Engineered cellular pathways for programmed autoregulation of differentiation

• US 8,685,720 B2
• Filed: 11/01/2007
• Issued: 04/01/2014
• 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, Lemischka, Ihor, Schaniel, Christoph, Miller, Miles, Guye, Patrick, Purnick, Priscilla E. M.
• Primary Examiner(s): Marvich, Maria
• Application Number:
  US12/312,197
• Publication Number:
  US 20100285584A1
• Time in Patent Office: 2,343 Days
• Field of Search: None
• US Class Current: 435/320.1
• 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, wherein the one or more vectors comprise:

a) a first nucleotide sequence comprising a promoter operably linked to a nucleic acid sequence encoding a first cell-cell communication molecule,

b) a second nucleotide sequence comprising a promoter operably linked to a nucleic acid sequence encoding a second cell-cell communication molecule that binds with said first cell-cell communication molecule to produce a first binding molecule,

c) a third nucleotide sequence comprising a promoter operably linked to a nucleic acid sequence encoding a first cell fate regulator protein that induces differentiation of a first cell type into a second cell type that expresses a marker protein of said second cell type,

d) a fourth nucleotide sequence comprising a promoter operably linked to a nucleic acid sequence encoding a first repressor,

e) a fifth nucleotide sequence comprising a promoter operably linked to a nucleic acid sequence encoding 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,

f) a sixth nucleotide sequence comprising a promoter operably linked to a nucleic acid sequence encoding a second repressor,

g) a seventh nucleotide sequence comprising a promoter operably linked to a nucleic acid sequence encoding a third repressor,

h) a eight nucleotide sequence comprising a promoter operably linked to a nucleic acid sequence encoding a second activator,

i) a ninth nucleotide sequence comprising a promoter operably linked to a nucleic acid sequence encoding 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,

j) a tenth nucleotide sequence comprising a promoter operably linked to a nucleic acid sequence encoding a fifth repressor,

k) an eleventh nucleotide sequence comprising a promoter operably linked to a nucleic acid sequence encoding 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,

1) a twelfth nucleotide sequence comprising a promoter operably linked to a nucleic acid sequence encoding 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, and

m) a thirteenth nucleotide sequence comprising a promoter operably linked to a nucleic acid sequence encoding a second cell fate regulator protein that is operably linked to a cell type specific promoter of said second cell type, wherein the second regulator induces 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 wherein said fifth repressor represses expression of said twelfth nucleic acid sequence, and wherein said first cell-cell communication molecule and said second cell-cell communication molecule are selected from the group consisting of,

(1) the first cell-cell communication molecule comprising LuxI and the second cell-cell communication molecule comprising LuxR,

(2) the first cell-cell communication molecule comprising LuxR and the second cell-cell communication molecule comprising LuxI,

(3) the first cell-cell communication molecule comprising CinI and the second cell-cell communication molecule comprising CinR,

(4) the first cell-cell communication molecule comprising CinR and the second cell-cell communication molecule comprising CinI,

(5) the first cell-cell communication molecule comprising RhlI and the second cell-cell communication molecule comprising RhlIR, and

(6) the first cell-cell communication molecule comprising RhlIR and the second cell-cell communication molecule comprising RhlI.

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

n) a fourteenth nucleic acid sequence encoding a third cell fate regulator protein that is operably linked to a cell type specific promoter of said second cell type, that induces 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 cell fate regulator protein and said third cell fate regulator protein.

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

o) a fifteenth nucleic acid sequence encoding a third cell-cell communication molecule, wherein said sixth repressor represses expression of said third cell-cell communication molecule, and

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

4. The composition of claim 3, wherein said second binding molecule induces expression of said second activator.

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

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