Genome Editing for Thalassemia CAF Patient-Family Conference 21 June 2014 Daniel E. Bauer, MD PhD

Disclosures Consultant for Editas Medicine

Genetics: each cell carries a genome ASHG.org

The genome is composed of DNA, 3 billion positions education-portal.com

The genome includes lots of DNA … education-portal.com

… with many genes …

… and even more non-coding DNA. education-portal.com

Genome editing

Genome editing tools are sequence-specific nucleases van der Oost. Science (2013) 339:768. Genome editing tools have two features: 1)Recognize specific DNA sequences (i.e. specific genes or non-coding elements)

Genome editing tools are sequence-specific nucleases van der Oost. Science (2013) 339:768. Genome editing tools have two features: 1)Recognize specific DNA sequences (i.e. specific genes or non-coding elements)

Genome editing tools are sequence-specific nucleases van der Oost. Science (2013) 339:768. Genome editing tools have two features: 1)Recognize specific DNA sequences (i.e. specific genes or non-coding elements)

Genome editing tools are sequence-specific nucleases van der Oost. Science (2013) 339:768. Genome editing tools have two features: 1)Recognize specific DNA sequences (i.e. specific genes or non-coding elements) 2)Cut DNA (“nuclease”), then a scar is left behind

Genome editing: cleavage repair can either disrupt original sequence or replace it with a new copy NEB.com

Genome editing: cleavage repair can either disrupt original sequence or replace it with a new copy NEB.com “delete”

Genome editing: cleavage repair can either disrupt original sequence or replace it with a new copy NEB.com “delete”“copy and paste”

Two strategies for genetic therapy: gene addition and genome editing Fischer. Nature (2014) 510:226.

Two strategies for genetic therapy: addition and editing Gene addition: Feasible with existing technology; clinical trials ongoing. Early trial results appear exciting. Challenges: 1. Will enough of the added gene be made in the cells with the integration? Will enough of the blood stem cells have the added gene? 2. Is the benefit durable? Will the added gene continue to function over days, weeks, months, years, decades? 3. Is the added gene safe? Will its semi-random integration into the genome change the function of other genes in the genome? Fischer. Nature (2014) 510:226.

Two strategies for genetic therapy: addition and editing Gene editing: Promise of permanent repair of the underlying disease-causing mutation. Promise of specific beneficial change at the intended genomic site (e.g.  - globin gene) without impacting remainder of genome. Challenges: 1. Technology is in a relatively early stage and needs to be further developed. 2. Can enough cells be edited to have therapeutic impact? 3. Will the editing be exquisitely specific, or will other regions of the genome aside from the target be affected? Fischer. Nature (2014) 510:226.

Thalassemia is caused by mutations of the  or  -globin genes

Many different mutations of the  -globin gene cause  -thalassemia

The problem in  -thalassemia is too much  -globin relative to  -globin

 -globin (fetal hemoglobin) can functionally substitute for  -globin (adult hemoglobin)

BCL11A enhancer determines fetal hemoglobin level Hardison and Blobel. Science (2013) 342:206.

BCL11A enhancer determines fetal hemoglobin level Hardison and Blobel. Science (2013) 342:206.

BCL11A enhancer determines fetal hemoglobin level Hardison and Blobel. Science (2013) 342:206.

Collect blood stem cells from patient with  -thalassemia Introduce sequence-specific nucleases to disrupt BCL11A enhancer Reinfuse modified blood stem cells to patient The vision: mimicking common protective genetic variation for therapeutic benefit

Collect blood stem cells from patient with  -thalassemia Introduce sequence-specific nucleases to disrupt BCL11A enhancer Reinfuse modified blood stem cells to patient Potential benefits: Loss of BCL11A expression in red blood cells causing increased fetal hemoglobin Spares BCL11A expression in other blood cells Modification would be permanent Survival advantage of cells (would outcompete unmodified cells) Compared to gene addition, no semi-random insertion of material into the genome, and no need for lifelong expression of foreign sequences The vision: mimicking common protective genetic variation for therapeutic benefit

Collect blood stem cells from patient with  -thalassemia Introduce sequence-specific nucleases to disrupt BCL11A enhancer Reinfuse modified blood stem cells to patient Potential benefits: Loss of BCL11A expression in red blood cells causing increased fetal hemoglobin Spares BCL11A expression in other blood cells Modification would be permanent Survival advantage of cells (would outcompete unmodified cells) Compared to gene addition, no semi-random insertion of material into the genome, and no need for lifelong expression of foreign sequences Potential risks: Genome modification at sites other than the intended target Preparation (“conditioning”) therapy for stem cell transplant (shared risk of both gene addition and genome editing; potentially much less toxic than for “allotransplant” (from related or unrelated donor) The vision: mimicking common protective genetic variation for therapeutic benefit

Summary  -thalassemia results from mutations in  -globin, a single gene within a large genome Gene addition adds a copy of  -globin by semi- random integration into the genome –Currently being tested in early-phase clinical trials –Challenges include: durable high-level expression; ensuring other important genes are not disrupted due to integration Genome editing offers the promise of precise and permanent genome modification to mimic protective genetic variation (e.g. at BCL11A) or to repair  -globin –Challenges include: effective delivery of genome editing tools to cells to achieve efficient target disruption/repair; ensuring modification is limited to intended target

Acknowledgments Boston Children’s Ellis Neufeld David Williams David Nathan Jennifer Eile Alan Cantor Bill Pu Dana-Farber Cancer Institute GC Yuan Luca Pinello Broad Institute Feng Zhang Neville Sanjana Ophir Shalem Boston Children’s Hospital Stuart Orkin Jian Xu Vijay Sankaran Sophia Kamran Matthew Canver Carrie Lin Abhishek Dass Yuko Fujiwara Zhen Shao E. Crew Smith Cong Peng Hojun Li Montreal Heart Institute Guillaume Lettre Samuel Lessard Stanford University Matthew Porteus Richard Voit University of Washington John Stamatoyannopoulos Peter Sabo Jeff Vierstra

Feasibility The vision: mimicking common protective genetic variation for therapeutic benefit ASH 2013 Abstracts #434 and Slide courtesy of Sangamo BioSciences.

Feasibility The vision: mimicking common protective genetic variation for therapeutic benefit Tebas et al. NEJM (2014) 370:901.