(PROJECT 2- CHEN) The hypothesis to be tested in this project is that transplant of gene-modified HSPC and T-cells give rise to repopulating cells exhibiting normal differentiation. The other projects proposed in this program investigate genetic modifications of HSPC and/or T-cell populations to either resist infection from HIV-1 (Project 4: Symonds), provide anti-HIV-1 effector activity (Project 3: Kitchen), or improve the efficiency of engraftment (Project 1: An). We propose to track behavior of individual transduced cells within the population of transplanted and repopulating cells by using vector integration sites (VIS) as a marker to uniquely identify individual repopulating clones and assess the behavior of these clones for repopulation characteristics. Repopulation of normal non-gene modified HSPC and T-cells results from the additive contributions of hundreds to thousands of stem and progenitor cells which give rise to clones of repopulating mature blood cells. Classicly, it was thought that these repopulating cells are relatively homogeneous in regards to features such as longevity and differentiation, however the recent studies tracking the behavior of individual HSPC clones in mice and our recent clonal tracking studies of HSPC in non-human primates demonstrate that these stem and progenitor cells represent complex mixtures of cells having diverse functional properties. Heterogeneous populations of HSPC differ in regards to multiple factors, including relative contribution to repopulation, self-renewal, commitment to differentiation lineage, and longevity. Gene modification with transgenes which further alter the genetic properties of these cells introduces additional possibilities for inadvertent and potentially adverse behavior changes. Clonal analysis of gene-modified HSPC can address the likelihood of successful HSPC genetic therapy for HIV-1 disease. For example, it is important to have long- term repopulation by many gene-modified HSPC clones (polyclonal) capable of multilineage differentiation, particularly into HIV target cells, myeloid and lymphoid cell, and well represented in all primary and secondary lymphoid tissues. Analysis depends upon having a high-throughput technology that can accurately quantitate and sensitively detect clones. To that end, we have adapted the genomic pyrosequencing technology for this purpose and published a validated high throughput assay which enables a quantitative and sensitive enumeration of individual VIS and, in combination with intensive bioinformatics computation, determines the relative frequencies of progeny derived from individual HSPC within a given tissue sample. This project will utilize humanized mice and non-human primate tissue samples derived from Projects 1 and 3 for clonal tracking.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program--Cooperative Agreements (U19)
Project #
5U19AI117941-05
Application #
9691131
Study Section
Special Emphasis Panel (ZAI1)
Project Start
Project End
2021-04-30
Budget Start
2019-05-01
Budget End
2020-04-30
Support Year
5
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Khamaikawin, Wannisa; Shimizu, Saki; Kamata, Masakazu et al. (2018) Modeling Anti-HIV-1 HSPC-Based Gene Therapy in Humanized Mice Previously Infected with HIV-1. Mol Ther Methods Clin Dev 9:23-32
Carrillo, Mayra A; Zhen, Anjie; Zack, Jerome A et al. (2017) New approaches for the enhancement of chimeric antigen receptors for the treatment of HIV. Transl Res 187:83-92
Zhen, Anjie; Peterson, Christopher W; Carrillo, Mayra A et al. (2017) Long-term persistence and function of hematopoietic stem cell-derived chimeric antigen receptor T cells in a nonhuman primate model of HIV/AIDS. PLoS Pathog 13:e1006753
Suryawanshi, Gajendra W; Xu, Song; Xie, Yiming et al. (2017) Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow. J Vis Exp :
Zhen, Anjie; Carrillo, Mayra A; Kitchen, Scott G (2017) Chimeric antigen receptor engineered stem cells: a novel HIV therapy. Immunotherapy 9:401-410
Peterson, Christopher W; Haworth, Kevin G; Burke, Bryan P et al. (2016) Multilineage polyclonal engraftment of Cal-1 gene-modified cells and in vivo selection after SHIV infection in a nonhuman primate model of AIDS. Mol Ther Methods Clin Dev 3:16007
Kitchen, Scott G; Zack, Jerome A (2016) Engineering HIV-Specific Immunity with Chimeric Antigen Receptors. AIDS Patient Care STDS 30:556-561
Pernet, Olivier; Yadav, Swati Seth; An, Dong Sung (2016) Stem cell-based therapies for HIV/AIDS. Adv Drug Deliv Rev 103:187-201
Ali, Ayub; Kitchen, Scott G; Chen, Irvin S Y et al. (2016) HIV-1-Specific Chimeric Antigen Receptors Based on Broadly Neutralizing Antibodies. J Virol 90:6999-7006
Zhen, Anjie; Kamata, Masakazu; Rezek, Valerie et al. (2015) HIV-specific Immunity Derived From Chimeric Antigen Receptor-engineered Stem Cells. Mol Ther 23:1358-1367