Gene therapy for recessive dystrophic epidermolysis bullosa using genetically corrected autologous keratinocytes

Patent metadata

Field	Value
Publication number	US-2024067926-A1
Application number	US-202318501837-A
Country	US
Kind code	A1
Filing date	Nov 3, 2023
Priority date	Jan 4, 2016
Publication date	Feb 29, 2024
Grant date	—

Abstract

Methods are provided for the cell-based delivery of collagen VII for the treatment of Epidermolysis Bullosa and corneal erosion. The disclosure also provides a composition and a pharmaceutical composition comprises, comprise, or alternatively consist essentially of, or yet further consist of a keratinocyte sheet or a corneal cell sheet.

First claim

1 . A method for treating Recessive Dystrophic Epidermolysis Bullosa (RDEB) in a human patient having one or more mutations in both copies of a human collagen VII A1 (Col7A1) gene and suffering from RDEB, the method comprising:
(a) subjecting the human patient to one or more tests selected from replication competent retrovirus (RCR), COL7A1-sensitive cytotoxic T-cells, or a combination thereof; (b) obtaining from the human patient a population of skin cells comprising keratinocytes; (c) isolating a population of keratinocytes comprising the one or more mutations in the co/7A1 gene from the population of skin cells; (d) transducing the isolated population of keratinocytes ex vivo with a retrovirus comprising a genetic construct encoding a functional human collagen VII (COL7A1) protein to generate a population of genetically corrected keratinocytes that meet specified pre-release criteria for viral transduction efficiency (VTE) and proviral genome copy number (PGCN); (e) culturing the genetically corrected keratinocytes in a first and a second culture media to form an autologous COL7A1 corrected keratinocyte sheet; (f) subjecting the autologous corrected keratinocyte sheet to one or more pre-release tests selected from a group consisting of VTE test, PGCN test, and replication competent retrovirus (RCR) test; and (a) transplanting a graft of the autologous corrected keratinocyte sheet to a skin wound bed of the human patient.

2 .- 3 . (canceled)

4 . The method of claim 1 , wherein the retrovirus is a LZRSE-virus or a Gibbon Ape Leukemia Virus (GALV)-pseudotyped virus.

5 . The method of claim 1 , wherein the genetic construct comprises nucleotides 409 to 11849 of SEQ ID NO: 1 or nucleotides 1001 to 11157 of SEQ ID NO: 1.

6 . The method of claim 1 , wherein the specified pre-release criteria for viral transduction efficiency (VTE) is >50% and the proviral genome copy number (PGCN) is less than 1.5.

7 . The method of claim 1 , wherein the wound is free of non-corrected wound bed keratinocytes.

8 . The method of claim 1 , further comprising cauterizing the wound bed of the human patient to ablate the non-corrected wound bed keratinocytes prior to transplanting the graft of the autologous corrected keratinocyte sheet.

9 .- 10 . (canceled)

11 . The method of claim 1 , wherein the keratinocyte sheet is subject to one or more tests selected from a group consisting of sterility test, endotoxin test, mycoplasma test, gram stain sterility test, LEAES viability test, cytotoxic T cell assay, anti-C7 LH24 mAb characterization, electron microscopy, immuno-electron microscopy, immunofluorescence staining, C7 expression, and AF analysis.

12 . The method of claim 1 , wherein the one or more pre-release tests are RCR test.

13 . The method of claim 1 , wherein the keratinocyte sheet is placed on an acellular matrix, a collagen matrix, or a biocompatible mesh.

14 . The method of claim 13 , wherein the biocompatible mesh is made of thermoplastic resin, polyethylene, ultra-high molecular weight polyethylene, high molecular weight polyolefin, uncoated monofilament polypropylene, polyether ether ketone, polyethylene terephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, nylon, silicon, or any combination thereof.

15 . (canceled)

16 . The method of claim 1 , wherein the isolated population of skin cells comprises stem cells.

17 . The method of claim 16 , further comprising differentiating the stem cells to keratinocytes.

18 - 67 . (canceled)

68 . The method of claim 1 further comprising subjecting the transplanted human patient to one or more tests selected from RCR test, COL7A1-sensitive cytotoxic T-cell assay, or a combination thereof.

69 . The method of claim 68 , wherein a blood sample from the transplanted patient is subjected to one or more tests selected from RCR test, COL7A1-sensitive cytotoxic T-cell assay, or a combination thereof at about 1, 3, 6, or 12 months after transplantation.

70 . The method of claim 1 , wherein the retrovirus is selected from Moloney murine leukemia virus (MoMLV), human immunodeficiency virus (HIV), AKR murine leukemia virus (AKR), feline immunodeficiency virus (FIV), or avian leukosis virus (ALV).

71 . The method of claim 1 , wherein the retrovirus is a MoMLV.

72 . The method of claim 1 , wherein the genetic construct further comprises a promoter driving COL7A1 expression.

73 . The method of claim 72 , the promoter is a MoMLV LTR promoter.

74 . The method of claim 72 , wherein the retrovirus is a MoMLV and the promoter is a MoMLV LTR promoter.

75 . The method of claim 1 , wherein the genetic construct comprises nucleotides 1001 to 11157 of SEQ ID NO: 1.

76 . The method of claim 6 , wherein the average PGCN is less than 1.5, 1, or 0.5.

77 . The method of claim 6 , wherein the average PGCN is less than or equal to 1.5.

78 . The method of claim 6 , wherein the average PGCN is greater than or equal to 0.5 and less than or equal to 1.5.

79 . The method of claim 1 , wherein the first culture medium comprises a keratinocyte medium.

80 . The method of claim 1 , wherein the second culture medium comprises Dulbecco's Modified Eagle Medium, F12 medium, and/or at least 10% fetal bovine serum.

81 . The method of claim 1 , wherein transducing the isolated population of keratinocytes ex vivo is repeated at least twice.

Assignees

• Univ Leland Stanford Junior
• Us Gov Veterans Affairs

Inventors

• Siprashvili Zurab
• Nguyen Ngon T
• Marinkovich M Peter
• Tang Jean
• Lane Alfred T
• Khavari Paul A

Classifications

• C12N5/0629 Primary

  Keratinocytes; Whole skin · CPC title

• A61K48/0058 Primary

  Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct · CPC title

• A61K48/0075

  characterised by an aspect of the delivery route, e.g. oral, subcutaneous · CPC title

• A61K48/0091

  Purification or manufacturing processes for gene therapy compositions · CPC title

• C12N2510/00

  Genetically modified cells · CPC title