Abstract

The new deep sequencing methods offer tremendously improved means of characterizing genetic diversity. The Sequencing and Viral Evolution Core, under the direction of Dr. F. Bushman, will perform deep sequencing to support the IPCP team. The core will analyze viral populations in the presence and absence of therapy to monitor the effects of treatment, characterize the effects of zinc-finger nucleases on genomic DMA, and quantify lentiviral vector integration sites to monitor for possible genotoxicity. The Bushman laboratory has published extensively on applications of deep sequencing, and will adapt these methods to support the proposed projects. The effects of the gag and pol epitope targeting will be assessed by quantifying longitudinal changes in viral quasispecies that accumulate selectively in the presence of pressure from therapy. For HIV viruses replicating in cells with the CXCR4 and CCR5 knockouts, deep sequencing will be used to characterize viral envelope sequences present prior to therapy and their evolution under therapy. Deep sequencing will be used to characterize the genomic structure at the site of gene deletion mediated by zinc finger nucleases and scan for possible associated chromosomal abnormalities. Deep sequencing will also be used to follow integration site selection by lentiviral vectors, and monitor for possible genotoxicity due to vector integration.
Our specific aims are as follows:
Specific Aim 1 : DNA barcoding and pyrosequencing for analysis of viral mutations in response to gag and pol epitope targeting (Projects 1 and 2), and zinc finger mediated CXCR4 and CCR5 knockout in CD4 T cells (Projects 2 and 3).
Specific Aim 2 : Longitudinal analysis of lentiviral vector integration sites using pyrosequencing (Projects 1 and 2).

Public Health Relevance

The new 454/Roche pyrosequencing method allows up to 100,000,000 bases of DNA sequence to be determined in a single one day run. The core will use this method to evaluate the effectiveness of therapy and monitor for possible adverse events.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program--Cooperative Agreements (U19)
Project #
5U19AI082628-04
Application #
8375248
Study Section
Special Emphasis Panel (ZAI1-CCH-A)
Project Start
Project End
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
4
Fiscal Year
2012
Total Cost
$212,170
Indirect Cost
$69,840

  Institution

Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104

  Publications

Richardson, Max W; Guo, Lili; Xin, Frances et al. (2014) Stabilized human TRIM5? protects human T cells from HIV-1 infection. Mol Ther 22:1084-1095
Didigu, Chuka A; Wilen, Craig B; Wang, Jianbin et al. (2014) Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection. Blood 123:61-9
Cameron, Brian J; Gerry, Andrew B; Dukes, Joseph et al. (2013) Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells. Sci Transl Med 5:197ra103
Maier, Dawn A; Brennan, Andrea L; Jiang, Shuguang et al. (2013) Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5. Hum Gene Ther 24:245-58
Richardson, Max W; Jadlowsky, Julie; Didigu, Chuka A et al. (2012) Kruppel-like factor 2 modulates CCR5 expression and susceptibility to HIV-1 infection. J Immunol 189:3815-21
Didigu, Chukwuka A; Doms, Robert W (2012) Novel approaches to inhibit HIV entry. Viruses 4:309-24
Scholler, John; Brady, Troy L; Binder-Scholl, Gwendolyn et al. (2012) Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Sci Transl Med 4:132ra53
Wilen, Craig B; Wang, Jianbin; Tilton, John C et al. (2011) Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases. PLoS Pathog 7:e1002020
Cannon, Paula; June, Carl (2011) Chemokine receptor 5 knockout strategies. Curr Opin HIV AIDS 6:74-9
Mukherjee, Rithun; Plesa, Gabriela; Sherrill-Mix, Scott et al. (2010) HIV sequence variation associated with env antisense adoptive T-cell therapy in the hNSG mouse model. Mol Ther 18:803-11

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