Homologous recombination in multipotent adult progenitor cells

Patent metadata

Field	Value
Publication number	US-2018272000-A1
Application number	US-201715709144-A
Country	US
Kind code	A1
Filing date	Sep 19, 2017
Priority date	Nov 27, 2002
Publication date	Sep 27, 2018
Grant date	—

Abstract

The invention relates to methods of altering gene expression by homologous recombination in a multipotent adult progenitor cell (MAPC). In particular, methods of producing a recombinant MAPC, of correcting a genetic defect in a mammal, of providing a functional and/or therapeutic protein to a mammal, and of transforming a MAPC are provided. MAPCs containing an erogenous DNA as well as recombinant MAPCs and their differentiated progeny are also provided.

First claim

1 - 20 . (canceled)

21 . A method of altering gene expression in a cell, the method comprising:
a) introducing, by nucleoporation, a polynucleotide into isolated expanded human non-embryonic, non-germ cells, with a transfection efficiency up to 14% and without toxicity to the cells, the cells having undergone at least 10-40 cell doublings in culture, wherein the cells are multipotent adult progenitor cells characterized in that they are non-embryonic, non-germ cells that express telomerase, are not transformed, and have a normal karyotype, the polynucleotide comprising a sequence of interest; and b) culturing the nucleoporated cells produced in step a) under conditions sufficient to express the sequence of interest, such that the resulting cells have altered gene expression.

22 . The method of claim 21 wherein the polynucleotide also comprises a sequence homologous to a genomic DNA sequence in the cells and the polynucleotide is homologously recombined in the cells.

23 . The method of claim 21 or 22 wherein the non-embryonic, non-germ cells further express one or more of oct4, rex-1, rox-1, or sox-2.

24 . The method of claim 21 or 22 wherein the non-embryonic, non-germ cells can differentiate into at least one cell type of at least two of the endodermal, ectodermal, and mesodermal embryonic lineages.

25 . The method of claim 24 wherein the non-embryonic, non-germ cells further express one or more of oct4, rex-1, rox-1, or sox-2.

26 . The method of claim 24 wherein the non-embryonic, non-germ cells can differentiate into at least one cell type of each of the endodermal, ectodermal, and mesodermal embryonic lineages.

27 . The method of claim 26 wherein the non-embryonic, non-germ cells further express one or more of oct4, rex-1, rox-1, or sox-2.

28 . The method of claim 21 or 22 , further comprising combining the non-embryonic, non-germ cells produced in step b) with a pharmaceutically-acceptable carrier.

29 . The method of claim 21 or 22 , wherein the non-embryonic, non-germ cells are cultured at a density of about 500 cells/cm 2 -1500 cells/cm 2 .

30 . The method of claim 29 , wherein the non-embryonic, non-germ cells are cultured at a density of about 500 cells/cm 2 .

31 . The method of any of claims 21 - 30 , wherein the non-embryonic, non-germ cells are isolated from bone marrow.

Assignees

• Verfaillie Catherine M
• Lakshmipathy Uma
• Abt Holding Co
• Univ Minnesota

Inventors

• Verfaillie Catherine M
• Lakshmipathy Uma

Classifications

• A61K48/005 Primary

  characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered · CPC title

• C12N15/1082

  Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors · CPC title