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California State University, Northridge CALIFORNIA STATE UNIVERSITY, NORTHRIDGE Characterization of Chromosomal Alterations Using a Zinc-Finger Nuclease Targeting the Beta-globin Gene Locus in Hematopoietic Stem/Progenitor Cells A thesis submitted in partial fulfillment of the requirements For the degree of Master of Science in Biology By Joseph D. Long May 2017 The thesis of Joseph Long is approved: Dr. Jonathan Kelber Date Dr. Virginia Vandergon Date Dr. Cindy S. Malone, Chair Date California State University, Northridge ii Acknowledgements First, I want to thank Dr. Cindy Malone for making this opportunity and ultimately this work possible. The experiences I have had and everything that I have learned from Dr. Malone and at UCLA through the Bridges program have been invaluable to becoming a better scientist and person. While the work presented here was done through the Kohn Lab at UCLA, it was based on a solid foundation of skills learned in Dr. Malone’s lab and in courses taught by Dr. Virginia Vandergon and Dr. Jonathan Kelber at CSUN. This foundation enabled me to achieve a higher level of scientific development at UCLA that I would not have been able to reach otherwise. Second, I want to thank Dr. Donald Kohn, Dr. Zulema Romero and Dr. Caroline Kuo for making this work possible and for their incredible help and advice throughout the course of this project. Also, I want to thank everyone else in the Kohn lab for all of their contributions to this project. iii Table of Contents Signature Page…………………………………………….………………………...….....ii Acknowledgments…………………………………..………...………………….....……iii List of Figures………………......…………………………………………………...…....vi List of Abbreviations……………………………………………...…………………….viii Abstract…………………………………………………….….………………………….ix Introduction………………………………………...………………..……...........1 Materials and Methods..............................................................................4 Cell Culture..................................................................................4 Cel-1 Surveyor Nuclease Assay...........................................................4 Rearrangement Detection by PCR........................................................4 Rearrangement Detection by ddPCR.....................................................5 Table 1. PCR primers to amplify HBB and HBD Nuclease Cleavage Sites........6 Table 2. Probes and Primers used for ddPCR...........................................6 Table 3. Oligo Sequences for High-Throughput Sequencing.........................7 Sanger Sequencing of PCR-Amplifiied Rearrangement Events in CFUs...........8 High-Throughput Sequencing.............................................................8 Statistical Analysis..........................................................................9 Results…………………………………………………………………...……………….10 Detection of gene rearrangements between beta- and delta-globin genes using PCR amplification……………………………………………….….….….….….11 iv Gene Rearrangements dependent on cleavage at both HBB and HBD rather than gene conversion............................................................................16 Chromosomal Rearrangements Observed using CD34+ Cells from Bone Marrow from Donors with Sickle Cell Disease..................................................22 High-Throughput Sequencing to Further Characterize the Rearrangement Events.......................................................................................27 Discussion………………………………………………………………………………..33 References...........................................................................................38 v List of Figures and Tables Table 1: PCR primers to amplify HBB and HBD nuclease cleavage sites………...….......6 Table 2: Probes and primers used for droplet digital PCR…...…………………..……….6 Table 3: Oligo sequences for high throughput sequencing ...…………………..………....7 Figure 1: Potential rearrangement events occurring as the result of on-target cleavage in HBB and off-target cleavage in HBD…………………………………………..10 Figure 2: Detection of rearrangement events by PCR in CB CD34+ cells treated with 1ug of ZFN mRNA …………………………………………………………………….12 Figure 3: Detection of rearrangement events by PCR in CFU from CD34+ cells treated with 1ug of ZFN mRNA ………………………………………………………14 Figure 4: Representative examples of Sanger sequencing alignments for each rearrangement event in CFUs from SCD BM CD34+ cells treated with 1 ug of ZFNs mRNA …………………………………………………………………..15 Figure 5: Schematic showing the binding sites of both ZFN pairs, 88/01 and 55/58 at the HBB and HBD locus …………………………………………………………..18 Figure 6: Allelic disruption measured by the surveyor nuclease assay at HBB and HBD in samples treated with ZFNs 88/01 or ZFNs 55/58……………………………...19 Figure 7: Detection of rearrangement events by ddPCR in CD34+ cells from CB using ZFNs with (55/58) or without (88/01) off-target cleavage at the HBD………..20 Figure 8: Detection of rearrangement events by ddPCR in pooled K562s and BM CD34+ cells ……………………………………………………………………………21 vi Figure 9: Detection of rearrangement events by ddPCR in bulk cells and CFUs derived from SCD BM CD34+cells ……………………………………………………23 Figure 10: Allelic disruption in SCD BM CD34+ samples detected by surveyor nuclease assay………………………………………………………………………….24 Figure 11: Representative ddPCR plots for CFU sample rearrangement detection to show mono vs. bi-allelic modification determination ……………………………..25 Figure 12: High throughput sequencing of HBB rearrangements in ZFN treated SCD BM CD34+ cells …………………………………………………………………28 Figure 13: Detection of rearrangement events by high-throughput sequencing in CD34+ cells treated with 1ug of ZFN mRNA ……………………………………….29 Figure 14: Low frequency Self-Self translocation event detected by high throughput sequencing …………………………………………………………………...31 Figure 15: Low frequency of rearrangement events connecting beta-globin to other non- beta or delta globin loci detected by high throughput sequencing …………..32 vii List of Abbreviations HSPC: Hematopoietic Stem/Progenitor Cell CFU: Colony Forming Unit SCD: Sickle Cell Disease BM: Bone Marrow CB: Cord Blood HBB: Beta-Globin Gene HBD: Delta-Globin Gene HBG: Gamma-Globin Gene shRNA: Short-Hairpin RNA ZFN: Zinc-Finger Nuclease DSB: Double Stranded Break NHEJ: Non-Homologous End Joining TALEN: Transcription Activator-Like Effector Nuclease IDLV: Integrase Defective Lentiviral Vector ddPCR: Droplet Digital PCR CRISPR: Clustered Regularly Interspaced Short Palindromic Repeat Cas9: CRISPR Associated Protein 9 gRNA: single-guide RNA HDR: Homology Directed Repair viii Abstract Characterization of Chromosomal Alterations Using a Zinc-Finger Nuclease Targeting the Beta-globin Gene Locus in Hematopoietic Stem/Progenitor Cells By Joseph Long Master of Science in Biology The use of engineered nucleases for targeted gene correction of the sickle mutation in hematopoietic stem and progenitor cells (HSPCs) combined with a homologous DNA donor template can result in targeted gene correction. However, due to a high sequence homology existing between the beta- and delta-globin genes, off-target cleavage events have been observed at delta-globin when using endonucleases targeted to the sickle mutation. Moreover, the introduction of multiple double stranded breaks by endonucleases has the potential to induce chromosomal alterations such as translocations, deletions, and inversions. We have developed a Droplet Digital PCR assay to characterize the frequency of deletions, inversions and translocations between the beta- and delta-globin paralogs when delivering these nucleases. Pooled CD34+ cells and colony forming units (CFUs) from SCD BM donors were treated with nuclease only or nuclease with a DNA donor template (as an integrase defective lentiviral vector or as a ix single stranded oligonucleotide); and then analyzed for each of the potential chromosomal rearrangements. We observed that in both pooled and CFU samples, the intergenic β-δ-globin deletion was the most frequent event, followed by the inversion of the intergenic fragment, and with the interchromosomal translocation as the least frequent. These findings demonstrate the need to develop site-specific endonucleases with high specificity to avoid unwanted chromosomal alteration x Introduction Sickle cell disease (SCD) is one of the most common autosomal recessive disorders worldwide, caused by an adenine to thymine transversion in the sixth codon of the beta-globin gene (HBB) (Ingram 1957). As a result of this mutation, the SCD red blood cells become adhesive and non-deformable, leading to vaso-occlusive events, organ crises, and chronic pain compromising the quality of life and lifespan of the patients. Current therapies are based on palliative treatment for painful crises, and allogeneic bone marrow (BM) transplantation is the only available cure. Autologous stem cell gene therapy has become a promising approach that avoids the immunologic limitations of allogeneic hematopoietic stem/progenitors cell (HSPC) transplantation (Bolanos-Meade and Brodsky 2009). Current gene therapy strategies focus on gene addition by integrating viral vectors of an anti-sickling HBB (Pawliuk et al 2001; Levasseur et al 2003; Romero and Urbinati 2013), a gamma-globin gene (HBG) (Persons, Hargrove et al. 2003; Perumbeti et al 2009) or shRNA to the HBG repressor BCL11A (Guda et al. 2015). However, gene addition by viral vectors has the potential risk of genotoxicity (Cavazzana-Calvo, Hacein-Bey et al. 2000; Hacein-Bey-Abina, Von Kalle et al. 2003; Ott, Schmidt et al. 2006) and may be limited by gene expression variegation or silencing (Challita and
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