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  -chain Ex6TGA 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

pGKNeopAFRT TGA Exon 6 I-Sce1 BspH1 CCAGTAAAAGGAACAAACAATGTCTCTTAGGAAGGAACAAAAGTACT... GGTCATTTTCCTTGTTTGTTACAGAGAATCCTTCCTTGTTTTCATGA... Wild Type / 14:19 I-AniI CCAGTAAAAGGAACAAACAATATCCCTATTGTCCCATTAAAAGTACT... GGTCATTTTCCTTGTTTGTTATAGGGATAACAGGGTAATTTTCATGA... I-SceI XSCID Common  -chain Ex6TGA Knock-in Models

XSCID Common  -chain Knock-in Model Status Report: 1. I-SceI common  -chain Ex6 TGA 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 Ex6 TGA knock-in Construct made, ES cells being screened. I-AniI XSCID engineering in progress.

XSCID Common  -chain Knock-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 Lines 1. 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 Certo and 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 BMature B Stem Cell Pro-BPre-B Pre-BCR Hardy Fractions: A-CDEFIIIFIIFI B220 CD43 IgM IgD Bone MarrowSpleen Btk XLA Btk XID/Btk-/- Btk-/Tec- XID/Tec -

Yeast surface Aga1p s s s s Aga2p I-AniI HA 3’-ACTCCTCCAAAGAGACATT 5’-TGAGGAGGTTTCTCTGTAA Biotin Ani-wt: T G A G G A G G T T T C T C T G T A A m-XID: A G T G C C T G T T T C T C T T G A C m-wt: C –7 –6 –5 -4 –3 –2 – Design/display/sorting I-AniI HE’s for XID site I-Anil Yeast surface display Yupeng Wang Jordan 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 A m-XID: A G T G C C T G T T T C T C T T G A C m-wt: C –7 –6 –5 -4 –3 –2 – 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

pGKNeopALox TGA Exon 6 I-Ani1 BspH1 CCAGTAAAAGGAACAAACAATGTCTCTTAGGAAGGAACAAAAGTACT... GGTCATTTTCCTTGTTTGTTACAGAGAATCCTTCCTTGTTTTCATGA... I-Ani1 Recognition Sequence (or I-Sce1 or WT 14/19 I-Ani1)

pGKNeopAFRT TGA Exon 6 I-Sce1 BspH1 CCAGTAAAAGGAACAAACAATGTCTCTTAGGAAGGAACAAAAGTACT... GGTCATTTTCCTTGTTTGTTACAGAGAATCCTTCCTTGTTTTCATGA... Wild Type / 14:19 I-AniII-AniI CCAGTAAAAGGAACAAACAATGTCTCTTTGGAGGAGTCAAAAGTACT... GGTCATTTTCCTTGTTTGTTACAGAGAAACCTCCTCAGTTTTCATGA... CCAGTAAAAGGAACAAACAATATCCCTATTGTCCCATTAAAAGTACT... GGTCATTTTCCTTGTTTGTTATAGGGATAACAGGGTAATTTTCATGA... I-SceI XSCID Common  -chain Ex6TGA Knock-in Models

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 (5644): Smith GR. Annu Rev Genet : Doolittle RF. Proc Natl Acad Sci USA : Arakawa H., et al. Science (5558): Wang L., et al. Proc Natl Acad Sci USA (48): Mahadevaiah SK., et al. Nat Genet (3):271-6