Inherited diseases such as SCID-X1, WAS, ADA, ALD and beta-thalassemia have been treated successfully using therapeutic gene transfer into hematopoietic stem cells. However, not all patients have benefited equally. There have been several adverse events in which integration of gene therapy vectors near cellular proto-oncogenes was associated with upregulation of transcription and leukemia, and in some subjects the gene therapy did not effectively ameliorate the underlying condition. We have carried out extensive studies of cell populations during human gene correction using vector integration site distribution data to infer the behavior of progenitor cells. We developed novel deep sequencing and bioinformatic methods for this purpose, yielding a wealth of data on successful and unsuccessful outcomes. Using these data and ongoing long term monitoring, we can address a new set of questions on the population biology of transduced cells, both to understand more fully the optimal strategies for human gene correction, and to understand development of the human hematopoietic system itself. We propose to acquire and analyze data on integration site distributions during long term patient monitoring. To expand our picture of blood cell development, we will also acquire and analyze data on VDJ recombination products in T-cells. In our first Aim, we will address the following hypotheses: 1) vectors with intact LTRs (including transcriptional enhancers) are much more active in driving expansion of cell clones than vectors without them, but some types of events with all vectors are consistent with insertional activation and vector driving~ 2) long term progenitor function differs depending on disease state, vector type, conditioning regimen and clinical condition of the subject~ 3) most transduced progenitor cells are in fact committed not just to the lymphoid or myeloid lineages, but even more specifically within each~ 4) progenitors are contributing cells to the periphery episodically, and this is further magnified by proliferation of antigen responsive cells and 5) stem cell activity wanes over time, but only very slowly. In our second Aim, which adds in TCR data, we will investigate the following further hypotheses: 1) TCR-beta repertoire sizes after SCID-X1 or WAS gene correction approach those observed in healthy adults~ 2) CD4+ and CD8+ T-cell repertoires contain mostly different TCR-beta recombination products, indicating mostly independent recombination and commitment~ 3) the number of cell divisions can be estimated from these data that link progenitors and mature T-cells~ 4) proliferation in response to antigen involves only a specific subset of progenitors and TCRs~ 5) TCR sequencing can be a sensitive marker for clonal expansion in leukemia and successful eradication of the leukemic clone by chemotherapy. Thus the proposed study will provide unique data useful both in optimizing gene therapy methods and understanding human hematopoiesis, and also provide a mechanism for supporting FDA-mandated long-term follow up of gene-corrected subjects.

Public Health Relevance

Stem cell gene therapy can be curative for individuals suffering from inherited diseases such as SCID-X1, WAS, beta thalassemia, and CGD. The proposed project will use integrated gene therapy vectors as markers to track long-term outcome over multiple successful trials, allowing investigation of the cell biology of the human blood cell system using integration sites as lineage tracers. The project will also analyze VDJ recombination products in T-cells from the same individuals using deep sequencing, allowing quantitation of the growth and function of progenitor cells and descendant T- cells in humans.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1-IMM-N (52))
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Johnson, David R
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University of Pennsylvania
Schools of Medicine
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Berry, Charles C; Ocwieja, Karen E; Malani, Nirav et al. (2014) Comparing DNA integration site clusters with scan statistics. Bioinformatics 30:1493-500
Manganaro, Lara; Pache, Lars; Herrmann, Tobias et al. (2014) Tumor suppressor cylindromatosis (CYLD) controls HIV transcription in an NF-?B-dependent manner. J Virol 88:7528-40
Hacein-Bey-Abina, Salima; Pai, Sung-Yun; Gaspar, H Bobby et al. (2014) A modified ?-retrovirus vector for X-linked severe combined immunodeficiency. N Engl J Med 371:1407-17
Zhong, Li; Malani, Nirav; Li, Mengxin et al. (2013) Recombinant adeno-associated virus integration sites in murine liver after ornithine transcarbamylase gene correction. Hum Gene Ther 24:520-5
Schneider, William M; Brzezinski, Jonathon D; Aiyer, Sriram et al. (2013) Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag. Proc Natl Acad Sci U S A 110:9487-92
Sadelain, Michel; Papapetrou, Eirini P; Bushman, Frederic D (2012) Safe harbours for the integration of new DNA in the human genome. Nat Rev Cancer 12:51-8
Berry, Charles C; Gillet, Nicolas A; Melamed, Anat et al. (2012) Estimating abundances of retroviral insertion sites from DNA fragment length data. Bioinformatics 28:755-62
Negre, Olivier; Fusil, Floriane; Colomb, Charlotte et al. (2011) Correction of murine ýý-thalassemia after minimal lentiviral gene transfer and homeostatic in vivo erythroid expansion. Blood 117:5321-31
Brady, Troy; Roth, Shoshannah L; Malani, Nirav et al. (2011) A method to sequence and quantify DNA integration for monitoring outcome in gene therapy. Nucleic Acids Res 39:e72
Roth, Shoshannah L; Malani, Nirav; Bushman, Frederic D (2011) Gammaretroviral integration into nucleosomal target DNA in vivo. J Virol 85:7393-401

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