gene repair in murine hematopoietic stem cells (ngec component 6) aim 1: develop and test a murine...
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Gene repair in murine hematopoietic stem cells
(NGEC Component 6)
• Aim 1: Develop and test a murine X-linked severe combined immunodeficiency (XSCID) model for I-SceI (or engineered I-AniI gene) repair.– Additional model- GFP/FOXP3 knock-in model
• Aim 2: Develop and test non-integrating lentiviral (NIL) vectors for concurrent HE and repair template delivery.
• Aim 4: Engineer Ani-I for Btk gene repair (in XID/Tec-/-
model of human X-linked agammaglobulinemia; XLA).
XSCID Common-chainEx6TGA
Knock-in Models
Overall Goals:
Proof of Concept for Gene Repair in vivo using gold standard HE and marked selective advantage for normal lymphoid cells
Proof of Concept for HE engineering in conjunction with in vivo gene repair
Plan: generation and testing of common -chain Ex6TGA X-SCID models Jordan Jarjour
c-Containing Receptors
ILIL--2R2R ILIL--4R4R ILIL--7R7R ILIL--9R9R ILIL--15R15R
c
IL-2R
IL-2R
IL-4R IL-7R IL-9R IL-2R
IL-15R
intracellular
extracellular
ILIL--21R21R
IL-21R
c-Containing Receptors
ILIL--2R2R ILIL--4R4R ILIL--7R7R ILIL--9R9R ILIL--15R15R
c
IL-2R
IL-2R
IL-4R IL-7R IL-9R IL-2R
IL-15R
intracellular
extracellular
ILIL--21R21R
IL-21R
Oct2001
c c c c c
Common Gamma Chain (CD132) 9kb8000 bp
Exon 1
Exon2
Exon 3
Exon 4 Exon 5Exon 6
Exon 7
Exon 8
Bam HI (1380) Bam HI (5433)
Bsp HI (1243)
Bsp HI (3878)Hin dIII (54)
Hin dIII (7366)Nhe I (2644)
Nhe I (7971)
pGK Neo pAFRT FRT
TGA
Exon 6
I-Sce1
BspH1
CCAGTAAAAGGAACAAACAATGTCTCTTAGGAAGGAACAAAAGTACT... GGTCATTTTCCTTGTTTGTTACAGAGAATCCTTCCTTGTTTTCATGA...
Wild Type / 14:19 I-AniI
CCAGTAAAAGGAACAAACAATATCCCTATTGTCCCATTAAAAGTACT... GGTCATTTTCCTTGTTTGTTATAGGGATAACAGGGTAATTTTCATGA...
I-SceI
XSCID Common-chainEx6TGA
Knock-in Models
XSCID Common-chainKnock-in Model
Status Report:
1. I-SceI common -chain Ex6TGA X-SCID knock-in Construct made, ES clone isolated, injected, and agouti (=X-linked)
pups obtained from initial breeding. Very strong likelihood of germline transmission
2. WT common- -chain Ex6TGA knock-in Construct made, ES cells being screened. I-AniI XSCID engineering in progress.
UW01, Southern to confirm 1C4 targeting,Hind III digest, Probe 5’
1 kb
1C
4W
t ES
L-H
ind
III
1 kb
1C
4W
t ES
L-H
ind
III
10 kb
8 kb
6 kb
5 kb
4 kb
23 kb
9.6 kb
6.6 kb
4.4 kb
10 kb
8 kb
6 kb
5 kb
4 kb
ethidium Southern
UW01, Southern to confirm 1C4 targeting,Hind III digest, Probe 5’
1 kb
1C
4W
t ES
L-H
ind
III
1 kb
1C
4W
t ES
L-H
ind
III
10 kb
8 kb
6 kb
5 kb
4 kb
23 kb
9.6 kb
6.6 kb
4.4 kb
10 kb
8 kb
6 kb
5 kb
4 kb
ethidium Southern
XSCID Common-chainKnock-in Model
Proof of Concept: Gene Repair in vivo in following NIL infection of purified hematopoietic stem cells (HSC).
Initial plans:
Benchmarking NIL-driven DNA Repair in ES or Lymphoid Cell Lines1. Recovery of -chain (CD132) surface expression (flow cytometry)
2. Test NIL vectors and conversion tract requirement
3. Off-site cutting and genomic instability
Additional Model for In vivo Testing: I-SceI/GFP/Foxp3 Knock-in
Foxp3 deficiency results in severe autoimmunity in humans (IPEX) and in mice (“scurfy”) due to lack of Treg cells
Foxp3: X-chromosome–encoded forkhead transcription factor Required for generation and maintenance of Treg cells
Jordan Jarjour and Yupeng Wang
Status: Construct completed, ES clones being isolated
Non-integrating lentiviral (NIL) vectors for concurrent HE and repair template delivery.
Mike Certoand Vector Core
NIL vectors generated using mutant packaging construct: psPAX2(int-) Integrase mutated to an inactive form via D64V amino acid substitution)
Btk and B cell development
Immature B Mature BStem Cell Pro-B Pre-B
Pre-BCR
Hardy Fractions: A-C D E FIII FII FI
B220CD43 IgM IgD
Bone Marrow Spleen
Btk
XLABtk
XID/Btk-/-
Btk-/Tec-XID/Tec -
Yeast surface
Aga1ps
ss
s
Aga2p
I-AniI
HA
3’-ACTCCTCCAAAGAGACATT5’-TGAGGAGGTTTCTCTGTAABiotin
Ani-wt: T G A G G A G G T T T C T C T G T A Am-XID: A G T G C C T G T T T C T C T T G A C
m-wt: C
-10 -9 -8 –7 –6 –5 -4 –3 –2 –1 +1 +2 +3 +4 +5 +6 +7 +8 +9
Design/display/sorting I-AniI HE’s for XID site
I-Anil Yeast surface displayYupeng WangJordan Jarjour
WT I-AniI is 11/19 nucleotide match for human and murine Btk:
Ani-wt: T G A G G A G G T T T C T C T G T A Am-XID: A G T G C C T G T T T C T C T T G A C
m-wt: C
-10 -9 -8 –7 –6 –5 -4 –3 –2 –1 +1 +2 +3 +4 +5 +6 +7 +8 +9
Oligos: 1bp -10A-8T-5C-4T+6T+7G+9C-8C
Oligos: 2bp -10A –8T+6T +7G-10A –8C
Oligos: 3bp -6C –5C –4T+6T +7G +9C
Oligos: 5bp -10A –8T -6C –5C –4T-10A –8C -6C –5C –4T
Oligos: 8bp mXID and mWT
Design/displaying/sorting I-AniI HE’s for XID site
Based on computer designed enzyme, using error-prone PCR to generate random mutation in I-Anil DNA binding domain and sorting high binding affinity AniI by MACS and FACS.
Yupeng Wang
Common Gamma Chain (CD132) 9kb8000 bp
Exon 1
Exon2
Exon 3
Exon 4 Exon 5Exon 6
Exon 7
Exon 8
Bam HI (1380) Bam HI (5433)
Bsp HI (1243)
Bsp HI (3878)Hin dIII (54)
Hin dIII (7366)Nhe I (2644)
Nhe I (7971)
pGK Neo pALox Lox
TGA
Exon 6
I-Ani1
BspH1
CCAGTAAAAGGAACAAACAATGTCTCTTAGGAAGGAACAAAAGTACT... GGTCATTTTCCTTGTTTGTTACAGAGAATCCTTCCTTGTTTTCATGA...
I-Ani1 Recognition Sequence (or I-Sce1 or WT 14/19 I-Ani1)
Common Gamma Chain (CD132) 9kb8000 bp
Exon 1
Exon2
Exon 3
Exon 4 Exon 5Exon 6
Exon 7
Exon 8
Bam HI (1380) Bam HI (5433)
Bsp HI (1243)
Bsp HI (3878)Hin dIII (54)
Hin dIII (7366)Nhe I (2644)
Nhe I (7971)
pGK Neo pAFRT FRT
TGA
Exon 6
I-Sce1
BspH1
CCAGTAAAAGGAACAAACAATGTCTCTTAGGAAGGAACAAAAGTACT... GGTCATTTTCCTTGTTTGTTACAGAGAATCCTTCCTTGTTTTCATGA...
Wild Type / 14:19 I-AniII-AniI
CCAGTAAAAGGAACAAACAATGTCTCTTTGGAGGAGTCAAAAGTACT... GGTCATTTTCCTTGTTTGTTACAGAGAAACCTCCTCAGTTTTCATGA... CCAGTAAAAGGAACAAACAATATCCCTATTGTCCCATTAAAAGTACT... GGTCATTTTCCTTGTTTGTTATAGGGATAACAGGGTAATTTTCATGA...
I-SceI
XSCID Common-chainEx6TGA
Knock-in Models
Common Gamma Chain (CD132) 9kb1379 bp
I-AniI
Exon 5 Exon 6 Exon 7
LoxP
Premature Stop Codon
Common Gamma Chain (CD132) 9kb1379 bp
I-SceI
Exon 5 Exon 6 Exon 7
LoxP
Premature Stop Codon
Common Gamma Chain (CD132) 9kb1379 bp
Wild Type
Exon 5 Exon 6 Exon 7
LoxP
Premature Stop Codon
Figure 3: Mouse SCID model for gene correction in IL2R-deficient strains. Each strain, generated by homologous recombination, carries a premature stop codon at the beginning of Exon 6 which will abrogate surface expression of the -chain which is a component in multiple cytokine receptors required for efficient hematopoiesis. In the absence of the -chain, development of both B- and T-lymphocytes is blocked at an early stage. Strain a) and b) carry the engineered HE target sites recognized by I-AniI and I-SceI placed immediately upstream of the splice acceptor site. Strain c) retains the wild-type sequence, which is a 14/19 near-consensus target sequence for I-AniI cleavage. These mouse strains will allow for proof of concept type experiments within an optimized system for analyzing gene repair, as well as providing an in-vivo target for evaluating the ability, in relation to highly efficient natural HEs, to successfully engineer an artificially evolved HE capable of gene repair.
a)
b)
c)
References:
1. Hacein-Bey-Abina S., et al. Science. 2003. 302(5644):415-9
2. Smith GR. Annu Rev Genet. 2001. 35:243-74
3. Doolittle RF. Proc Natl Acad Sci USA. 1993. 90:5379-81
4. Arakawa H., et al. Science. 2002. 295(5558):1301-6
5. Wang L., et al. Proc Natl Acad Sci USA. 2004. 101(48):16745-9
6. Mahadevaiah SK., et al. Nat Genet. 2001. 27(3):271-6