Gene repair in murine hematopoietic stem cells (NGEC Component 6) Aim 1: Develop murine X-linked severe combined immunodeficiency (XSCID) models for I-SceI.

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Gene repair in murine hematopoietic stem cells (NGEC Component 6) Aim 1: Develop murine X-linked severe combined immunodeficiency (XSCID) models for I-SceI vs. engineered I-AniI gene repair. Aim 2: Develop and test non-integrating lentiviral (NIL) vectors for concurrent HE and repair template delivery. Aim 3: Test gene repair using NIL vectors in primary cells from XSCID mice. Aim 4: Engineer Ani-I for Btk gene repair (in XID/Tec-/- model of human X-linked agammaglobulinemia; XLA). Aim 5: Test gene repair in XID mice in the presence vs. absence of the cis-linked selectable marker, MGMTP140K. Alex, Jordan, and Byoung Yupeng

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

XSCID Common  -chain Knock-in Models Proof of Concept: Gene Repair in vivo in following NIL infection of purified hematopoietic stem cells (HSC). Testing NIL-driven DNA Repair in HSC and In vitro B Lymphoid Cell Lines 1. Model % WT HSC required for phenotypic correction in vivo- Alex/Jordan 3. Test NIL vectors in vitro and in vivo using Sce-XSCID model- Alex/Jordan/Byoung 4. Expand, phenotype and backcross Ani-XSCID model- Alex 2. Establish quantitative assay of repair frequency- Alex

Non-integrating lentiviral (NIL) vectors for concurrent HE and repair template delivery. Byoung Ryu and Vector Core NIL vectors generated using mutant packaging construct: psPAX2(int-) Integrase mutated to an inactive form via D64V amino acid substitution)

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

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

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