part B2 was added to the clinical trial to investigate the additional injection of E-CEL UVEC around the opening after suturing. The treatment protocols in Parts B1 and B2 of the clinical trial (both involving the injection of E-CEL UVEC cells along the curettage-treated fistula tract without fistula incision) may shorten the time to fistula tract healing and reduce the need for more invasive and painful fistula incision surgery or seton placement. Percutaneous E-CEL UVEC cell injection with fistula curettage (Part B1) is a simple outpatient procedure that causes minimal damage to the fistula tract without damaging the anal sphincter structure. The procedure performed in Part B2 of the clinical trial is also less invasive than fistula incision, reduces the risk of anal sphincter damage, and heals faster because it does not create a new surgical wound, thus enabling outpatient treatment. Administering EC to one or more sides of the fistula tract closes the tract and reduces the likelihood of recurrence. Simultaneous administration of EC to the internal opening of the fistula promotes the healing of the mucosal epithelium, leading to accelerated healing and further improved resistance to recanalization / recurrence of the fistula tract.

[0108] [Table 2-1] [Table 2-2] [Table 2-3]

[0109] Example 3 E4ORF1 for non-healing enterofacial fistulas + HUVEC clinical trial In an adult male patient with chronic, non-healing small bowel-cutaneous anterior abdominal wall fistula (two fistula scars and two skin openings 2-3 cm apart), the following clinical replacement was performed. Up to 2 liters of intestinal contents were released from the fistula daily from the abdomen, causing a severe decline in quality of life.

[0110] The patient was transported to the operating room and injected with E-CEL UVEC under sterile conditions. The patient was given a moderate sedative and placed in a supine position. An imaging system was used that allowed for the superposition of images of the fistula tract obtained from a preoperative CT scan onto a cone-beam CT (cbCT) fluoroscopy screen. The fistula site was cleaned by removing excess granulation tissue on the skin side.

[0111] The internal opening of the fistula was surgically closed using image guidance. Again using image guidance, E-CEL UVEC cells were injected from a syringe using a 25G needle, first into the internal opening at a rate of 15 × 10 6 Inject individual living cells, then along the entire length of the soft tissue surrounding the fistula, along the fistula tubule, 15 × 10 6 Cells were injected in all directions around the fistula tract at a dose of live cells / cm². After the procedure, a sterile dressing was applied to the abdomen, and the patient recovered from anesthesia and was observed in the recovery area.

[0112] The patient showed an excellent response to the initial treatment. The patient tolerated the initial infusion of E-CEL UVEC cells into the enterofistula without complications or side effects. One of the two external fistulas closed after treatment, and the other showed significant proliferation of healthy granulation tissue. Fistula discharge also decreased dramatically.

[0113] Following the success of the initial administration, a second dose of E-CEL UVEC cells was administered 6 weeks later.

[0114] Subsequent additional administrations further reduced the discharge from the fistula, and the size of the external opening gradually decreased. When treatment was temporarily interrupted due to administrative issues, the patient experienced a slight increase in fistula discharge. This increase subsided upon resumption of treatment, and the size of the external opening of the fistula and the amount of discharge from the fistula further decreased. The amount of exudate from the fistula is currently less than approximately 200 mL per day, and the fistula opening has shrunk to a diameter of approximately 1-2 mm. With continued treatment, the fistula is expected to close completely.

[0115] List of references 1. Panes, J., et al., Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn's disease: a phase 3 randomized, double-blind controlled trial. Lancet, 2016. 388(10051): p. 1281-90. 2. 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. 3. Gori, JL, et al., Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates. Stem Cells Transl Med, 2016. 4. Poulos, M.G., et al., Endothelial transplantation rejuvenates aged hematopoietic stem cell function. J Clin Invest, 2017. 127(11): p. 4163-4178. 5. 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. 6. Steele, S.R., et al., Practice parameters for the management of perianal abscess and fistula-in-ano. Dis Colon Rectum, 2011. 54(12): p. 1465-74. 7. Abramowitz, L., et al., The outcome of fistulotomy for anal fistula at 1 year: a prospective multicentre French study. Colorectal Dis, 2016. 18(3): p. 279-85. 8. Hall, J.F., et al., Outcomes after operations for anal fistula: results of a prospective, multicenter, regional study. Dis Colon Rectum, 2014. 57(11): p. 1304-8. 9. Rizzo, J.A., A.L. Naig, and E.K. Johnson, Anorectal abscess and fistula-in-ano: evidence-based management. Surg Clin North Am, 2010. 90(1): p. 45-68, Table of Contents. 10. Whiteford, M.H., et al., Practice parameters for the treatment of perianal abscess and fistula-in-ano (revised). Dis Colon Rectum, 2005. 48(7): p. 1337-42. 11. Cavanaugh, M., N. Hyman, and T. Osler, Fecal incontinence severity index after fistulotomy: a predictor of quality of life. Dis Colon Rectum, 2002. 45(3): p. 349-53. 12. Vogel, J.D., et al., Clinical Practice Guideline for the Management of Anorectal Abscess, Fistula-in-Ano, and Rectovaginal Fistula. Dis Colon Rectum, 2016. 59(12): p. 1117-1133. 13. Abbas, M.A., C.H. Jackson, and P.I. Haigh, Predictors of outcome for anal fistula surgery. Arch Surg, 2011. 146(9): p. 1011-6.

Claims

1. A method for treating gastrointestinal (GI) fistulas in human subjects, the method comprising: applying E4ORF1 to a human subject having a GI fistula. ＋ Human umbilical vein endothelial cells (E4ORF1) ＋ The method involves administering a composition containing HUVEC, wherein (a) about 5 × 10 ６ ~About 200×10 ６ Total E4ORF1 ＋ HUVEC, or (b) approximately 5 × 10 per 1 cm of fistula length ６ ~Approx. 120×10 ６ E4ORF1 ＋ The method comprising administering HUVEC to a subject by local administration to the site of the GI fistula, thereby treating the GI fistula in the subject.

2. The method according to claim 1, wherein the subject is also treated by performing a fistula incision.

3. A method of treating a GI fistula in a human subject, the method comprising administering to a human subject having a GI fistula an effective amount of a composition comprising E4ORF1 ＋ by local administration to the site of the GI fistula, wherein the subject is not treated by fistulotomy, thereby treating the GI fistula in the subject, said method.

4. (a) Approximately 5 x 10 ６ ~About 200×10 ６ Total E4ORF1 ＋ HUVEC, or (b) approximately 5 × 10 per 1 cm of fistula length ６ ~Approx. 120×10 ６ E4ORF1 ＋ The method according to claim 3, wherein HUVEC is administered to the subject.

5. (a) Approximately 7.5 x 10 ６ ~Approx. 30×10 ６ Total E4ORF1 ＋ HUVEC, or (b) approximately 7.5 × 10 per 1 cm of fistula length ６ ~Approx. 30×10 ６ E4ORF1 ＋ The method according to claim 4, wherein HUVEC is administered to the subject.

6. (a) Approximately 15 x 10 ６ ~Approx. 30×10 ６ Total E4ORF1 ＋ HUVEC, or (b) approximately 15 × 10 per 1 cm of fistula length ６ E4ORF1 ＋ The method according to claim 5, wherein HUVEC is administered to the subject.

7. E4ORF1 ＋ HUVEC administration along the length of the GI fistula E4ORF1 ＋ The method according to any of the prior claims, comprising arranging HUVEC.

8. E4ORF1 ＋ The method according to any of the preceding claims, wherein the GI fistula is curettaged before administering HUVEC.

9. E4ORF1 ＋ HUVEC administration is performed along the length of the curettage GI fistula E4ORF1 ＋ The method according to claim 8, comprising distributing HUVEC.

10. E4ORF1 ＋ HUVEC administration, E4ORF1 ＋ Distribute half of the HUVEC along one side of the curettage GI fistula, and E4ORF1 ＋ The method according to claim 8, comprising distributing half of the HUVEC along the opposite side of the curettage GI fistula.

11. The method according to any of the preceding claims, further comprising surgically closing the internal opening of a GI fistula.

12. The method according to any of the preceding claims, further comprising surgically closing the internal opening of the GI fistula using sutures.

13. E4ORF1 around the surgically closed internal opening of the GI fistula ＋ The method according to claim 12, further comprising administering HUVEC submucosally.

14. Around the surgically closed internal opening of the GI fistula, approximately 15 × 10 ６ ~Approx. 30×10 ６ Individual E4ORF1 ＋ The method according to claim 13, further comprising administering HUVEC submucosally.

15. E4ORF1 for the target ＋ The method according to any of the preceding claims, comprising administering HUVEC multiple times, wherein the time interval between administrations is approximately two weeks to approximately twelve weeks.

16. The method according to claim 15, wherein the time interval between administrations is approximately 4 weeks to approximately 10 weeks.

17. The method according to claim 16, wherein the time interval between administrations is approximately 6 weeks to approximately 8 weeks.

18. E4ORF1 for the target ＋ The method according to any one of claims 15 to 17, wherein the administration of HUVEC is repeated once.

19. E4ORF1 for the target ＋ The method according to any one of claims 15 to 17, wherein the administration of HUVEC is repeated twice.

20. E4ORF1 for the target ＋ The method according to any one of claims 15 to 17, wherein the administration of HUVEC is repeated three times.

21. E4ORF1 for the target ＋ The method according to any one of claims 15 to 17, wherein the administration of HUVEC is repeated four times.

22. The method according to any of the preceding claims, wherein the GI fistula is an intestinal cutaneous fistula.

23. The method according to any of the preceding claims, wherein the GI fistula is an enterojejunal fistula.

24. The method according to any of the preceding claims, wherein the GI fistula is an anal fistula, anorectal fistula, anorovaginal fistula, appendiceal fistula, biliary fistula, cecal fistula, colonic fistula, colorectal fistula, cocovesical fistula, duodenal fistula, duodenocolonic fistula, enterovesical fistula, esophageal fistula, gallbladder fistula, gastrostomy, gastrocolic fistula, gastrojejunal fistula, ileal fistula, jejunal fistula, oral fistula, pancreatic fistula, rectal fistula, rectovaginal fistula, or salivary gland fistula.

25. The method according to any of the preceding claims, wherein the GI fistula is an anal fistula.

26. The composition is E4ORF1 ＋ The method according to any of the preceding claims, comprising HUVEC 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.

27. The method according to claim 26, wherein the composition comprises human serum albumin (HSA).

28. The method according to claim 26 or claim 27, wherein the composition comprises dextran 40.

29. The method according to any one of claims 26 to 28, wherein the composition comprises up to about 0.25% DMSO.

30. The method according to any one of claims 26 to 29, wherein the composition comprises HSA, dextran 40, and up to about 0.25% DMSO.

31. The method according to any one of the preceding claims, wherein the composition is administered locally using an injection needle.

32. The method according to any of the preceding claims, wherein the composition is administered locally under image guidance.

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