Stem Cells Gene Therapy Zoran Galic Ph.D . UCLA School of Medicine 310/825-4671 zgalic@ucla.edu

1. Stem Cells Gene Therapy ZoranGalic Ph.D. UCLA School of Medicine 310/825-4671 zgalic@ucla.edu

2. Gene Therapy Gene transfer to human cells in order to restore, modify or enhance specific cellular functions • Characterize a genetic defect at molecular/gene level • Use a suitable viral or non-viral vector to introduce the correct copy of the defective gene into affected cells (ex vivo or in vivo) • Introduce the modified cells back into the patients (for ex vivo gene transfer)

3. What is a Stem Cell? • Self-renews (long-lived); • A single cell can differentiate into multiple, functional cell types A self-renew B stem cell stem cell C differentiated cells

4. Adult Stem Cells • Stem cells in tissues respond to demands of growth or repair. • Adult stem cells exist in: • Bone marrow • Skin • Prostate • Mammary gland • Liver • Skeletal muscle • Heart muscle • Dental pulp • Colon • Brain • Fat tissue

5. Why use stem cells for gene therapy? A self-renew B Stem cells are a self-renewing population of cells and thus may reduce or eliminate the need for repeated gene therapy stem cell stem cell C differentiated cells

6. Stem Cell-based Gene Therapy The Food and Drug Administration (FDA) has not yet approved any human gene therapy product for “sale”. > 50 gene therapy clinical trials in the United States using human stem cells (for cancer, HIV and single-gene disease treatments). The only type of human stem cell successfully used in gene therapy trials so far is the hematopoietic stem cell. However, several other types of stem cells are being studied as gene-delivery-vehicle candidates (mesenchymal stem cells, myoblasts, osteoblasts, and neural stem cells). The National Institute of Health- Stem Cell Information (http://stemcells.nih.gov/info/scireport/chapter11.asp)

7. Why use hematopoietic stem cells (HSC) for gene therapy? HSC are easy to: -obtain(circulating blood or bone marrow from adults or the umbilical cord blood of newborn infants), -identify/manipulate(cell markers known/variety of reagents available), -introduce back into patients(simple infusion).

8. Hematopoietic Stem Cell (HSC) Gene Therapy Anti-HIV Gene Myeloid stem cell Lymphoid stem cell Bone Marrow HSC B progenitor Basophil progenitor T progenitor Eosinophil progenitor Myelomonocytic progenitor Erythroid progenitor Megakaryoblast DP Thymocyte Monocyte NK Cell Megakaryocyte CD8+ T cell CD4+ T cell Red blood cells Platelets Eosinophil Basophil Neutrophil B cell Macrophage

9. Nature Reviews Cancer3, 477-488 (2003);

10. Map of Retrovirus and Retroviral Vector A. Retrovirus y+ 5’ LTR gag pol env 3’ LTR D A B. Retroviral Vector y+ 5’ LTR Exogenous gene (s) 3’ LTR D E/P ATG Stop Vector RNA AAAA Therapeutic Protein

11. Retroviral Vector Particle Exogenous Gene(s) Human Gene LTR LTR RT Int GAG POL ENV Ecotropic Ampho GALV RD114 VSV-G Pro

13. Retroviral gene-transfer scheme Retroviral gene-transfer scheme. Molecules on the vector envelope interact with cell-surface receptors before the viral envelope is removed and the core, including viral mRNA, enters the cytoplasm. After reverse transcription, the double-stranded DNA circularizes to an episomal form. The pre-integration-complex DNA contains two LTR sequences and the therapeutic gene. The LTR sequences encode the promoter, repressor and enhancer regions that regulate retroviral gene expression. A purine-rich sequence upstream of the 3’ LTR directs reverse transcription; whereas specific sequences near the ends of each LTR are essential for genomic integration. For MLV to access chromosomes, the nuclear membrane must be dissolved (e.g. during mitosis). Once integrated into the host genome, transcription of the therapeutic gene ensues. TRENDS in Biotechnology Vol.23 No.12, 2005

14. Summary of current gene-therapy protocols in humans Summary of current gene-therapy protocols in humans. Harvested or mobilized HSCs are CD34 enriched and cultured with fibronectin, cytokines and retroviral supernatant for one to two days (lentiviral vectors) or three to four days [murine leukemia virus (MLV)-based vectors]. Autologously transplanted cells migrate to the marrow and initiate hematopoiesis. Flow cytometry and molecular biology methods can be used to evaluate the procedure's efficiency. TRENDS in Biotechnology Vol.23 No.12, 2005

15. GMP Level Closed System Gene Transduction and Cell Processing Apheresis Final Infusion Product Cytomate Gene Transfer Product Isolex Culture Isolex

16. RNA interference RNA interference (RNAi) also called post transcriptional gene silencing (PTGS), is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Ribozymes-RNA catalytic molecules that have the ability to cleave RNA at a specific sequence.

17. NATURE MEDICINE VOLUME 15 (3) 2009 Phase 2 gene therapy trial of an anti-HIV ribozymein autologous CD34+ cells Ronald T Mitsuyasu1, Thomas C Merigan2, Andrew Carr3, Jerome A Zack4, Mark A Winters2, Cassy Workman5, Mark Bloch6, Jacob Lalezari7, Stephen Becker8, Lorna Thornton8, Bisher Akil9, Homayoon Khanlou10, Robert Finlayson11, Robert McFarlane12, Don E Smith13, Roger Garsia14,David Ma3, Matthew Law15, John M Murray15,16, Christof von Kalle17,18, Julie A Ely19, Sharon M Patino19, Alison E Knop19, Philip Wong19, Alison V Todd19, Margaret Haughton19, Caroline Fuery19, Janet L Macpherson19, Geoff P Symonds19, Louise A Evans19, Susan M Pond19 & David A Cooper3,15.

18. Complementary Complementary Flanking Sequence Flanking Sequence Hammerhead Ribozyme (Specificity is determined by the base pairing of the complementary arms) Cleavage site 3' 5' GGAGCCAGUAGAUCCUAA TARGET RNA RIBOZYME CUAGGAUU CUCGGUCA 5' 3' C A U G A A G U A G C G A U G C Catalytic G C Domain • RNA, hybridising arms • True enzymes-catalytic domain • Nucleophilic attack after GUA A G U G Haseloff & Gerlach, 1988

19. How Might anti-HIV Ribozyme Work? Mature retroviral particle Ribozyme • Cleavage of tat region • Sustained ribozyme expression in the relevant cell types • Inhibition of HIV replication • Enhanced cell viability in vitro Attachment CD4 Assembly Fusion Chemokine receptor Uncoating     RT inhibitor  Translation Structural proteins Reverse Transcription   Integrated proviral DNA Transcription  Multply spliced mRNAs  Preintegration complex Nucleus

20. Control Ribozyme Pre-Clinical Results • Cleavage in test-tube • Sustained ribozyme expression • Inhibition of HIV replication in T-Cell lines and PBMC • Enhanced cell viability • No development of resistance Sun et al (1994) PNAS 91, 9715; Sun et al (1995) PNAS 92, 7272; Wang et al(1998) Hum Gene Ther 9, 1283

21. HIV-Infected individual Phase I Trial - CD34+ Stem Cells UCLA Drs Amado, Mitsuyasu G-CSF Unpurified precursor blood cells Infuse transduced cells • Results • Ten patients enrolled • Gene transfer successful • No safety concerns to date • Analysis from 0 to 3 years • Lineages containing and expressing the ribozyme in all patients • Persistence dependenton CD34+ cell dose CD34+ progenitor cells RIBOZYME Transduction CONTROL

22. Clinical Trials: anti-HIV Ribozyme Phase I 10 adult patients treated with anti-HIV tat/vprribozyme plus control vector transduced into autologous CD34+ cells; Therapeutic gene and control were present long-term (3 years); Transduced genes were present in naïve T cells, however the number of transduced cells was low.

23. Phase II • 3 centers involved; 74 total patients enrolled (19 treated at UCLA); • The numbers of transgenic peripheral blood cells were higher than in the Phase I clinical trial, but still very low (0.01% to 0.38%), indicating poor HSC engraftment; • Very modest antiviral effect.

24. Conclusions: • Ribozyme/stem cell-delivered gene therapy is safe and has antiviral • activity, albeit modest; • Large number of subjects can be treated similarly at several distinct • clinical sites, indicating that this type of approach can be scaled up to treat • substantial numbers of patients; • These trials show the potential of the gene therapy approach for the treatment • of HIV-1. • Areas for Maximizing Effect: • number of gene-containing cells in the peripheral blood and lymphatic tissue; • apparent efficacy of anti-HIV agent; • improve relatively laborious cell processing.

25. Downregulation of CCR5 expression as a therapeutic approach to HIV infection • Important points: • Naturally occurring CCR5 mutations/deletions do not cause any obvious functional • defects in the affected individuals; • Experimental data suggest that downregulation of CCR5 expression on target cells • profoundly reduces the rate of HIV infection; • The case of the “Berlin patient”.