ENGINEERED CELLULAR PATHWAYS FOR PROGRAMMED AUTOREGULATION OF DIFFERENTIATION

• US 20100285584A1
• Filed: 11/01/2007
• Published: 11/11/2010
• Est. Priority Date: 11/03/2006
• Status: Active Grant

• Current Assignee: The Trustees of Princeton University (Princeton University)
• Original Assignee: The Trustees of Princeton University (Princeton University)
• Inventors: Weiss, Ron, Schaniel, Christoph, Purnick, Priscilla, Guye, Patrick, Miller, Miles, Lemischka, Ihor
• Granted Patent: US 8,685,720 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2800/40 Systems of functionally co-operating vectors
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/003 tet inducible
• CPC Class Codes: C12N 2830/005 repressible enhancer/promoter combination, e.g. KRAB
• CPC Class Codes: C12N 2830/006 tet repressible
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/15 chimeric enhancer/promoter combination
• CPC Class Codes: C12N 2830/55 from bacteria
• CPC Class Codes: C12N 2830/85 mammalian

Abstract

The present invention provides compositions and methods for programming mammalian cells to perform desired functions. In particular, the present invention provides compositions and methods for programming stem cells to differentiate into a desired cell type. A quorum sensing systems that regulates the expression of cell fate regulators is introduced into mammalian host cells, such as stem cells. The quorum sensing systems generally comprises vectors that express the components of a bacterial quorum sensing pathway, including proteins which catalyze the synthesis of an autoinducer and a gene encoding a regulatory partner of the autoinducer, and vectors in which genes encoding cell fate regulators are operably linked to a promoter induced by the autoinducer/regulatory partner complex. The system can also comprise vectors in which genes encoding additional cell fate regulators are operably linked to a promoter that is induced by a factor synthesized in response to a first stage of differentiation, so that a second stage of differentiation is triggered.

Claims

1. A composition comprising one or more mammalian vectors that comprise:

a) a first nucleic acid sequence capable of producing a first cell-cell communication molecule,

b) a second nucleic acid sequence capable of producing a second cell-cell communication molecule that is capable of binding with said first cell-cell communication molecule to produce a first binding molecule,

c) a third nucleic acid sequence capable of producing a third cell-cell communication molecule, and

d) a fourth nucleic acid sequence capable of producing a fourth cell-cell communication molecule that is capable of binding with said first cell-cell communication molecule to produce a second binding molecule.

2. The composition of claim 1, wherein said one or more vectors further comprises:

a fifth nucleic acid sequence capable of producing a first repressor, and

a sixth nucleic acid sequence capable of producing a first activator, wherein

i) said first repressor protein represses expression of said first activator, and

ii) said first activator activates expression of

1) said first repressor, and

2) said first activator.

3. The composition of claim 2, wherein said one or more vectors further comprises:

e) a seventh nucleic acid sequence capable of producing a second repressor,

f) a eighth nucleic acid sequence capable of producing a third repressor,

g) a ninth nucleic acid sequence capable of producing a second activator,

h) an tenth nucleic acid sequence capable of producing a fourth repressor, wherein

i) said second repressor represses expression of said third repressor,

ii) said third repressor represses expression of said second activator,

iii) said second activator activates expression of said fourth repressor, and

iv) said first activator activates expression of said second repressor.

4. The composition of claim 3, wherein said one or more vectors further comprises:

i) a eleventh nucleic acid sequence capable of producing a fifth repressor, and

j) a twelfth nucleic acid sequence capable of producing a sixth repressor, wherein

i) said fifth repressor represses expression of said sixth repressor and of said first cell-cell communication molecule,

ii) said sixth repressor represses expression of said fifth repressor,

iii) said fourth repressor represses expression of said fifth repressor,

iv) said second binding molecule induces expression of said second activator, and

v) said sixth repressor represses expression of said third cell-cell communication molecule.

5. The composition of claim 4, wherein said one or more vectors further comprises:

k) a thirteenth nucleic acid sequence capable of producing a seventh repressor, wherein

i) said first binding molecule induces expression of said seventh repressor, and

ii) said seventh repressor represses expression of said fourth repressor.

6. The composition of claim 5, wherein said one or more vectors further comprises:

l) a fourteenth nucleic acid sequence capable of producing a protein that inhibits cell growth, wherein said second binding molecule induces expression of said protein that inhibits cell growth.

7. The composition of claim 6, wherein said one or more vectors further comprises:

m) a fifteenth nucleic acid sequence capable of producing a first cell fate regulator protein that is capable of inducing differentiation of a first cell type into a second cell type that expresses a marker protein of said second cell type, wherein said fifth repressor represses expression of said first cell fate regulator protein.

8. The composition of claim 7, wherein said one or more vectors further comprises:

n) a sixteenth nucleic acid sequence capable of producing a second cell fate regulator protein that is operably linked to a cell type specific promoter of said second cell type, and that is capable of inducing differentiation of said second cell type into a third cell type, wherein expression of said first cell fate regulator protein causes expression of said second cell fate regulator protein.

9. The composition of claim 8, wherein said one or more vectors further comprises:

o) a seventeenth nucleic acid sequence capable of producing a third cell fate regulator protein that is operably linked to a cell type specific promoter of said second cell type, and that is capable of inducing differentiation of said second cell type into said third cell type, wherein expression of said first cell fate regulator protein causes expression of said second and third cell fate regulator proteins.

10. The composition of claim 1, wherein said one or more vectors further comprises:

e) a fifth nucleic acid sequence capable of producing a repressor and operably linked to a tissue specific promoter, and

f) a sixth nucleic acid sequence capable of producing an apoptosis protein, wherein expression of said repressor represses expression of said apoptosis protein.

94. A method for altering the number of differentiable cells and the number of differentiated cells in a cell population, comprising:

a) providing;

i) a population comprising mammalian differentiable cells and differentiated cells, and

ii) the composition of claim 1, wherein said first, second, third and fourth cell-cell communication molecules are exogenous to said differentiable and differentiated mammalian cells, and

b) treating said population with said composition under conditions such that said one or more vectors are introduced into said differentiable cells and said differentiated cells to produce a population of transgenic cells comprising transgenic differentiable cells and transgenic differentiated cells, wherein

i) a decrease in the number of said transgenic differentiable cells in said transgenic population causes an increase in the proliferation of said transgenic differentiable cells,

ii) an increase in the number of said transgenic differentiable cells in said transgenic population causes a decrease in the proliferation of said transgenic differentiable cells,

iii) a decrease in the number of said transgenic differentiated cells in said transgenic population causes

1) an increase in the differentiation of said transgenic differentiable cells, and

2) an increase in proliferation of said transgenic differentiable cells, and

iv) an increase in the number of said transgenic differentiated cells in said transgenic population causes a decrease in the differentiation of said transgenic differentiable cells.

125. A mammalian cell produced by the method of claim 94.

11-27. -27. (canceled)

28. A composition comprising one or more mammalian vectors that comprise:

a) a first nucleic acid sequence capable of producing a first cell-cell communication molecule,

b) a second nucleic acid sequence capable of producing a second cell-cell communication molecule that is capable of binding with said first cell-cell communication molecule to produce a first binding molecule, and

c) a third nucleic acid sequence capable of producing a first cell fate regulator protein that is capable of inducing differentiation of a first cell type into a second cell type that expresses a marker protein of said second cell type.

29. The composition of claim 28, wherein said one or more vectors further comprises:

d) a fourth nucleic acid sequence capable of producing a first repressor, and

e) a fifth nucleic acid sequence capable of producing a first activator, wherein

i) said first repressor represses expression of said first activator, and

ii) said first activator activates expression of;

1) said first repressor, and

2) said first activator.

30. The composition of claim 29, wherein said one or more vectors further comprises:

f) a sixth nucleic acid sequence capable of producing a second repressor,

g) a seventh nucleic acid sequence capable of producing a third repressor,

h) a eight nucleic acid sequence capable of producing a second activator,

i) a ninth nucleic acid sequence capable of producing a fourth repressor, wherein

i) said second repressor represses expression of said third repressor,

ii) said third repressor represses expression of said second activator,

iii) said second activator activates expression of said fourth repressor, and

iv) said first activator activates expression of said second repressor.

31. The composition of claim 30, wherein said one or more vectors further comprises:

j) a tenth nucleic acid sequence capable of producing a fifth repressor, and

k) an eleventh nucleic acid sequence capable of producing a sixth repressor, wherein

i) said fifth repressor represses expression of said sixth repressor and of said first cell-cell communication molecule,

ii) said sixth repressor represses expression of said fifth repressor, and

iii) said fourth repressor represses expression of said fifth repressor.

32. The composition of claim 31, wherein said one or more vectors further comprises:

l) a twelfth nucleic acid sequence capable of producing a seventh repressor, wherein

i) said first binding molecule induces expression of said seventh repressor, and

ii) said seventh repressor represses expression of said fourth repressor.

33. The composition of claim 31, wherein said fifth repressor represses expression of said twelfth nucleic acid sequence.

34. The composition of claim 32, wherein said one or more vectors further comprises:

m) a thirteenth nucleic acid sequence capable of producing a second cell fate regulator protein that is operably linked to a cell type specific promoter of said second cell type, and that is capable of inducing differentiation of said second cell type into a third cell type, wherein expression of said first cell fate regulator protein causes expression of said second cell fate regulator protein and differentiation of said second cell type into said third cell type.

35. The composition of claim 34, wherein said one or more vectors further comprises:

n) a fourteenth nucleic acid sequence capable of producing a third cell fate regulator protein that is operably linked to a cell type specific promoter of said second cell type, that is capable of inducing differentiation of said second cell type into said third cell type, wherein expression of said first cell fate regulator protein causes expression of said second and third cell fate regulator proteins.

36. The composition of claim 35, wherein said one or more vectors further comprises:

o) a fifteenth nucleic acid sequence capable of producing a third cell-cell communication molecule, and

p) a sixteenth nucleic acid sequence capable of producing a fourth cell-cell communication molecule that is capable of binding with said first cell-cell communication molecule to produce a second binding molecule.

37. The composition of claim 36, wherein said second binding molecule induces expression of said second activator, and said sixth repressor represses expression of said third cell-cell communication molecule.

38. The composition of claim 36, wherein said one or more vectors further comprises:

q) a seventeenth nucleic acid sequence capable of producing a protein that inhibits cell growth, wherein said second binding molecule induces expression of said protein that inhibits cell growth.

39. The composition of claim 28, wherein said one or more vectors further comprises:

d) a fourth nucleic acid sequence capable of producing a repressor and operably linked to a tissue specific promoter,

e) a fifth nucleic acid sequence capable of producing an apoptosis protein, wherein expression of said repressor represses expression of said apoptosis protein, and wherein the absence of expression of said repressor induces expression of said apoptosis protein.

106. A method for altering the number of differentiated cells in a cell population, comprising:

a) providing;

i) a population comprising mammalian differentiable cells and differentiated cells, and

ii) the composition of claim 28, wherein said first and second cell-cell communication molecules are exogenous to said differentiable and differentiated mammalian cells, and

b) treating said population with said composition under conditions such that said one or more vectors are introduced into said differentiable cells and said differentiated cells to produce a population of transgenic cells comprising transgenic differentiable cells and transgenic differentiated cells, wherein

i) a decrease in the number of said transgenic differentiated cells in said transgenic population causes an increase in the differentiation of said transgenic differentiable cells, and

ii) an increase in the number of said transgenic differentiated cells in said transgenic population causes a decrease in the differentiation of said transgenic differentiable cells.

126. A mammalian cell produced by the method of claim 106.

40-62. -62. (canceled)

63. A composition comprising one or more mammalian vectors that comprise:

a) a first nucleic acid sequence capable of producing a first cell-cell communication molecule,

b) a second nucleic acid sequence capable of producing a second cell-cell communication molecule that is capable of binding with said first cell-cell communication molecule to produce a binding molecule, and

c) a third nucleic acid sequence capable of producing a protein that inhibits cell growth, wherein said binding molecule induces expression of said second cell-cell communication molecule and of said protein that inhibits cell growth.

64-93. -93. (canceled)

95-105. -105. (canceled)

107-124. -124. (canceled)