Employing gene trapping for the correction of the Gp91phox (Nox2) mutation in chronic granulomatous disease (X-CGD)
Final Report Abstract
Retroviruses are widely used to transduce and stably express genes of interest in hematopoietic stem cells (HSC). Several clinical trials employing retroviruses for the ex vivo transduction of healthy genes into hematopoietic stem cells from patients with monogenic blood disorders provided definite proof for curative potential of gene therapy. However, these trials also showed that retroviral gene therapy can cause leukemia by the insertional activation of cellular oncogenes. To address this issue, we developed a gene trap approach for transducing therapeutic genes into hematopoietic stem cells. Gene traps are devoid of promoter- and enhancer elements and therefore likely to be less genotoxic than conventional gene therapy vectors. We could show that disease causing gene mutations can be corrected by healthy copy transgenes delivered to hematopoietic cells by gene trap vectors and that the gene trapped cells can be expanded in vitro and in vivo by using a growth factor receptor activating dimerization strategy and severely immunodeficient SCID-X1 mice as bone marrow transplant recipients, respectively. Future experiments will combine gene trapping with CRISPR/Cas9 genome editing to directly insert gene trap gene therapy cassettes into “safe harbor” loci to thus completely circumvent the insertional mutagenesis problem encountered with most randomly integrating gene therapy vectors.
Publications
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(2011) Forward and Reverse Genetics through Derivation of Haploid Mouse Embryonic Stem Cells. Cell Stem Cell 9: 563-574
Elling U, Taubenschmid J, Wirnsberger G, O'Malley R, Demers SP, Vanhaelen Q, Shukalyuk AI, Schmauss G, Schramek D, Schnütgen F, von Melchner H, Ecker JR, Stanford WL, Zuber J, Stark A, Penninger JM
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(2011) Gene therapy of chronic granulomatous disease: the engraftment dilemma. Mol Ther 19:28-35
Grez M, Reichenbach J, Schwäble J, Seger R, Dinauer MC, Thrasher AJ
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(2011) High throughput gene trapping and postinsertional modifications of gene trap alleles. Methods 53:347-355
Schnütgen F, Ehrmann F, Ruiz-Noppinger P, von Melchner H
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(2011) Physiological regulation of transgene expression by a lentiviral vector containing the A2UCOE linked to a myeloid promoter. Gene Ther 19:1018-1029
Brendel C, Müller-Kuller U, Schultze-Strasser S, Stein S, Chen-Wichmann L, Krattenmacher A, Kunkel H, Dillmann A, Antoniou MN, Grez M
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(2011) Resources for proteomics in mouse embryonic stem cells. Nat Methods 8:103-104
Schnütgen F, Ehrmann F, Poser I, Hubner NC, Hansen J, Floss T, deVries I, Wurst W, Hyman A, Mann M, von Melchner H
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(2012) The mammalian gene function resource: the International Knockout Mouse Consortium. Mamm Genome 23: 580-586
Bradley A, Anastassiadis K, Ayadi A, Battey JF, Bell C, Birling MC, Bottomley J, Brown SD, Burger A, Bult CJ, Bushell W, Collins FS, Desaintes C, Doe B, Economides A, Eppig JT, Finnell RH, Fletcher C, Fray M, Frendewey D, Friedel RH, Grosveld FG, Hansen J, Herault Y, Hicks G, Horlein A, Houghton R, Hrabe de Angelis M, Huylebroeck D, Iyer V, de Jong PJ, Kadin JA, Kaloff C, Kennedy K, Koutsourakis M, Lloyd KC, Marschall S, Mason J, McKerlie C, McLeod MP, von Melchner H, Moore M, Mujica AO, Nagy A, Nefedov M, Nutter LM, Pavlovic G, Peterson JL, Pollock J, Ramirez-Solis R, Rancourt DE, Raspa M, Remacle JE, Ringwald M, Rosen B, Rosenthal N, Rossant J, Ruiz Noppinger P, Ryder E, Schick JZ, Schnütgen F, Schofield P, Seisenberger C, Selloum M, Simpson EM, Skarnes WC, Smedley D, Stanford WL, Stewart AF, Stone K, Swan K, Tadepally H, Teboul L, Tocchini-Valentini GP, Valenzuela D, West AP, Yamamura K, Yoshinaga Y, Wurst W
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(2013) Gene Targeting and Site-Specific Recombination in Mouse ES Cells. Methods Enzymol 533:133-155
Anastassiadis K, Schnütgen F, von Melchner H, Stewart AF
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(2013) Human miR223 promoter as a novel myelo-specific promoter for chronic granulomatous disease gene therapy. Hum Gene Ther Methods 24(3):151-9
Brendel C, Hänseler W, Wohlgensinger V, Bianchi M, Tokmak S, Chen-Wichmann L, Kuzmenko E, Cesarovic N, Nicholls F, Reichenbach J, Seger R, Grez M, Siler U
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(2013). Alpharetroviral Vector-mediated Gene Therapy for X- CGD: Functional Correction and Lack of Aberrant Splicing. Mol Ther 21:648-661
Kaufmann KB, Brendel C, Suerth JD, Mueller-Kuller U, Chen-Wichmann L, Schwäble J, Pahujani S, Kunkel H, Schambach A, Baum C, Grez M
