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Gene repair in murine hematopoietic stem cells (NGEC Component 6)

Alex, Jordan, and Byoung. Yupeng. 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.

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Gene repair in murine hematopoietic stem cells (NGEC Component 6)

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  1. Alex, Jordan, and Byoung Yupeng 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.

  2. TGA Lox pGK Neo pA Lox Exon 6 I-Ani1 GGTCATTTTCCTTGTTTGTTACAGAGAATCCTTCCTTGTTTTCATGA... CCAGTAAAAGGAACAAACAATGTCTCTTAGGAAGGAACAAAAGTACT... I-Ani1 Recognition Sequence (or I-Sce1 or WT 14/19 I-Ani1) BspH1

  3. XSCID Common g-chain Knock-in Models 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 2. Establish quantitative assay of repair frequency- Alex 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 Proof of Concept: Gene Repair in vivo in following NIL infection of purified hematopoietic stem cells (HSC).

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

  5. XSCID Common g-chainEx6TGA Knock-in Models TGA FRT pGK Neo pA FRT Exon 6 I-Sce1 GGTCATTTTCCTTGTTTGTTATAGGGATAACAGGGTAATTTTCATGA... CCAGTAAAAGGAACAAACAATATCCCTATTGTCCCATTAAAAGTACT... I-SceI Wild Type / 14:19 I-AniI GGTCATTTTCCTTGTTTGTTACAGAGAAACCTCCTCAGTTTTCATGA... GGTCATTTTCCTTGTTTGTTACAGAGAATCCTTCCTTGTTTTCATGA... CCAGTAAAAGGAACAAACAATGTCTCTTTGGAGGAGTCAAAAGTACT... CCAGTAAAAGGAACAAACAATGTCTCTTAGGAAGGAACAAAAGTACT... I-AniI BspH1

  6. XSCID Common g-chainEx6TGA Knock-in Models TGA FRT pGK Neo pA FRT Exon 6 I-Sce1 GGTCATTTTCCTTGTTTGTTATAGGGATAACAGGGTAATTTTCATGA... CCAGTAAAAGGAACAAACAATATCCCTATTGTCCCATTAAAAGTACT... I-SceI Wild Type / 14:19 I-AniI GGTCATTTTCCTTGTTTGTTACAGAGAATCCTTCCTTGTTTTCATGA... CCAGTAAAAGGAACAAACAATGTCTCTTAGGAAGGAACAAAAGTACT... BspH1

  7. a) b) c) Figure 3: Mouse SCID model for gene correction in IL2Rg-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 g-chain which is a component in multiple cytokine receptors required for efficient hematopoiesis. In the absence of the g-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. 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

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