The development of leukemia has been observed in 5 out of 19 SCID-Xl patients treated by stem cell gene therapy. In all 5 patients the development of leukemia was due to insertional mutagenesis, i.e. the retrovirus- mediated activation of nearby proto-oncogenes. Thus, the careful study of integration sites and the contribution of individual clones to repopulation will be of crucial importance for all gene therapy studies, and the FDA has mandated the careful monitoring of retroviral integration sites in all clinical gene therapy studies. The Vector Integration and Tracking Core D will provide all projects a centralized facility to efficiently identify foamy virus (FV) vector integration sites in complex biological DNA samples from in vitro or in vivo studies. We will use recently developed improved non-restriction (nr)LAM-PCR. The core will be able to process a variety of samples from murine, dog, or human to generate DNA for shuttle vector or PCR- based methods. For nrLAM-PCR, as little as lOng of DNA can be used to carry out FV vector integration site amplification and extended processing in preparation for sequencing. The samples processed by Core D will be compatible with both Sanger-based sequencing for pilot experiments and new sequencing methodologies, such as pyrosequencing, for deep sequencing to identify all of the amplifiable FV vector integration sites in a given sample. Core D will also provide assistance to all projects to design primers specific to FV vector integration sites to allow for DNA-based real time (RT)-PCR tracking to assess the contribution for individual clones that are deemed important for further investigation. In addition, Core D will provide a centralized facility to analyze integration sites via a common gateway interface (CGI)-PERL web server. Current versions of the human (hg19), dog (canFam2) and mouse (mm9) genomes will be supported. The Core will also provide support to investigators through PERL programs to correlate FV vector integration sites with data from published databases including proto-oncogene TSS, microarray data and over- represented gene classes. The bioinformatics component will also generate random datasets for all three genomes, human, mouse and dog, to evaluate over-represented gene classes near vector proviruses.

Public Health Relevance

The focus of this PPG is to test the efficacy and safety of the most appropriate retrovirus vector system, FV vectors for the treatment of SCID-Xl. An indispensible aspect of retrovirus-based safety analysis is integration site profiling and characterization of the surrounding genomic elements. The specific aims of Core D will provide a careful standardized approach defining the safety of the current and improved FV vectors developed in Projects 1-3.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program Projects (P01)
Project #
5P01AI097100-03
Application #
8712355
Study Section
Special Emphasis Panel (ZAI1)
Project Start
Project End
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Seattle Children's Hospital
Department
Type
DUNS #
City
Seattle
State
WA
Country
United States
Zip Code
98109
Humbert, Olivier; Chan, Frieda; Rajawat, Yogendra S et al. (2018) Rapid immune reconstitution of SCID-X1 canines after G-CSF/AMD3100 mobilization and in vivo gene therapy. Blood Adv 2:987-999
Browning, D L; Everson, E M; Leap, D J et al. (2017) Evidence for the in vivo safety of insulated foamy viral vectors. Gene Ther 24:187-198
Singh, Swati; Khan, Iram; Khim, Socheath et al. (2017) Safe and Effective Gene Therapy for Murine Wiskott-Aldrich Syndrome Using an Insulated Lentiviral Vector. Mol Ther Methods Clin Dev 4:1-16
Nalla, Arun K; Trobridge, Grant D (2016) Prospects for Foamy Viral Vector Anti-HIV Gene Therapy. Biomedicines 4:
Browning, Diana L; Trobridge, Grant D (2016) Insulators to Improve the Safety of Retroviral Vectors for HIV Gene Therapy. Biomedicines 4:
Browning, Diana L; Collins, Casey P; Hocum, Jonah D et al. (2016) Insulated Foamy Viral Vectors. Hum Gene Ther 27:255-66
Bii, Victor M; Trobridge, Grant D (2016) Identifying Cancer Driver Genes Using Replication-Incompetent Retroviral Vectors. Cancers (Basel) 8:
Adair, Jennifer E; Waters, Timothy; Haworth, Kevin G et al. (2016) Semi-automated closed system manufacturing of lentivirus gene-modified haematopoietic stem cells for gene therapy. Nat Commun 7:13173
Nalla, Arun K; Williams, Theodore F; Collins, Casey P et al. (2016) Lentiviral vector-mediated insertional mutagenesis screen identifies genes that influence androgen independent prostate cancer progression and predict clinical outcome. Mol Carcinog 55:1761-1771
Humbert, Olivier; Gisch, Don W; Wohlfahrt, Martin E et al. (2016) Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T-cells. Mol Ther 24:1237-46

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