CRYOPRESERVED ENDOTHELIAL CELL COMPOSITIONS

Abstract

The present invention provides compositions comprising high densities of endothelial cells (such as human umbilical vein endothelial cells) in freezing media, methods of producing such compositions, and methods of using such compositions in the preparation of therapeutic endothelial cell compositions. Such compositions and methods provide numerous advantages including eliminating the requirement to remove cryopreservatives before administration of therapeutic endothelial cell compositions to human subjects and requiring minimal manipulations and human interventions before use in therapeutic methods.

Claims

1. A composition comprising E4ORF1+endothelial cells (ECs) at a density of from about 50 million cells per ml to about 150 million cells per ml in a freezing medium comprising an effective amount of a cryopreservative.

2. The composition of claim 1, wherein the ECs are human umbilical vein endothelial cells (HUVEC s).

3. The composition of claim 1 or claim 2, wherein the endothelial cells (ECs) are at a density of from about 75 million cells per ml to about 125 million cells per ml.

4. The composition of claim 1 or claim 2, wherein the endothelial cells (ECs) are at a density of about 100 million cells per ml.

5. The composition of any of the preceding claims further comprising human serum albumin (HSA).

6. The composition of any of the preceding claims further comprising about 10% human serum albumin (HSA).

7. The composition of any of the preceding claims wherein the cryopreservative is selected from the group consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, and glycerol.

8. The composition of any of the preceding claims wherein the cryopreservative is dimethyl sulfoxide.

9. The composition of any of the preceding claims comprising from about 5% to about 10% dimethyl sulfoxide.

10. The composition of any of the preceding claims comprising about 10% dimethyl sulfoxide.

11. The composition of any of the preceding claims wherein the freezing medium comprises an endothelial growth medium.

12. The composition of any of the preceding claims, wherein the ECs comprise a recombinant nucleotide sequence that encodes an adenovirus E4ORF1 protein operatively linked to a heterologous promoter.

13. The composition of claim 12, wherein the nucleotide sequence is within a vector.

14. The composition of claim 13, wherein the vector is a retroviral vector.

15. The composition of claim 14, wherein the retroviral vector is a lentiviral vector.

16. The composition of claim 15, wherein the retroviral vector is a Maloney murine leukemia virus (MMLV) vector.

17. The composition of any of the preceding claims, wherein the E4ORF1 is human adenovirus type 5 E4ORF1.

18. The composition of any of the preceding claims, wherein the ECs do not comprise an entire adenovirus E4 region.

19. The composition of any of the preceding claims, wherein the ECs do not comprise do not comprise an E4ORF2, E4ORF3, E4ORF4, E4ORF5 or E4ORF6 coding sequence or amino acid sequence.

20. A composition comprising (a) E4ORF1+ECs at a density of about 100 million cells per ml, (b) endothelial growth media, (c) about 5 to about 10% DMSO, and (d) about 10% HSA.

21. A composition comprising (a) E4ORF1+HUVECs at a density of about 100 million cells per ml, (b) endothelial growth media, (c) about 5 to about 10% DMSO, and (d) about 10% HSA.

22. A composition according to any of the preceding claims, further comprising hematopoietic stem cells or hematopoietic progenitor cells.

23. The composition of claim 22, wherein the hematopoietic stem cells or hematopoietic progenitor cells are from bone marrow.

24. The composition of claim 22, wherein the hematopoietic stem cells or hematopoietic progenitor cells are from peripheral blood.

25. The composition of claim 22, wherein the hematopoietic stem cells or hematopoietic progenitor cells are from amniotic fluid.

26. The composition of claim 22, wherein the hematopoietic stem cells or hematopoietic progenitor cells are from umbilical cord blood.

27. The composition of any of the preceding claims wherein the composition is in a freezing container.

28. The composition of claim 27, wherein the freezing container is a cryovial.

29. The composition of claim 27, wherein the freezing container is a cryobag.

30. The composition of claim 27, wherein the freezing container is adapted for aseptic transfer of its contents to a patient in a closed system.

31. A composition according to any of the preceding claims for use in the preparation of a therapeutic composition for administration to a human subject.

32. Use of a composition according to any of the preceding claims in the preparation of a therapeutic composition for administration to a human subject.

33. A method of preparing a therapeutic composition for administration to a human subject, the method comprising diluting a composition according to any of the preceding claims with a physiological saline solution, wherein the EC cell concentration after dilution is from about 3 million cells per ml to about 5 million cells per ml, thereby preparing a therapeutic composition for administration to a human subject.

34. The method of claim 33, wherein the physiological saline comprises dextran 40 and HSA at amounts such that, after dilution, the therapeutic composition comprises about 8% dextran and about 4% HSA.

35. The method of claim 33, wherein the method does not comprise any centrifugation steps.

36. The method of claim 33 or 34, wherein the method does not comprise removing the freezing medium or cyropreservative.

37. A method of freezing endothelial cells, the method comprising: a. suspending endothelial cells (ECs) at a density of from about 50 million cells per ml to about 150 million cells per ml in a freezing medium, wherein the freezing medium comprises an effective amount of a cryopreservative, thereby creating a freezing composition, and b. subjecting the freezing composition to a gradual decrease in temperature to about 80 C. to 90 C.

38. The method of claim 37, wherein in step (b) the temperature is decreased at a rate of about 1 C. per minute.

39. The method of claim 37 or claim 38, comprising subsequently transferring the freezing composition to liquid nitrogen.

40. The method of any of claims 37-39, wherein the endothelial cells (ECs) are at a density of from about 75 million cells per ml to about 125 million cells per ml.

41. The method of any of claims 37-39, wherein the endothelial cells (ECs) are at a density of about 100 million cells per ml.

42. The method of any of claims 37-41, wherein the ECs are human umbilical vein endothelial cells (HUVECs).

43. The method of any of claims 37-42, wherein the freezing medium comprises human serum albumin (HSA).

44. The method of any of claims 37-43, wherein the freezing medium comprises about 10% human serum albumin (HSA).

45. The method of any of claims 37-44, wherein the cryopreservative is selected from the group consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, and glycerol.

46. The method of any of claims 37-45, wherein the cryopreservative is dimethyl sulfoxide.

47. The method of any of claims 37-46, wherein the freezing medium comprises from about 5% to about 10% dimethyl sulfoxide.

48. The method of any of claims 37-47, wherein the freezing medium comprises about 10% dimethyl sulfoxide.

49. The method of any of claims 37-48, wherein the freezing medium comprises a cell culture medium.

50. The method of any of claims 37-49, wherein the freezing medium comprises an endothelial growth medium.

51. The method of any of claims 37-50, wherein the endothelial cells (ECs) are adenovirus E4ORF1+ECs.

52. The method of any of claims 37-51, wherein the ECs comprise a recombinant nucleotide sequence that encodes an adenovirus E4ORF1 protein.

53. The method of claim 52, wherein the wherein the nucleotide sequence is operatively linked to a heterologous promoter.

54. The method of claim 52 or 53, wherein the nucleotide sequence is within a vector.

55. The method of claim 54, wherein the vector is a retroviral vector.

56. The method of claim 55, wherein the retroviral vector is a lentiviral vector.

57. The method of claim 55, wherein the retroviral vector is a Maloney murine leukemia virus (MMLV) vector.

58. The method of any of claims 51-57, wherein the E4ORF1 is human adenovirus type 5 E4ORF1.

59. The method of any of claims 51-58, wherein the ECs do not comprise an entire adenovirus E4 region.

60. The method of any of claims 51-59, wherein the ECs do not comprise do not comprise an E4ORF2, E4ORF3, E4ORF4, E4ORF5 or E4ORF6 coding sequence or amino acid sequence.

61. The method of any of claims 37-60, wherein the freezing composition further comprises hematopoietic stem cells or hematopoietic progenitor cells.

62. The method of claim 61, wherein the hematopoietic stem cells or hematopoietic progenitor cells are from bone marrow.

63. The method of claim 61, wherein the hematopoietic stem cells or hematopoietic progenitor cells are from peripheral blood.

64. The method of claim 61, wherein the hematopoietic stem cells or hematopoietic progenitor cells are from amniotic fluid.

65. The method of claim 61, wherein the hematopoietic stem cells or hematopoietic progenitor cells are from umbilical cord blood.

66. The method of any of claims 37-65, wherein the freezing composition is in a freezing container.

67. The method of claim 66, wherein the freezing container is a cryovial.

68. The method of claim 66, wherein the freezing container is a cryobag.

69. The method of claim 66, wherein the freezing container is adapted for aseptic transfer of its contents to a patient in a closed system.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1. Graph showing total cell counts of E4ORF1+HUVECs at 0, 2, 4, 6, 24, 48 and 72 hours post-thaw. Cells were frozen at a concentration of 110.sup.8 (i.e., 100 million) cells per ml in 2 ml cryovials using the rate-controlled freezing program described in Example 1. Initial refers to pre-freeze data.

[0036] FIG. 2. Graph showing viability of E4ORF1+HUVECs at 0, 2, 4, 6, 24, 48 and 72 hours post-thaw. Cells were frozen at a concentration of 110.sup.8 cells per ml in 2 ml cryovials using the rate-controlled freezing program described in Example 1. Initial refers to pre-freeze data.

[0037] FIG. 3. Graph showing viable cell counts of E4ORF1+HUVECs at 0, 2, 4, 6, 24, 48 and 72 hours post-thaw. Cells were frozen at a concentration of 110.sup.8 cells per ml in 2 ml cryovials using the rate-controlled freezing program described in Example 1. Initial refers to pre-freeze data.

[0038] FIG. 4. Graph showing viable cell recovery of E4ORF1+HUVECs at 0, 2, 4, 6, 24, 48 and 72 hours post-thaw. Cells were frozen at a concentration of 110.sup.8 cells per ml in 2 ml cryovials using the rate-controlled freezing program described in Example 1.

[0039] FIG. 5. Bar charts showing percentage viability (left panel) and percentage recovery (right panel) of E4ORF1+HUVECs frozen at either 1.310.sup.7 (i.e., 13 million) cells/ml or 110.sup.8 (i.e., 100 million) cells/ml, as indicated, in in a freezing medium comprising 5% DMSO and 20% human serum albumin (HSA) in 2-mL cryovials using the HUVEC freezing program described in Example 1. Cells were cryopreserved for at least 24 hours in liquid nitrogen before thawing, diluting at a 1:20 ratio in a dilution buffer containing 8.3% dextran and 4.2% HSA without any centrifugation/pelleting or rinsing to remove cryopreservative.

DETAILED DESCRIPTION

[0040] The Summary of the Invention and Claims sections of this patent disclosure describe the main embodiments of the 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, and are intended to be, combined in various different ways. Such combinations of the specific embodiments described herein are intended to fall within the scope of the present invention

[0041] Definitions & Abbreviations

[0042] 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 frm 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.

[0043] 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.

[0044] 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).

[0045] Units, prefixes, and symbols are denoted in their Systeme 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.

[0046] Wherever embodiments are described with the language comprising, otherwise analogous embodiments described in terms of consisting of and/or consisting essentially of are included.

[0047] As used herein, the terms about and approximately, when used in relation to numerical values, mean within + or 10% of the stated value.

[0048] As used herein, the abbreviation EC(s) refers to endothelial cell(s).

[0049] 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).

[0050] As used herein, the abbreviation E4ORF1+ is used to refer to cells engineered to express E4ORF1. For example, the abbreviation E4ORF1+HUVECs is used to refer to human umbilical cord endothelial cells (HUVECs) engineered to express E4ORF1. E4ORF1+ cells contain a recombinant E4ORF1 nucleic acid molecule and express the E4ORF1 protein.

[0051] The term culturing as used herein, refers to the propagation of cells on or in media of various kinds. Co-culturing refers to the propagation of two or more distinct types of cells on or in media of various kinds.

[0052] As used herein the term effective amount refers to an amount of a specified agent (e.g., a cryopreservative) or a specified cell population (e.g. E4ORF1+HUVECs) that is sufficient to achieve one or more of the outcomes described herein. For example, an effective amount of a cryopreservative is an amount that results in effective cell freezing and effective cell recovery following freezing. An appropriate effective amount in any individual case may be determined empirically, for example using standard techniques known in the art. Furthermore, an effective amount may be determined using assays such as those described in the Examples section of this patent disclosure to assess effects on cell freezing and/or on recovery from cell freezing. For all of the embodiments described herein that involve compositions comprising, or methods of using, specified agents or specified cell populations, in some embodiments the amount of the agent(s) or cell population(s) is any effective amount. For example, if no specific amount is specified, the amount may be any effective amount.

[0053] The term engineered when used in relation to cells (typically endothelial cells such as HUVECs) herein refers to cells that have been engineered by man to result in the recited phenotype (e.g. E4ORF1.sup.+) or to express a recited nucleic acid molecule or polypeptide. 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 (e.g. by genetic modification), for example so that they express a polypeptide that they would not otherwise express.

[0054] The terms frozen and cryopreserved (and grammatical variations thereof) are used interchangeably herein, and are used in accordance with their usual meaning in the art.

[0055] The terms genetic modification and/or genetically modified and/or gene-modified refer to any addition, deletion, alteration or disruption of or to a nucleotide sequence or to a cell's genome or to a cell's content of genetic material. In some embodiments, the endothelial cells described herein may, in addition to being genetically modified to provide a nucleic acid molecule that encodes E4ORF1, may also comprise one or more other genetic modificationsas desired. The term genetic modification and the above related terms encompass both transient and stable genetic modification and encompass the use of various different gene delivery vehicles and methods including, but not limited to, transduction (viral mediated transfer of nucleic acid to a recipient, either in vivo or in vitro), transfection (uptake by cells of isolated nucleic acid), liposome mediated transfer and others means of gene delivery that are well known in the art.

[0056] As used herein the term isolated refers to a cell population, product, compound, or composition which is separated from at least one other cell population, product, compound, or composition with which it is associated in its usual state, such as in its naturally occurring state in the body of a living subject.

[0057] As used herein, the term recombinant refers to nucleic acid molecules that are isolated, generated and/or designed by man (including by a machine) using methods of molecular biology and genetic engineering (such as molecular cloning), and that either comprise nucleotide sequences that do not exist in nature, or are comprised within nucleotide sequences that do not exist in nature, or are provided in association with nucleotide sequences that they would not be associated with in nature, or that are provided in the absence of nucleotide sequences with which they would ordinarily be associated in nature. Thus, recombinant nucleic acid molecules are to be distinguished from nucleic acid molecules that exist in naturefor example in the genome of an organism. For example, a nucleic acid molecule that comprises a complementary DNA or cDNA copy of an mRNA sequence, without any intervening intronic sequences such as would be found in the corresponding genomic DNA sequence, would thus be considered a recombinant nucleic acid molecule. By way of a further example, a recombinant E4ORF1 nucleic acid molecule might comprise an E4ORF1 coding sequence operatively linked to a promoter and/or other genetic elements with which that coding sequence is not ordinarily associated in a naturally-occurring adenovirus genome, or in the absence of absence of nucleotide sequences with which it would ordinarily be associated in an adenovirus genome.

[0058] The term subject includes mammalssuch as humans and non-human primates, as well as other mammalian species including rabbits, rats, mice, cats, dogs, horses, cows, sheep, goats, pigs and the like. In some embodiments the subjects are mammalian subjects. In some embodiments the subjects are humans. In some embodiments the subjects are non-human primates.

[0059] The terms patient and human subject may be used interchangeably herein.

[0060] E4ORF 1

[0061] Several of the embodiments of the present invention described herein involve engineered endothelial cells (ECs) that are E4ORF1+. 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 endothelial cells.

[0062] 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 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. CA105991), 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 one embodiment the E4ORF1 sequence used is that having NCBI accession number AZR66741.1. In one embodiment the E4ORF1 sequence used is that having NCBI accession number AP 000232.1. In one embodiment the E4ORF1 sequence used is has the amino acid sequence

TABLE-US-00001 (SEQIDNO.1) MAAAVEALFVVLEREGAILPRQEGFSGVYVFFSPINFVIPPMGAVMLSL RLRVCIPPGYFGRFLALTDVNQPDVFTESYIMTPDMTEELSVVLFNHGD QFFYGHAGMAVVRLMLIRVVFPVVRQASNV.

[0063] In some embodiments the E4ORF1 polypeptide may have an amino acid sequence, or may 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 known in the art (for example as described in U.S. Pat. No. 8,465,732) or 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, 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.

[0064] In some embodiments E4ORF1 sequences are used without other sequences from the adenovirus E4 regionfor 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 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] The E4ORF1-encoding sequences can be introduced into endothelial cells 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, 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.

[0069] 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).

[0070] In some embodiments the compositions of the present invention comprise both E4ORF1+ and E4ORF1-negative endothelial cells. In some embodiments at least about 75% of the endothelial cells in the composition are E4ORF1+. In some embodiments at least about 80% of the endothelial cells in the composition are E4ORF1+. In some embodiments at least about 85% of the endothelial cells in the composition are E4ORF1+. In some embodiments at least about 90% of the endothelial cells in the composition are E4ORF1+. In some embodiments at least about 95% of the endothelial cells in the composition are E4ORF1+. In some embodiments at least about 98% of the endothelial cells in the composition are E4ORF1+. In some embodiments at least about 99% of the endothelial cells in the composition are E4ORF1+.

[0071] In some embodiments the compositions of the present invention comprise endothelial cells that, on average across all of the endothelial cells in the composition, comprise less than one copy of a genomically integrated E4ORF1 coding sequence per cell. In some embodiments the compositions of the present invention comprise endothelial cells that, on average across all of the endothelial cells in the composition, comprise about one copy of a genomically integrated E4ORF1 coding sequence per cell. In some embodiments the compositions of the present invention comprise endothelial cells that, on average across all of the endothelial cells in the composition, comprise more than one copy of a genomically integrated E4ORF1 coding sequence per cell. In some embodiments the compositions of the present invention comprise endothelial cells that, on average across all of the endothelial cells in the composition, comprise about 0.7 copies of a genomically integrated E4ORF1 coding sequence per cell. In some embodiments the compositions of the present invention comprise endothelial cells that, on average across all of the endothelial cells in the composition, comprise about 0.8 copies of a genomically integrated E4ORF1 coding sequence per cell. In some embodiments the compositions of the present invention comprise endothelial cells that, on average across all of the endothelial cells in the composition, comprise about 0.9 copies of a genomically integrated E4ORF1 coding sequence per cell. In some embodiments the compositions of the present invention comprise endothelial cells that, on average across all of the endothelial cells in the composition, comprise about 1.0 copies of a genomically integrated E4ORF1 coding sequence per cell. In some embodiments the compositions of the present invention comprise endothelial cells that, on average across all of the endothelial cells in the composition, comprise about 1.1 copies of a genomically integrated E4ORF1 coding sequence per cell. In some embodiments the compositions of the present invention comprise endothelial cells that, on average across all of the endothelial cells in the composition, comprise about 1.2 copies of a genomically integrated E4ORF1 coding sequence per cell. In some embodiments the compositions of the present invention comprise endothelial cells that, on average across all of the endothelial cells in the composition, comprise about 1.3 copies of a genomically integrated E4ORF1 coding sequence per cell. In some embodiments the compositions of the present invention comprise endothelial cells that, on average across all of the endothelial cells in the composition, comprise about 1.4 copies of a genomically integrated E4ORF1 coding sequence per cell. In some embodiments the compositions of the present invention comprise endothelial cells that, on average across all of the endothelial cells in the composition, comprise about 1.5 copies of a genomically integrated E4ORF1 coding sequence per cell.

[0072] In some embodiments the presence of E4ORF1 coding sequences 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. Pat. No. 8,465,732). In some of such embodiments the results of any of such assays can be compared between batches of E4ORF1+endothelial cells (e.g., between a test batch and a control batch having known E4ORF1 properties), for example to assess consistency and/or to make any adjustments based thereon.

[0073] The handling, manipulation, and expression of E4ORF1 sequences in endothelial cells 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.,

[0074] 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. Additional aspects relevant to the handling and expression of E4ORF1 sequences in endothelial cells are described in U.S. Pat. No. 8,465,732, the contents of which are hereby incorporated by reference.

[0075] Endothelial Cells

[0076] In some embodiments the endothelial cells (ECs) described herein can be derived from any suitable source of vascular endothelial cells known in the art. In some embodiments the endothelial cells are primary endothelial cells. In some embodiments the endothelial cells are mammalian cells, such as human or non-human primate cells, or rabbit, rat, mouse, goat, pig, or other mammalian cells. In some embodiments the endothelial cells are primary human endothelial cells. In some embodiments the endothelial cells are umbilical vein endothelial cells (UVECs), such as human umbilical vein endothelial cells (HUVECs). In some embodiments the endothelial cells are adipose ECs. In some embodiments the endothelial cells are skin ECs. In some embodiments the endothelial cells are cardiac ECs. In some embodiments the endothelial cells are kidney ECs. In some embodiments the endothelial cells are lung ECs. In some embodiments the endothelial cells are liver ECs. In some embodiments the endothelial cells are bone marrow ECs. Other suitable endothelial cells that can be used include those described previously as being suitable for E4ORF1-expression in U.S. Pat. No. 8,465,732, the contents of which are hereby incorporated by reference.

[0077] In some embodiments the endothelial cells are gene-modified such that they comprise one or more genetic modifications. For example, in some embodiments the endothelial cells are engineered to express E4ORF1. In some embodiments the endothelial cells may also be engineered to express ETV2. Indeed, in each of the embodiments described throughout this patent disclosure where the ECs express E4ORF1, the ECs may also express ETV2. Similarly, in some embodiments the endothelial cells may also be engineered to express BMP4. Indeed, in each of the embodiments described throughout this patent disclosure where the ECs express E4OR1, the ECs may also express BMP4.

[0078] Furthermore, in some embodiments the ECs described herein may comprise a corrected version of a gene known to be involved in, or suspected of being involved in, a disease or disorder that affects endothelial cells, or any other gene, such as a therapeutically useful gene, that it may be desired to provide in endothelial cells or administer or deliver using engineered endothelial cells.

[0079] Methods of Use

[0080] In some embodiments, the compositions described herein can be used in various therapeutic methods, or can be used in the preparation of therapeutic compositions which can in turn be used in various therapeutic methods. Such therapeutic methods may comprise any methods for which the administration of ECs (such as HUVECs) to a subject may be desired or beneficial. In carrying out such therapeutic methods the therapeutic compositions described herein can be administered to subjects using any suitable means known in the art, for example by injection (e.g. intravenous injection, intramuscular injection, subcutaneous injection, local injection), by infusion (e.g. by intravenous infusion, subcutaneous infusion, local infusion), or by surgical implantation. The therapeutic compositions can be administered in a single dose or in multiple doses. The skilled artisan will be able to select a suitable route of administration and a suitable schedule of administration depending on the particular situation.

[0081] In some embodiments the therapeutic compositions described herein may comprise, or be administered together with, compositions comprising one or more additional cell types. Such additional cell types may be, for example stem or progenitor cells, such as hematopoietic stem cells, hematopoietic progenitor cells, c-kit+Scal+ hematopoietic stem cells, lymphoid progenitor cells, CD4-CD8-CD44+CD25-ckit+ cells, early thymic progenitors, CD4-CD8-CD44+CD25-ckit- cells or DN1 cells.

[0082] Cell Culture & Cryopreservation Methods

[0083] Methods of culturing cells are well known in the art and any suitable cell culture methods can be used. For example, ECs can be cultured using methods known to be useful for culturing other endothelial cells, or, methods known to be useful for culturing E4ORF1-expressing endothelial cells, for example as described in U.S. Pat. No. 8,465,732, the contents of which are hereby incorporated by reference. In some embodiments the ECs can be cultured in the absence of serum, or in the absence of exogenous growth factors, or in the absence of both serum and exogenous growth factors.

[0084] Exemplary cryopreservation protocols are described hereinincluding in the Examples section of this patent disclosure. In addition, several methods for cryopreservation of ECs (including HUVECs) are known in the art and can be used in connection with the present invention. In particular, reference is made to the EC/HUVEC cryopreservation methods described in Lehle et al., Cryopreservation of human endothelial cells for vascular tissue engineering. Cryobiology 50 (2005) 154-161; Lehle et al., Identification and Reduction of Cryoinjury in Endothelial Cells: A First Step toward Establishing a Cell Bank for Vascular Tissue Engineering. Tissue Engineering Volume 12, Number 12, 2006; Lonza; Clonetics Endothelial Cell SystemTechnical Information & Instructions, www.lonza.com; 2018, Technical Information & Instructions; Marquez-Curtis et al., Beyond membrane integrity: Assessing the functionality of human umbilical vein endothelial cells after cryopreservation. Cryobiology 72 (2016) 183-190; Pegg., Cryopreservation of vascular endothelial cells as isolated cells and as monolayers. Cryobiology 44 (2002) 46-53; Polchow et al., Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks. Journal of Translational Medicine 2012 10:98; Puzanov et al., New Approach to Cryopreservation of Primary Noncultivated Human Umbilical Vein Endothelium in Biobanking. Biopreservation And Biobanking; Volume 16, Number 2, 2018; Sultani, A. B. et al. Improved Cryopreservation of Human Umbilical Vein Endothelial Cells: A Systematic Approach. Sci. Rep. 6, 34393; (2016); Reardon et al. Investigating membrane and mitochondrial cryobiological responses of HUVEC using interrupted cooling protocols. Cryobiology 71 (2015) 306-317; and von Bomhard A. et al., (2016) Cryopreservation of Endothelial Cells in Various Cryoprotective Agents and MediaVitrification versus Slow Freezing Methods. PLoS ONE 11(2) the contents of each of which are hereby incorporated by reference.

[0085] Kits

[0086] The present invention also contemplates kits comprising the compositions described herein, or for preparing the compositions described herein, and/or for carrying out any of the methods described herein. Such kits may contain any of the components described herein, including, but not limited to, nucleotide sequences (for example those encoding E4ORF1), ECs (such as HUVECs), populations of E4ORF1+ECs (such as E4ORF1+HUVECs), means or compositions for detection of ECs (such as HUVECs) or the proteins or nucleic acid molecules expressed therein, (e.g. nucleic acid probes, antibodies, etc.), freezing media, cryopreservatives, HSA, dextran (e.g. dextran40), cryovials, cryobags, or any combination thereof. All such kits may optionally comprise instructions for use. A label may accompany the kit and may include any writing or recorded material (which may be electronic or in computer readable form) providing instructions or other information for use of the kit contents. For example, in some embodiments such kits may comprise a composition comprising E4ORF1+HUVECs in freezing media in a cryovial or cryobag and instructions for the thawing, dilution, and/or clinical use thereof.

[0087] Certain aspects of the present invention may be further described with reference to the following non-limiting Examples.

EXAMPLES

Example 1

High Density Freezing of E4ORF1+HUVECs

[0088] Experiments were performed to assess the recovery and viability of E4ORF1+HUVECs that had been cryopreserved using a controlled-rate freezing program, and using various different freezing containersincluding some containers that were adapted for direct dilution and aseptic delivery of cells to patients in a closed system.

[0089] E4ORF1+HUVECs were pelleted and then suspended at a concentration of either about 13 million (1.310 7) cells per ml or 100 million (1.010.sup.8) cells per ml in a freezing medium comprising 5% DMSO and 20% human serum albumin (HSA).

[0090] In some experiments about 0.5 mls (0.57 mls) of this cell suspension was transferred to each of several 2 ml cryovials. In some experiments about 1 ml of this cell suspension was transferred to each of several 2 ml or 5 ml cryovials or to cryobags. In some of these experiments Crystal Zenith cryovials manufactured by Daikyo or Briostor or Transfer/Freezing Bag Sets manufactured by Pall Medical were usedeach of which is adapted for aseptic delivery of thawed cell products to patients in a closed system.

[0091] A rate-controlled freezing program was utilized to freeze the E4ORF1+HUVECs in freezing mediumthe details of which are provided in Table 1 below.

TABLE-US-00002 TABLE 1 Controlled Rate Freezing Program Program Protocol HUVEC 1. Rate Controlled Freezer powered on and cools Freezing chamber to 4 C. at a rate of 1 C. per minute. Program 2. Cells placed in the chamber, along with a probe (which is placed in a blank cryovial). 3. Rate Controlled Freezer cools at a rate of 1 C. per minute until the probe is at a temperature of 4 C. 4. Rate Controlled Freezer cools at a rate of 1 C. per minute until probe is at a temperature of 4 C. 5. Rate Controlled Freezer cools at a rate of 25 C. per minute until chamber is at a temperature of 40 C. 6. Rate Controlled Freezer warms at a rate of 10 C. per minute until chamber is at a temperature of 12 C. 7. Rate Controlled Freezer cools at a rate of 1 C. per minute until probe is at a temperature of 40 C. 8. Rate Controlled Freezer cools at a rate of 10 C. per minute until probe is at a temperature of 90 C. 9. Cells are removed from Rate Controlled Freezer chamber and immediately transferred to LN2 storage.

[0092] After executing the rate-controlled freezing program, frozen cells were stored in liquid nitrogen (LN2) for at least 24 hours (1 days) to 96 hours (4 days).

[0093] The E4ORF1+HUVECs were then thawed and diluted in a dextran- and HSA-containing dilution buffer (comprising dextran 40 8.3% HSA 4.2%) to yield a diluted cell concentration of approximately 3.4 million cells per ml.

[0094] Recovery of viable cells was assessed either immediately post-thaw (i.e. at 0 hours post-thaw) or after the cells had been maintained at room temperature for 2, 4, 6, 24, 48 or 72 hours post-thaw using standard methods involving staining cells with trypan blue and counting cells using a hemocytometer.

[0095] FIGS. 1-4 present, in graphical form, the total cell counts (FIG. 1), viability (FIG. 2), absolute viable cell counts (FIG. 3) and percentage viable cell recovery (FIG. 4) of E4ORF1+HUVECs at 0, 2, 4, 6, 24, 48 and 72 hours post-thaw when the cells were frozen using the rate-controlled freezing program in 2 ml cryovials (initial in the Figures refers to pre-freeze viability). The post-thaw viability was stable, (see FIG. 2), unexpectedly showing virtually no drop in viability over the course of the experiment.

[0096] The percentage viability and percentage recovery of E4ORF1+HUVECs frozen at either 1.310.sup.7 cells/ml or 110.sup.8 cells/ml in in a freezing medium comprising 5% DMSO and 20% human serum albumin (HSA) in either standard screw-cap cryovials, CZ cryovials, or cryobags (Briostor Transfer/Freezing Bag Set) at either both 2-mL or 5-mL sizes HUVEC freezing program, was determined. Cells were cryopreserved for at least 24 hours in liquid nitrogen before thawing, diluting at a 1:20 ratio in a dilution buffer containing 8.3% dextran and 4.2% HSA without any centrifugation or rinsing to remove cryopreservative (as described above), subsequently assessing cell number/viability (as described above).

[0097] The results are shown in FIG. 5A-B and Table 2.

TABLE-US-00003 TABLE 2 Screw Cap CZ Vial Screw Cap CZ vial 1.3 10.sup.7/ml 1.3 10.sup.7/ml 1 10.sup.8/ml 1 10.sup.8/ml Viability 98 96 97 98 Recovery 102 105 78 96

[0098] The data from these studies showed:

[0099] (a) that surprisingly high levels of viability and viable cell recovery could be achieved (with either freezing protocol) even when E4ORF1+HUVECs were frozen at ultra-high cell density, with no observed decrease in viability/recovery when increasing cell densities from about 10 million cells per ml to about 100 million cells per ml,

[0100] (b) that E4ORF1+HUVECs frozen at ultra-high density can be diluted directly, without the need for removal of cryopreservatives, to generate a useable cell therapy product containing an appropriate amount and concentration of E4ORF1+HUVECs in a buffer suitable for administration to a human subjectall without any significant loss of viability, and (c) that the HUVEC freezing program we describe can be used to maintain post-thaw viability of E4ORF1+HUVECs.

Example 2

E4ORF1+HUVECs Frozen at High Density Can be Thawed, Diluted and Safely Administered to Human Subjects

[0101] A Phase I clinical trial was performed to assess the safety of administration of E4ORF1+HUVECs to human subjects. Subjects with chemosensitive lymphomas eligible for high dose therapy-autologous hematopoietic cell transplantation (HDT-AHCT) were enrolled.

[0102] The E4ORF1+HUVECs used in the clinical trial were supplied to clinical trials sites frozen (using methods as described herein) at a concentration of 100 million cells per ml (110 8 cells per ml) in a serum-free, CGMP manufactured, freezing medium (CryoStor CS5) supplemented with Human Serum Albumin (HSA) and DMSO, such that the final concentration of HSA was 10% and the final concentration of DMSO was 5%.

[0103] At the clinical trial sites, the cells were thawed and diluted in a dilution medium (using methods as described herein)without removal of cryopreservativeto yield a therapeutic composition comprising E4ORF1+HUVEC cells (about 510.sup.6 cells per ml), Dextran40 (8.3%), HSA (4.3%) and DMSO (0.25%) in saline.

[0104] The therapeutic composition was then administered intravenously to human subjects, after AHCT, in dose-escalated cohorts receiving either 510.sup.6, 1010.sup.6 or 2010.sup.6 cells/kg, either as a single or divided dose (in the case of divided dosing, cells were administered on day 0 and then again two days later). Supportive care therapies were administered as per site institutional guidelines.

[0105] The primary objective of the clinical trial was to assess the safety of the administered therapeutic compositions. Secondary objectives included assessment of grade 3 adverse eventsusing the NCI-CTCAEv5.0 grading system. See, Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0, Published: Nov. 27, 2017, U.S. Department of Health and Human Services, National Institutes of Health, National Cancer Institute, and Freites-Martinez et al., Using the Common Terminology Criteria for Adverse Events (CTCAEVersion 5.0) to Evaluate the Severity of Adverse Events of Anticancer Therapies. Actas Dermosifiliogr (Engl Ed). 2021 January; 112(1):90-92. Under this system, Grade 1 indicates that the adverse event (AE) is mild, Grade 2 is moderate, Grade 3 is severe, Grade 4 is life-threatening, and Grade 5 is fatal (death related to the AE). Oral/gastrointestinal adverse events of grade 3 that were assessed included oral mucositis, nausea, vomiting or diarrhea.

[0106] Twenty-nine human subjects with systemic lymphoma were treated with a median follow up of 271 days (range 179, 566). Adverse events were generally mild/moderate and of the type and magnitude expected with HDT-AHCT. No maximum tolerated dose was established through dosing up to 2010 6 cells/kg because the treatment was well tolerated.

[0107] The results of this Phase I study indicated that these therapeutic compositionsprepared from high density frozen E4ORF1+HUVECs using the methods and compositions described hereincould be safely administered to human subjects.

REFERENCE LIST

[0108] ATTC; Animal Cell Culture Guide; 2014 www.atcc.org

[0109] Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0, Published: Nov. 27, 2017, U.S. Department of Health and Human Services, National Institutes of Health, National Cancer Institute.

[0110] De Loecher et al., Effects of Cell Concentration on Viability and Metabolic Activity During Cryopreservation, 1998, Cryobiology, Vol. 37(2), p. 103-109.

[0111] Freites-Martinez et al., Using the Common Terminology Criteria for Adverse Events (CTCAEVersion 5.0) to Evaluate the Severity of Adverse Events of Anticancer Therapies. Actas Dermosifiliogr (Engl Ed). 2021 January; 112(1):90-92.

[0112] Lehle et al., Cryopreservation of human endothelial cells for vascular tissue engineering. Cryobiology 50 (2005) 154-161

[0113] Lehle et al., Identification and Reduction of Cryoinjury in Endothelial Cells: A First Step toward Establishing a Cell Bank for Vascular Tissue Engineering. TISSUE ENGINEERING Volume 12, Number 12, 2006

[0114] Lonza; Clonetics Endothelial Cell System; Technical Information & Instructions; www.lonza.com; 2018

[0115] Technical Information & Instructions

[0116] Marquez-Curtis et al., Beyond membrane integrity: Assessing the functionality of human umbilical vein endothelial cells after cryopreservation. Cryobiology 72 (2016) 183e190

[0117] Pegg., Cryopreservation of vascular endothelial cells as isolated cells and as monolayers. Cryobiology 44 (2002) 46-53

[0118] Polchow et al.: Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks. Journal of Translational Medicine 2012 10:98.

[0119] Puzanov et al.: New Approach to Cryopreservation of Primary Noncultivated Human Umbilical Vein Endothelium in Biobanking. BIOPRESERVATION AND BIOBANKING; Volume 16, Number 2, 2018

[0120] Sultani, A. B. et al. Improved Cryopreservation of Human Umbilical Vein Endothelial Cells: A Systematic Approach. Sci. Rep. 6, 34393; (2016).

[0121] Reardon et al. Investigating membrane and mitochondrial cryobiological responses ofHUVEC using interrupted cooling protocols. Cryobiology 71 (2015) 306-317

[0122] U.S. Pat. No. 8,465,732.

[0123] von Bomhard A. et al., (2016) Cryopreservation of Endothelial Cells in Various Cryoprotective Agents and MediaVitrification versus Slow Freezing Methods. PLoS ONE 11(2).

***

[0124] The present invention is further described by the following claims.