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

Patent metadata

Field	Value
Publication number	US-2019382724-A1
Application number	US-201716066253-A
Country	US
Kind code	A1
Filing date	Jan 3, 2017
Priority date	Jan 4, 2016
Publication date	Dec 19, 2019
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 Epidermolysis Bullosa (EB) in a subject, the method comprising:
obtaining from the subject a population of skin cells; correcting the skin cells ex vivo by integration of a genetic construct encoding a full-length wild-type human collagen VII (COL7A1) protein; culturing the genetically corrected cells to form a keratinocyte sheet; and transplanting a graft of the keratinocyte sheet to a skin wound bed; wherein the skin cells are corrected by transduction with a virus comprising the genetic construct; wherein the keratinocyte sheet has an average proviral genome copy number (PGCN) no more than 3, and optionally less than 2, 1.5, 1, or 0.5.

2 . (canceled)

3 . The method of claim 1 , wherein the virus comprises retrovirus, AAV (adeno-associated virus), or lentivirus.

4 . The method of claim 3 , wherein the retrovirus is a LZRSE-virus.

5 . The method of claim 3 , wherein the retrovirus is GaIV-pseudotyped.

6 . The method of claim 1 , wherein the skin cells thus transduced meet pre-release criteria of virus transduction efficiency (VTE)>50%.

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

8 . The method of of claim 1 , wherein the wound is treated to ablate the non-corrected wound bed keratinocytes.

9 . The method of claim 1 , wherein the subject suffers from Recessive Dystrophic Epidermolysis Bullosa (RDEB).

10 . The method of claim 1 , wherein the subject is human.

11 . The method of claim 1 , wherein the keratinocyte sheet is subject to one or more tests selected from a group consisting of VTE test, PGCN test, sterility test, endotoxin test, mycoplasma test, gram stain sterility test, LEAES viability test, post-release test, RCR 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 11 , wherein the immunofluorescence staining comprises direct or indirect immunofluorescence staining.

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 . The method of claim 1 , wherein the skin cells comprise keratinocytes.

16 . The method of claim 1 , wherein the skin cells comprise stem cells.

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

18 . A method for treating corneal erosion in a subject, the method comprising:
obtaining from the subject a population of corneal cells; correcting the corneal cells ex vivo by integration of a genetic construct encoding full-length wild-type human collagen VII (COL7A1) protein; culturing the genetically corrected cells to form a corneal cell sheet; and transplanting a graft of the corneal cell sheet to a corneal surface.

19 - 42 . (canceled)

43 . A pharmaceutical composition for treating EB in a subject, the pharmaceutical composition comprising a keratinocyte sheet that comprises a population of skin cells transduced with a virus comprising a genetic construct encoding a full-length wild-type COL7A1 protein, wherein the keratinocyte sheet has an average PGCN no more than 3, and optional less than 2, 1.5, 1, or 0.5.

44 . A pharmaceutical composition of claim 43 , wherein the skin cells are differentiated from stem cells ex vivo integrated with the genetic construct encoding the functional COL7A1 protein.

45 . The pharmaceutical composition of claim 43 , wherein the skin cells are obtained from the subject.

46 . The pharmaceutical composition of claim 43 , wherein the subject suffers from RDEB.

47 . The pharmaceutical composition of claim 43 , wherein the subject is human.

48 - 67 . (canceled)

Assignees

• Univ Leland Stanford Junior
• U S Government As Represented By The Dept Of Veterans Affairs

Inventors

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

Classifications

• A61P27/02

  Ophthalmic agents · CPC title

• A61P17/00

  Drugs for dermatological disorders · CPC title

• C12N2760/16043

  viral genome or elements thereof as genetic vector · 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

• C12N5/0629 Primary

  Keratinocytes; Whole skin · CPC title