Engineered cellular pathways for programmed autoregulation of differentiation

• US 9,458,471 B2
• Filed: 02/06/2014
• Issued: 10/04/2016
• 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, Purnick, Priscilla, Schaniel, Christoph, Miller, Miles, Guye, Patrick
• Primary Examiner(s): Marvich, Maria
• Application Number:
  US14/174,475
• Publication Number:
  US 20140234957A1
• Time in Patent Office: 971 Days
• US Class Current: 1/1
• 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 recombinant mammalian vectors, wherein the one or more vectors comprise:

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

b) a second nucleic acid sequence comprising a second promoter that is operably linked to second DNA 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 nucleic acid sequence comprising a third promoter that is operably linked to a third DNA 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 nucleic acid sequence comprising a fourth promoter that is operably linked to a fourth DNA sequence encoding a first repressor, and

e) a fifth nucleic acid sequence comprising a fifth promoter that is operably linked to a fifth DNA 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.

2. A composition comprising one or more recombinant mammalian vectors, wherein the one or more vectors comprise:

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

b) a second nucleic acid sequence comprising a second promoter that is operably linked to second DNA 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 nucleic acid sequence comprising a third promoter that is operably linked to a third DNA 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 nucleic acid sequence comprising a fourth promoter that is operably linked to a fourth DNA sequence encoding a first repressor, and

e) a fifth nucleic acid sequence comprising a fifth promoter that is operably linked to a fifth DNA 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,

wherein said one or more vectors further comprises;

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

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

h) an eighth nucleic acid sequence comprising an eighth promoter that is operably linked to an eighth DNA sequence encoding a second activator,

i) a ninth nucleic acid sequence comprising a ninth promoter that is operably linked to a ninth DNA 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.

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

j) a tenth nucleic acid sequence comprising a tenth promoter that is operably linked to a tenth DNA sequence encoding a fifth repressor, and

k) an eleventh nucleic acid sequence comprising an eleventh promoter that is operably linked to an eleventh DNA 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.

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

l) a twelfth nucleic acid sequence comprising a twelfth promoter that is operably linked to a twelfth DNA 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.

5. The composition of claim 3, wherein said fifth repressor represses expression of said twelfth nucleic acid sequence.

6. A composition comprising one or more recombinant mammalian vectors, wherein the one or more vectors comprise:

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

b) a second nucleic acid sequence comprising a second promoter that is operably linked to second DNA 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 nucleic acid sequence comprising a third promoter that is operably linked to a third DNA 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 nucleic acid sequence comprising a fourth promoter that is operably linked to a fourth DNA sequence encoding a repressor, wherein said fourth promoter is a tissue specific promoter, and

e) a fifth nucleic acid sequence comprising a fifth promoter that is operably linked to a fifth DNA sequence encoding 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.