MODIFIED IPS CELLS HAVING A MUTANT FORM OF A HUMAN IMMUNODEFICIENCY VIRUS (HIV) CELLULAR ENTRY GENE
Methods and composition for generation of genetically modified induced pluripotent stem cells and hematopoietic cell derived therefrom are provided. For example, in certain aspects those cells comprise a modified gene structure related to HIV cellular entry, such as CCR5 mutants.
1 . An isolated induced pluripotent stem (iPS) cell having a modified gene structure comprising a mutant form of an HIV cellular entry gene selected from the group consisting of CCR5, CXCR4, CCR3, CCR2B, and CCR1, wherein said mutant form is a null genotype or encodes an inactive protein form.
2 . The isolated iPS cell of claim 1 , wherein said gene structure is modified by homologous recombination or nuclease targeting.
3 . The isolated iPS cell of claim 1 , wherein said modified gene structure comprises a CCR5 null genotype or a CCR5 mutant encoding an inactive form of CCR5 protein.
4 . The isolated iPS cell of claim 3 , wherein said CCR5 mutant is a 32 base-pair deletion in the coding region of wild-type CCR5 (CCR5 delta32).
5 . The isolated iPS cell of claim 3 , wherein said CCR5 mutant is a CCR5m303 mutant.
6 . The isolated iPS cell of claim 3 , wherein said isolated iPS cell is homozygous for said CCR5 mutant.
7 . The isolated iPS cell of claim 3 , wherein said iPS cell has CCR5 delta32 and CCR5m303 mutants.
8 . An isolated modified hematopoietic cell differentiated from the isolated iPS cell of claim 1 .
9 . The isolated iPS cell of claim 8 , wherein said isolated hematopoietic cell is a hematopoietic stem cell, a hematopoietic progenitor cell, a T lymphocyte, a B lymphocyte, a mast cell, or a macrophage.
10 . An in vitro method for making a modified iPS cell, comprising:
a) obtaining a somatic cell, wherein said somatic cell is from a subject having or at risk of having an HIV infection or disorder;
b) reprogramming said somatic cell to provide an induced pluripotent stem cell (iPS cell); and
c) modifying said iPS cell to provide a modified iPS cell having a modified gene structure comprising a mutant form of an HIV cellular entry gene selected from the group consisting of CCR5, CXCR4, CCR3, CCR2B, and CCR1.
11 . The in vitro method of claim 10 , further comprising: d) inducing differentiation of said modified iPS cell to provide a modified hematopoietic cell.
12 . The in vitro method of claim 10 , wherein said modifying comprises homologous recombination or nuclease targeting.
13 . The in vitro method of claim 10 , wherein said modified gene structure is introduced into said iPS cell by a vector.
14 . The in vitro method of claim 10 , wherein said modified gene structure comprises a CCR5 null genotype or a CCR5 mutant encoding an inactive form of CCR5.
15 . The in vitro method of claim 14 , wherein said modifying comprises replacing one or two endogenous CCR5 alleles with said CCR5 mutant.
16 . The in vitro method of claim 14 , wherein said modifying comprises replacing two endogenous CCR5 alleles with said CCR5 mutant.
17 . The in vitro method of claim 14 , wherein said CCR5 mutant is a 32 base-pair deletion in the coding region of wild-type CCR5 (CCR5 delta32).
18 . The in vitro method of claim 14 , wherein said CCR5 mutant is a CCR5m303 mutant.
19 . The in vitro method of claim 14 , wherein said modified iPS cell is homozygous for said CCR5 mutants.
20 . The in vitro method of claim 14 , wherein said modified iPS cell has CCR5 delta32 and CCR5m303 mutant.
21 . The in vitro method of claim 10 , wherein said subject is human.