1. CCR5 Gene Edited Cells Traffic to Viral Reservoir Tissues and Undergo SHIV-Dependent Positive Selection Chris Peterson Hans-Peter Kiem Lab Fred Hutchinson Cancer Research Center Seattle, WA, USA

2. Timothy Brown: Road to HIV Functional Cure HIV + Acute Myeloid Leukemia Patient Identification of HLA-matched, CCR5Δ32 homozygous bone marrow donor Chemo- and Radiotherapy Conditioning Allogeneic stem cell transplant Withdrawal of cART >9 years later: remains functionally cured

3. ZFN Editing of CCR5 in Pigtailed Macaque CD34 + Cells for Autologous Transplant Enrich CD34 + cells from primed bone marrow Culture 24hr, 37° ex vivo (TPO, SCF, FLT-3 Ligand) Electroporate cells with ZFN mRNA, recover overnight at 30°C Infuse into conditioned animal Animal receives myeloablative conditioning regimen consisting of 1,020 cGy total body irradiation

4. Two Experimental Schemes to Evaluate ΔCCR5 Gene Therapy SHIV Status? ΔCCR5 Transplant SHIV Challenge ΔCCR5-SHIV cART ΔCCR5 Transplant SHIV Infection SHIV-ΔCCR5 cART Withdrawal

5. Checklist For Cell Therapy- Based HIV Cure Strategies Can the cells be engineered? Do the cells engraft in vivo and protect against viral replication? Are the cells present at secondary tissue sites?

6. CCR5 Stem Cell Products Can Be Efficiently Generated from SHIV + and SHIV - Animals

7. ΔCCR5 CD4 + Subsets Undergo SHIV-Dependent Selection ΔCCR5-SHIV n = 4 SHIV-ΔCCR5 n = 4

8. ΔCCR5 Cells Traffic to Secondary Lymphoid Tissues ΔCCR5-SHIV n = 4 SHIV-ΔCCR5 n = 5

9. Checklist For Gene Therapy- Based HIV Cure Strategies Can the cells be engineered? Do the cells engraft in vivo and protect against viral replication? Are the cells present at secondary tissue sites?

10. What Happens to the Viral Reservoir?

11. QVOA: Transplantation Does Not Increase the Size of the Latent SHIV Reservoir No Transplant Transplant *Undetectable by QVOA: Plotted as 0.01 IUPM for Graphing Purposes

12. Engraftment of 5% ΔCCR5 Cells is Insufficient for cART-Independent Virus Suppression SHIVSHIV + cARTSHIV Transplant No Transplant wt CCR5 transplant ΔCCR5 Transplant

13. ΔCCR5 Cells Engraft Long-Term in vivo SHIV SHIV + cART

14. Primitive NHP CD34 + Cells are Edited Less Efficiently than More Differentiated Subsets CD45RA CD90 CD34 + Stem Cells Primitive EarlyCommitted Radtke…Kiem et al, Submitted

15. Conclusions and Future Directions ΔCCR5 stem cells and progeny engraft in blood and tissues in SHIV - and SHIV + animals Target primitive stem cells to increase percentage of ΔCCR5 cells in vivo Transplantation reduces, but does not eliminate the latent viral reservoir Maximize immune recovery following transplantation and prior to cART withdrawal ΔCCR5 CD4 + T-cells undergo SHIV-dependent positive selection Evaluate T-cell gene therapy and “offensive” means of clearing latently infected cells

16. Thank You… Kiem Lab Hans-Peter Kiem Sowmya Reddy Jasbir Kaur Willi Obenza Kevin Haworth Stefan Radtke Zach Norgaard Olivier Humbert Bish Paul Devikha Chandrasekaran Anne-Sophie Kuhlmann Martin Wohlfahrt Sarah Weitz Christina Ironside Shirley Chan Morgan Giese Don Gisch Stefanie Schmuck Megan Atkins Frieda Chan Abbi Helfer Teresa Einhaus Grace Choi Anne Lee Jacob Isenberg Eva Pokhrel Cynthia Shen Adair Lab, FHCRC Jerome Lab, FHCRC Steve DeRosa, FHCRC Hu Lab, U. Washington Sékaly Lab, Case Western Tae-Wook Chun Sangamo BioSciences Funding U19 AI096111 U19 HL129902 R01 AI080326 Animal Technicians: Kelvin Sze, Veronica Nelson, Erica Curry