US2013217117A1PendingUtilityA1
Pluripotent stem cells obtained by non-viral reprogramming
Est. expiryOct 24, 2028(~2.3 yrs left)· nominal 20-yr term from priority
C12N 2501/605C12N 2501/603C12N 2501/606C12N 5/0696C12N 2501/602C12N 2510/00C12N 2501/604C12N 2501/608C12N 15/85
60
PatentIndex Score
0
Cited by
0
References
0
Claims
Abstract
Methods for reprogramming primate somatic cells to pluripotency using an episomal vector that does not encode an infectious virus are disclosed. Pluripotent cells produced in the methods are also disclosed.
Claims
exact text as granted — not AI-modifiedWe claim:
1 . A population of pluripotent cells produced according to a method comprising the steps of:
introducing into a primate somatic cell at least one non-viral episomal vector that encodes at least one potency-determining factor under conditions sufficient to reprogram the cell into a pluripotent cell, the at least one vector encoding at least potency-determining factors OCT4 and SOX2, wherein the cell expresses the introduced OCT4 and SOX2 and further comprises SV40 T Antigen; and culturing the pluripotent cell until it divides into a population of pluripotent cells substantially free of any vector component associated with exposing the potency-determining factors to the somatic cell.
2 . The population of claim 1 , where the primate somatic cells are obtained from a fetal or post-natal primate individual.
3 . The population of claim 2 , wherein the primate is human.
4 . The population of claim 1 , wherein the pluripotent cells are genetically identical to a post-natal individual.
5 . The population of claim 1 , wherein at least one vector further encodes at least one potency-determining factor selected from the group consisting of LIN28, NANOG, c-Myc, and KLF4.
6 . The population of claim 1 , wherein a single vector encodes at least OCT4 and SOX2 and comprises at least one operably-positioned internal ribosome entry site.
7 . The population of claim 6 , wherein the single vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, SV40 T antigen, IRES2, and KLF4.
8 . The population of claim 6 , wherein the vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, c-Myc, IRES2, KLF4, a third promoter, NANOG, IRES2, and LIN28.
9 . The population of claim 1 , wherein a first vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, SV40 T antigen, IRES2, and KLF4, wherein a second vector comprises, in order, a third promoter, OCT4, IRES2, SOX2, a fourth promoter, NANOG, IRES2, and KLF4, and wherein a third vector comprises, in order, a fifth promoter, c-Myc, IRES 2, and LIN28, wherein the promoters need not be identical.
10 . The population of claim 9 , wherein each of the first, second, third, and fourth promoters is an elongation factor 1α (EF1α) gene promoter.
11 . The population of claim 1 , wherein a first vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, KLF4, IRES2, c-Myc, a third promoter, NANOG, IRES2, and LIN28, wherein a second vector comprises, in order, a fourth promoter, OCT4, IRES2, SOX2, a fifth promoter, SV40 T antigen, IRES2, and KLF4, wherein the promoters need not be identical.
12 . The population of claim 11 , wherein each of the first, third, fourth, and fifth promoters is an EF1α gene promoter and wherein the second promoter is a cytomegalovirus immediate early gene (CMV) promoter.
13 . The population of claim 1 wherein a first vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, NANOG, IRES2, and LIN28, wherein a second vector comprises, in order, a third promoter, OCT4, IRES2, SOX2, a fourth promoter, SV40 T antigen, IRES2, and KLF4, and wherein a third vector comprises, in order, a fifth promoter, OCT4, IRES2, SOX2, a sixth promoter, c-Myc, IRES 2, and KLF4, wherein the promoters need not be identical.
14 . The population of claim 1 , wherein at least one vector encodes a plurality of factors and at least one vector comprises at least one internal ribosomal entry site.
15 . The population of claim 1 , wherein OCT4 and SOX2 are human.
16 . The population of claim 1 , wherein LIN28, NANOG, c-Myc, and KLF4 are human.
17 . A population of pluripotent cells substantially cured of exogenously introduced vector components.
18 . The population of claim 17 , wherein the cells are primate cells.
19 . The population of claim 18 , wherein the primate cells are human cells.
20 . The population of claim 17 , wherein the human cells are genetically identical to cells of a post-natal primate individual.
TABLE 1
Reprogramming genes and translation elements
Accession # or
Gene Symbol
Abbr.
Source
SEQ ID NO
sequence
OCT4
O
hESC
1
NM_002701
SOX2
S
hESC
2
NM_003106
NANOG
N
hESC
3
NM_024865
LN28
L
hESC
4
NM_024674
c-Myc
M
hESC
5
NM_002467
KLF4
K
hESC
6
NM_004235
SV40 T
T
pBABE-puro
7
EF579667
SV40 LT p
TERT
TERT
pBABE-hygro-
8
NM_198253
hTERT
IRES1
1
pIRESpuro3
—
IRES2
2
pIRES2EGFP
—
F2A
F2A
(synthesized)
9
CMV
C
10
EF1α
E
11
EF2α
—
12
TABLE 2
Episomal constructs
#
Name
Size (bp)
1
pCEP4-EGFP
10984
2 b
pEP4-E-O2S(2)
13523
3 b
pEP4-E-M2K
14293
4 a
pCEP4-M2K
13643
5 b
pEP4-E-K2M
14268
6 a
pCEP4-K2M
13636
7 b
pEP4-E-N2K
13819
8 b
pEP4-E-T2K
15071
9 a
pCEP4-M2L
12852
10 b
pEP4-E-N2L
13020
11 b
pEP4-E-T2L
14284
12 c
pEP4-E-O2S-C-M2K
16038
13 c
pEP4-E-O2S-E-M2K
16680
14 c
pEP4-E-O2S-C-K2M
16010
15 c
pEP4-E-O2S-E-K2M
16652
16 c
pEP4-E-O2S-E-N2K
16206
17 c
pEP4-E-O2S-E-T2K
17458
18 c
pEP4-E-O2S-E-N2L
15415
19 c
pEP4-E-O2S-E-T2L
16679
20 c
pEP4-O2S-C-M2L
15247
21 c
pEP4-E-O2S-E-K2T
17474
22 c
pEP4-E-O2S-C-M2L-E-N2K
19956
23 c
pEP4-E-O2S-C-M2K-E-N2L
19956
24 c
pEP4-E-O2S-C-K2M-E-N2L
19949
25 c
pEP4-E-O2S-C-M2L-E-T2K
21220
26 c
pEP4-E-O2S-C-M2K-E-T2L
21220
27 c
pEP4-E-O2S-C-K2M-E-T2L
21213
28 c
pEP4-E-O2S-C-M2L-E-K2T
21224
a All linked gene cassettes were cloned into the pCEP4-EGFP between BamHI and NheI restriction sites.
b All linked gene cassettes plus the EF1α promoter were cloned into the pCEP4-EGFP between BamHI and SpeI (19) restriction sites.
c All expression cassettes were cloned into the pCEP4-EGFP between BamHI and NruI restriction sites.
TABLE 3
Combinations of episomal constructs tested for reprogramming activity
Equiv-
alent of
AP+
pCEP4-
Test
Morph.
colony/
EGF(μg)
#
Plasmids
μg
Changes
plate
EXPERIMENT 1
6.3
1
pEP4-E-O2S-C-M2K
9.2
+/−
0
6.3
2
pEP4-E-O2S-K2Neo
9.3
+/−
0
6.3
pCEP4-M2L
7.4
6.3
3
pEP4-E-O2S-E-N2K
9.3
+/−
0
6.3
pCEP4-M2L
7.4
6.3
4
pEP4-E-O2S-E-T2K
10
+++
0
6.3
pCEP4-M2L
7.4
6.3
5
pEP4-E-O2S-E-TERT2K
10.8
+/−
0
6.3
pCEP4-M2L
7.4
6.3
6
pEP4-E-O2S-C-M2L
8.7
+/−
0
6.3
pEP4-E-N2K
7.9
6.3
7
pEP4-E-O2S-C-M2L
8.7
+
0
6.3
pEP4-E-T2K
8.6
6.3
8
pEP4-E-O2S-C-M2L
8.7
+/−
0
6.3
pEP4-E-TERT2K
9.4
EXPERIMENT 2
3.3
1
pEP4-E-O2S-C-M2K
5.0
+/−
0
3.3
2
pEP4-E-O2S-E-M2K
5.0
+/−
0
3.3
3
pEP4-E-O2S-C-K2M
5.0
+/−
0
3.3
4
pEP4-E-O2S-E-K2M
5.0
+/−
0
2.5
5
pEP4-E-O2S(2)
3.0
+/−
0
2.5
pCEP4-M2K
3.0
2.5
6
pEP4-E-O2S(2)
3.0
+/−
0
2.3
pEP4-E-M2K
3.0
2.5
7
pEP4-E-O2S(2)
3.0
+/−
0
2.5
pCEP4-K2M
3.0
2.5
8
pEP4-E-O2S(2)
3.0
+/−
0
2.3
pEP4-E-K2M
3.0
1.7
9N
pEP4-E-O2S(2)
2.0
+/−
0
1.5
pEP4-E-N2K
2.0
1.7
pCEP4-M2L
2.0
+/−
0
1.7
10N
pEP4-E-O2S(2)
2.0
+/−
0
1.7
pEP4-E-N2L
2.0
1.7
pCEP4-M2K
2.0
1.7
11N
pEP4-E-O2S(2)
2.0
+/−
0
1.7
pEP4-E-N2L
2.0
1.5
pEP4-E-M2K
2.0
1.7
12N
pEP4-E-O2S(2)
2.0
+/−
0
1.7
pEP4-E-N2L
2.0
1.7
pCEP4-K2M
2.0
1.7
13N
pEP4-E-O2S(2)
2.0
+/−
0
1.7
pEP4-E-N2L
2.0
1.5
pEP4-E-K2M
2.0
2.3
14N
pEP4-E-O2S-E-N2K
3.5
+/−
0
2.1
pCEP4-M2L
2.5
2.5
15N
pEP4-E-O2S-E-N2L
3.5
+/−
0
2.1
pCEP4-M2K
2.5
2.5
16N
pEP4-E-O2S-E-N2L
3.5
+/−
0
1.9
pEP4-E-M2K
2.5
2.5
17N
pEP4-E-O2S-E-N2L
3.5
+/−
0
2.1
pCEP4-K2M
2.5
2.5
18N
pEP4-E-O2S-E-N2L
3.5
+/−
0
1.9
pEP4-E-K2M
2.5
EXPERIMENT 3
1.7
9T
pEP4-E-O2S(2)
2.0
++
0
1.4
pEP4-E-T2K
2.0
1.7
pCEP4-M2L
2.0
1.7
10T
pEP4-E-O2S(2)
2.0
+
0
1.5
pEP4-E-T2L
2.0
1.7
pCEP4-M2K
2.0
1.7
11T
pEP4-E-O2S(2)
2.0
+
0
1.5
pEP4-E-T2L
2.0
1.5
pEP4-E-M2K
2.0
1.7
12T
pEP4-E-O2S(2)
2.0
+/−
0
1.5
pEP4-E-T2L
2.0
1.7
pCEP4-K2M
2.0
1.7
13T
pEP4-E-O2S(2)
2.0
+/−
0
1.5
pEP4-E-T2L
2.0
1.5
pEP4-E-K2M
2.0
2.2
14T
pEP4-E-O2SET2K
3.5
+++
0
2.1
pCEP4-M2L
2.5
2.3
15T
pEP4-E-O2S-E-T2L
3.5
+
0
2.1
pCEP4-M2K
2.5
2.3
16T
pEP4-E-O2S-E-T2L
3.5
+
0
1.9
pEP4-E-M2K
2.5
2.3
17T
pEP4-E-O2S-E-T2L
3.5
+/−
0
2.1
pCEP4-K2M
2.5
2.3
18T
pEP4-E-O2S-E-T2L
3.5
+/−
0
1.9
pEP4-E-K2M
2.5
1.9
19
pEP4-E-O2S-E-T2K
3.0
+++
1
2.0
pEP4-E-O2S-E-N2K
3.0
1.7
pCEP4-M2L
2.0
EXPERIMENT 4
6
1
pEP4-E-O2S-C-M2K-E-
10.9
+/−
0
N2L
4
2
pEP4-E-O2S-C-M2K-E-
7.3
+++
0
N2L
2
pEP4-E-O2S-E-T2K
3.2
6
3
pEP4-E-O2S-C-K2M-E-
10.9
+/−
0
N2L
4
4
pEP4-E-O2S-C-K2M-E-
7.3
++
2
N2L
2
pEP4-E-O2S-E-T2K
3.2
3
5
pEP4-E-O2S-E-N2L
4.2
+/−
0
3
pEP4-E-O2S-E-M2K
4.6
3
6
pEP4-E-O2S-E-N2L
4.2
++
0
2
pEP4-E-O2S-E-T2K
3.2
3
pEP4-E-O2S-E-M2K
4.6
3
7
pEP4-E-O2S-E-N2L
4.2
+/−
0
3
pEP4-E-O2S-C-M2K
4.4
3
8
pEP4-E-O2S-E-N2L
4.2
+
0
2
pEP4-E-O2S-E-T2K
3.2
3
pEP4-E-O2S-C-M2K
4.4
3
9
pEP4-E-O2S-E-N2L
4.2
+/−
0
3
pEP4-E-O2S-E-K2M
4.5
3
10
pEP4-E-O2S-E-N2L
4.2
+/−
0
2
pEP4-E-O2S-E-T2K
3.2
3
pEP4-E-O2S-E-K2M
4.5
3
11
pEP4-E-O2S-E-N2L
4.2
+/−
0
3
pEP4-E-O2S-C-K2M
4.4
3
12
pEP4-E-O2S-E-N2L
4.2
+
0
2
pEP4-E-O2S-E-T2K
3.2
3
pEP4-E-O2S-C-K2M
4.4
2
13
pEP4-E-O2S-C-M2L-E-
3.9
+
0
T2K
4
pEP4-E-O2S-E-N2K
5.9
6
14
pEP4-E-O2S-C-M2K-E-
11.6
+
0
T2L
3
15
pEP4-E-O2S-C-M2K-E-
5.8
+
0
T2L
3
pEP4-E-O2S-E-N2K
4.4
6
16
pEP4-E-O2S-C-K2M-E-
11.6
+/−
0
T2L
3
17
pEP4-E-O2S-C-K2M-E-
5.8
+
0
T2L
3
pEP4-E-O2S-E-N2K
4.4
6
18
pEP4-E-O2S-C-M2L-E-
11.6
+/−
0
K2T
3
19
pEP4-E-O2S-C-M2L-E-
5.8
+/−
0
K2T
3
pEP4-E-O2S-E-N2K
4.4
3
20
pEP4-E-O2S-E-K2T
4.8
+/−
0
3
pEP4-E-O2S-E-N2K
4.4
2
pEP4-E-O2S-C-M2L
2.8
+/−: No or very few colonies with morphological change were observed (FIG. 4B).
+, ++ and +++: Different number (from less to more) of colonies with morphological change were observed.Join the waitlist — get patent alerts
Track US2013217117A1 — get alerts on status changes and closely related new filings.
We store only your email — no account needed. See our privacy policy.