CRISPR Therapeutics AG (CRSP) Earnings Call Transcript & Summary

I'm Taby Ahsan, Vice President of Cell and Gene Therapy at Operations at the City of Hope. I'm acting Chair for today's meeting. I'd like to welcome everyone to the 76th meeting of the Cellular Tissue and Gene Therapies Advisory Committee for the Center for Biologics Evaluation and Research at the Food and Drug Administration. Today's meeting will meet an open session to discuss and make recommendations on the BLA 125787 from Vertex Pharmaceuticals for exa-cel. The applicant is requested in indications for the treatment of sickle cell disease in patients 12 years and older with current vaso-occlusive crisis. So I'd like to welcome all the committee members, the participants and the public that's viewing remotely. Again, I want to remind committee members and participants to use the Raise Your Hand feature and turn on your camera when you have a question or comment to make, and then I can recognize you, and then you can be called out to speak. And so with that, I'd like to introduce Cicely Reese, the Designated Federal Officer for today's meeting to make administrative announcements, conduct roll call and read the conflict of interest statement.

Thank you, Dr. Ahsan. Good morning, everyone. I am Cicely Reese, and it is my honor to serve as the Designated Federal Officer for today's 76th Cellular, Tissue and Gene Therapies Advisory Committee meeting. On behalf of the FDA, the Center for Biologics Evaluation and Research and the committee, I am happy to welcome everyone for today's virtual meeting. Today, the committee is meeting an open session to discuss and make recommendations on Biologics License Application 125787 from Vertex Pharmaceuticals, Inc. Today's meeting and topic were announced in the Federal Register notice that was published on September 7, 2023. Next slide, please. At this time, I would like to acknowledge and thank my Division Director, Dr. -- Division of Scientific Advisors and Consultants, Dr. Prabha Atreya. My team whose contributions have been critical for preparing today's meeting. Those persons include Ms. Tonica Burke; Ms. LaShawn Marks; Ms. Joanne Lipkind and many others from the division who have provided helpful and administrative support and preparation of this meeting. Next slide, please. I would now like to acknowledge CBER leadership, including Dr. Peter Marks, Director of CBER, Dr. Celia Witten, Deputy Director of CBER, Dr. Nicole Verdun, the new Director of CBER's Office of Therapeutic Products and many other OTP staff who have been -- who will be serving as speakers and presenters during the day as indicated on the agenda. On behalf of DSAC, our sincere gratitude also goes to many CBER and FDA staff working very hard behind the scenes to ensure that today's virtual meeting will also be a successful one. I also thank all other FDA staff contributing to today's discussion, some of whom are present and others who may be joining the meeting at other times. Next slide, please. Please direct any press or media questions for today's meeting to FDA's Office of Media Affairs at fdaoma@fda.hhs.gov. I would like to thank the audiovisual team, Ms. Gretchen Carter, DeVante Stephenson and Derek Bonner for facilitating today's meeting. The transcriptionist for today's meeting is Ms. Debbie Delacroix, and we will begin today's meeting by taking a formal roll call for the committee members and temporary voting members. When it is your turn, please make sure you turn on your video camera and you are unmuted. Then state your first and last name, organization, expertise or role. And when finished, you may turn off your camera, so we may proceed to the next person. Please see the member roster slides -- next slide, please, in which we will begin with the chair. Dr. Ahsan, please go ahead and introduce yourself. Thank you.

Good morning. Thank you, Cicely. So I'm Taby Ahsan, I'm Vice President for Cell and Gene Therapy Operations at the City of Hope. My expertise is in biomedical engineering or I'm a bioengineer by training, particularly in the applications of stem cells, tissue engineering and [indiscernible] Immunotherapies.

Thank you. Next. Dr. Breuer.

My name is Chris Breuer, I'm the Director of the Regenerative Medicine Center at Nationwide Children's Hospital in Columbus, Ohio, and my expertise is in translational research and Regenerative Medicine. Thank you.

Thank you. Dr. Crombez.

I'm Eric Crombez, Chief Medical Officer at Ultragenyx. I've been working in the field of gene therapy for the past 9 years trained in pediatrics and genetics and I'll be serving as the industry representative.

Thank you. Dr. London. Good morning. I'm Wendy London. I'm a biostatistician from Dana-Farber/Boston Children's Hospital, the Director of Biostatistics within pediatric hemoc, and my expertise is in prognostic factors for neuroblastoma. And I've served as a study statistician on many trials for pediatric oncology and sickle cell disease.

Thank you. Next slide, please. Dr. Kathleen O'Sullivan-Fortin.

Hi. I'm Kathleen O'Sullivan-Fortin. I'm a patient advocate and the Co-Founder of ALD Connect, and I'll be serving as the consumer representative.

Thank you. Dr. Ott.

Good morning, everybody. My name is Melanie Ott. I'm the Director of the Gladstone Institute of Virology and a Professor of Medicine at UCSF in San Francisco. My expertise is in viral pathogenesis, viral vectors and delivery. Thank you.

Thank you. Dr. Wu.

Good morning, everyone. My name is Joe Wu. I'm the Director of the Stanford Cardiovascular Institute. I'm Professor of Medicine and Radiology. My expertise is in cardiac cell therapy, gene therapy and organ [indiscernible].

Thank you. Next slide, please. Next, we will do roll call of our temporary voting members starting with Dr. Robert Dracker.

Thanks for letting me attend this meeting. I am currently being Chairperson of Pediatric Advisory Committee for the FDA. I'm a pediatrician, hematologists, oncologists and transfusion medicine specialists, and I'm [indiscernible] New York and Medical Director of Summerwood Pediatrics and in Patient Care Medical Services. Thank you.

Ms. Hightower.

My name is Jasmine Hightower. I am a patient advocate for sickle cell. I am also a patient. I have a background in Masters in Social Work. And I am currently on the Board and on many sickle cell and rare disease advisory committees. And I will be your patient representative today.

Thank you very much. Dr. Komor.

I'm Alexis Komor. I'm an assistant professor of chemistry and biochemistry as well as the Deputy Director of the Sanford Stem Cell Innovation Center at the University of California, San Diego, and my expertise is in genome editing.

Thank you. Dr. Lee.

Good morning. My name is Lisa Lee. I serve as the Associate Vice President for Research and Innovation at Virginia Tech, where I also serve as a professor of Public Health. I'm trained in epidemiology and public health ethics, and I am serving today as the bioethicist for the panel.

Thank you. Next slide, please. Dr. Shapiro.

Good morning. My name is Amy Shapiro. I'm a pediatric hematologist oncologist. I am CEO and Medical Director of the Indiana Hemophilia and Thrombosis Center. My area of expertise is hemostasis, thrombosis, classical hematology, including sickle cell and clinical research. Thank you.

Thank you. Dr. Tisdale.

I'm John Tisdale. I am Chief of the Cellular and Molecular Therapeutics branch at NHLBI, and I've been working on transplant and gene therapy for sickle cell disease for now almost 30 years.

Thank you. Dr. Wolfe.

Good morning. I'm Scot Wolfe. I'm a professor at UMass Chan Medical School. My lab focuses on genome editing and off-target analysis. Excited to be here.

Thank you very much. And thank you to everyone. There are a total of 14 participants, 13 voting members and 1 nonvoting member. Thank you very much for your introductions. Now I will move to the conflict of interest statement. Before I begin reading the conflict of interest statement, I would just like to briefly mention a few housekeeping items related to today's virtual meeting format. For members, speakers, FDA staff and anyone else joining us in the Zoom room, please keep yourself on mute unless you are speaking to minimize feedback. If you have raised your hand and are called upon to speak by the Chair, Dr. Ahsan, please turn on your camera, unmute, state your name and speak slowly and clearly so that your comments are accurately recorded for transcription and captioning. Thank you. I will now proceed with the reading of the conflict of interest statement for the public record. Thank you. Dated October 23 -- October 31, 2023, FDA conflict of interest disclosure statement. Read for the public record by Cicely Reese, Designated Federal Officer division of Scientific Advisory consultants, DFO for this committee meeting. The Food and Drug Administration is convening virtually today, October 31, 2023, the 76th meeting of the cellular Tissue and Gene Therapies Advisory Committee under the authority of the Federal Advisory Committee Act of 1972. Dr. Taby Ahsan is serving as the acting chair for today's meeting. Today, October 31, 2023, the committee will meet an open session to discuss and make recommendations on biologics license application, BLA 125787 from Vertex Pharmaceuticals, Inc. For exagamglogene autotemcel or exa-cel, the applicant has requested an indication for the treatment of sickle cell disease in patients 12 years and older with recurrent vaso-occlusive crises. This topic is determined to be a particular matter involving specific parties or PMISP. With the exception of the industry representative member, all standing and temporary voting members of CTGTAC are appointed as special government employees or regular government employees from other agencies, and are subject to federal conflicts of interest laws and regulations. The following information on the status of this committee's compliance with federal ethics and conflict of interest laws include, but are not limited to, 18 USC Section 208 which is being provided to participants in today's meeting and to the public. Related to the discussions at this meeting, all members and RTE and SGE consultants of this committee have been screened for potential financial conflicts of interest of their own as well as those affiliated to them, including those of their spouse or minor children. And for the purposes of 18, USC -- 18 U.S. Code Section 208, their employers. These interests may include investments, consulting, expert witness testimony, contracts and grants, cooperative research and development agreements also called CRDAs, teaching speaking, writing, patents and royalties and primary employment. These may include interests that are current or under negotiation. FDA has determined that all members of this advisory committee, both regular and temporary members are in compliance with federal ethics and conflict of interest laws. Under 18 U.S. Code Section 208, Congress has authorized FDA to grant waivers to special government employees who have financial conflicts of interest when it is determined that the agency's need for a special government employee services outweighs the potential for conflict of interest created by the financial interest involved or when the interest of a regular government employee is not so substantial as to be deemed likely to affect the integrity of the services, which the government may expect from the employee. Based on today's agenda and all financial interest reported by committee members and consultants, 1 conflict of interest waiver was issued under 18 U.S. Code Section 208 in connection with this meeting. We have the following consultants serving as temporary voting members. Dr. Robert Dracker, Dr. Lisa Lee, Dr. Amy Shapiro, Dr. John Tisdale, Dr. Scot Wolfe, Dr. Alexis Komor and Ms. Jasmine Hightower. The following member has been issued a conflict of interest waiver for participation in today's meeting, Dr. Wendy London. The waiver is posted on the FDA website for public disclosure. Ms. Kathleen O'Sullivan-Fortin is serving as the consumer representative for this committee meeting. Consumer representatives are appointed special government employees and are screened and cleared prior to their participation in the meeting. They are voting members of the committee. We have 1 patient representative, namely Ms. Jasmine Hightower. Patient representatives are special government employees and are screened and cleared prior to their participation in the meeting. They are temporary voting members of the committee. Dr. Eric Crombez will serve as the industry representative for this meeting. Industry representatives are not appointed as special government employees and serve as nonvoting members of the committee. Industry representatives act on behalf of all regulated industry and bring general industry perspective to the committee. Disclosure of conflicts of interest for guest speakers follows applicable federal laws, regulations and FDA guidance. FDA encourages all meeting participants, including open public hearing speakers to advise the committee of any financial relationships that they may have with any affected firms, it's products and if known, its direct competitors. We would like to remind members, consultants and participants that if discussions involve any other products or firms not already on the agenda for which an FDA participant has a personal or imputed financial interest, the participant needs to inform the DFO and exclude themselves from such discussions and their exclusion will be noted for the record. This concludes my reading of the conflict of interest statement for the public record. At this time, I would like to hand over the meeting to Dr. Taby Ahsan. Thank you.

Thank you, Cicely. So to start off the meeting today, we're going to have a short FDA introduction, and that will be from Dr. Nicole Verdun, the Director of Office of Therapeutic Products. Dr. Verdun, could you turn your camera and unmute yourself please.

Yes. Good morning. My name is Nicole Verdun, and I'm the Super Office Director in the Office of Therapeutic Products in CBER, and I'm happy to be leading the office at such an exciting time. On behalf of FDA, CBER and the Office of Therapeutic Products, I would like to welcome you to the 76th meeting of the Cell, Tissue and Gene Therapies Advisory Committee. I would like to start by welcoming our committee members. Thank you for the time you've taken to review the materials provided in advance of the meeting in order to participate in the discussion today. I would also like to thank our invited speakers for sharing their expertise in the area of genome editing and associated genetic modifications in the morning session. I would like to thank members of the public who will be participating in the open public hearing and those that have submitted comments to the docket. Vertex Pharmaceuticals has submitted an application for exa-cel for the treatment of sickle cell disease in patients 12 years and older with recurrent vasal occlusive crises, developed using CRISPR/Cas9 gene editing technology to result in increased levels of fetal hemoglobin in recipients. We are here to discuss specifically the study and analysis of potential off-target genome editing with exa-cel and additional recommendations. As many of you know, sickle cell disease is a debilitating hemoglobinopathy with significant unmet medical need, and to carry a reduction in overall survival for those affected. In addition, curative options are significantly limited. I've had the pleasure of taking care of several sickle cell patients and admire the courageous and resilient patient community. I'm also reminded of the sickle cell disease patient-focused drug development program at FDA, in which we heard directly from patients and their caregivers, which highlighted the significant unmet need in this disease. It is an exciting time in cell and gene therapy that we are beginning to address some of its unmet need in a variety of diseases. Exa-cel has been studied for treatment of sickle cell disease with severe vaso-occlusive crisis and has shown efficacy and safety in this population. In today's discussion, we would like to focus the discussion specifically on the off-target analysis for genome editing for exa-cel. We appreciate the committee's review and for the discussion today. And I would like to turn it back over to Dr. Ahsan to start us off. Thank you, everyone.

Great. Thank you, Dr. Verdun. So at this point, we're going to have 2 guest speaker presentations, one on genetic editing and one on the off targets of genetic editing. At the end of those 2 presentations, we'll then take questions from both speakers. So at this point, I'd like to introduce Dr. Fyodor Urnov, Professor of the Department of Molecular and Cell Biology at UC Berkeley as well as the Director of Technology and Translation at the Innovative Genomics Institute at -- in Berkeley, California. Dr. Urnov, if you could turn on your camera, and unmute yourself, please.

Good morning. I'm honored to provide a survey of the scientific foundations of human genome editing for you today. Next slide, please. My disclosures, which as Dr. Reese just mentioned, were reviewed by the FDA prior to this meeting are shown here. I note my work as a paid consultant who work at pharmaceuticals on the exa-cel program. Next slide, please. I need to train the state of our field of gene editing today by sitting 20 years back. So at the time, the sole method for targeted genetic engineering in human cells was an approach called gene targeting, a schematic of which you can see here. And when used in cancer cells, it was inefficient, 1 in 400 cells acquired the desired gene knockout. It was also genotoxic. There are no count cells acquired a bunch of extra chromosomes in the process, and you can see them in the skygram on the right. Most importantly, it just didn't work in primary human cells, so there are no therapeutic applications they could even be imagined. Next slide please. Well, folks, I can say that here we are in 2023, and we are preferably in a whole new world, 7,000 references with the word Cas9 in PubMed and genome editing with Cas9 and other tools has been shown to work in every basic and applied research setting would have been tried as well as in clinical trials in blood stem cells, T cells, the liver and the eye. Next slide. And we owe this remarkable exponential scale-up and the use of editing to the 20-fold discovery by Jennifer Doudna, here at UC Berkeley and Emmanuelle Charpentier of how a remarkable bacterial enzyme, Cas9 is naturally routed to foods target and how it can be reprogrammed. Now since 2012, the toolbox of editing has been expanded by invention of new ways to change DNA in living cells, for example, repairing point mutations, such as the work on base editing from the label data blue, and that has markedly accelerated the growth of editing as well. Next slide. Now while celebrating the truly magnificent impact that this work has had, I want to note that genome editing has a 3-decade history. Its core principle was established by Maria Jasin at Memorial Sloan Kettering in 1994. And then extensive work in the 2000s built a toolbox and editing of native human genes using earlier generation programmable nucleases. My own work on human genome editing for therapeutic purposes began 21 years ago. And in my remarks today, I will use that extended perspective to showcase key scientific features of editing that have still the test of time and remain relevant for the discussion today. Next slide. In 2005, in an important collaboration with Matt Porteus, my colleagues at Sangamo Therapeutics, and I demonstrate that the efficient repair of a point mutation at a mutational hotspots in a native gene in a human cell. So we then proposed the term genome editing to highlight the fact that the method requires an engineered enzyme, a genome editor, which binds the DNA target in the cell in an investigator specified way and then drives an enzymatic reaction that results in genetic change at that target. 2 enduring concepts emerged from that work. First, as all enzymes, genome editors followed by chemical principles that can be studied, understood and that inform their in-cell action. However, in contrast to enzymes reacting with substrates and test tubes, genome editors act on the genome in its living form. The biology of the cell is the prism to which genome editors act. Next slide. And look, this factor is not surprising. The most widely used genome editor, the enzyme Cas9 from a bacterial adaptive immune response system evolve to function in bacteria, and work by [indiscernible] followed by that of others described principles of repurposing it for genome editing in eukaryotic cells, which, as we all know, it's a very different biophysical environment kind of bacterial one. Now let me showcase for you the human genome editing relevant differences that emerged by focusing on Cas9 itself. Next slide. So this remarkable enzyme uses an RNA molecule it carries, shown in orange in the crystal structure on the upper left to recognize and distort a DNA double helix, which is shown here in blue and black. And then it forms a striking intermediate before creating a double-strand break in the DNA. Now its key feature was discovered by Martin Jinek here at UC Berkeley when he was in Jennifer Doudna's lab. And they found that this recognition mechanism is driven by a bullion logic using an AND operator. The protein Cas9 has to bind to a specific DNA sequence that has an awkward name and I'm sorry, some of the nomenclature in my field is not user-friendly. So this specific motif is called protospacer adjacent motif or PAM. And you can see it in the gray on the lower left. So once that happens, the RNA component of the complex takes over, a 20 nucleotide stretch of that RNA then pairs with just one strand in the DNA double helix and in the structure on the upper left, it's shown in blue. And it uses pairing rules for that, which we've known since 1953. And so once this complex forms, Cas9 can cleave both strands of the DNA target and creates a double-strand break and then releases. Okay. So that's the structural part. But the reason we're here today is this amazing 2012 experiment shown on the right. An analysis of this pairing mechanism led Martin and Jennifer to propose that if you change this 20-nucleotide stretch, to match a given such stretch in a DNA sequence every time flagged by the famous PAM, of course, you can create a double-strand break on demand, and this was beautiful. So when Cas9 was armed with 1 of 5 different such kind RNAs, each matching a different stretch on the piece of make DNA, then incubated with that DNA and analyzed on a gel, a pattern emerged that prove the notion that Cas9 can be programmed to induce a double-strand break on demand using pairing rules that are simple. I can explain them to my 7-year-old daughter, and that simplicity was incredibly empowering. Next slide. Because you see this discovery supercharged to 20 previous years in developing double-strand break genome editing, I alluded to earlier. So at the level of the introductory biology class, such brakes in human cells are resolved by 1 of 2 pathways. And joining that puts the two ends back together and homology directed preparer, which uses unidirectional transfer of genetic information from a related DNA molecule typically a sister chromatid to heal a break. Next slide. Genome editing is a collaboration with these 2 pathways. In the absence of the repair template, repeated cycles of cleavage by the editor result in small insertions and deletions of the target. If a repair template is provided in certain cell types under some circumstances, a mutation can be repaired or an entire transgene can be inserted at the site of the double-strand break. Next slide. Okay. It is key to appreciate that this schematic is a simplification. The cellular machinery for end joining and for homology-directed repair is elaborate, putting it mildly. And it imposes a lot of context specific. So dependent on DNA sequence of cell types and cell cycle state rules on the outcomes of editing. Next slide. However, complex the machinery in practical terms, this end joining based genome editing approach gives us small insertions and deletions and in certain settings, other arrangements, including larger deletions, and you will hear a lot more about this shortly from Dr. Bauer. Next slide. But let us examine these small indels first. As you see on the left, a key finding is whether using finger nucleases on the CCR5 gene or Cas9 on the same gene, the resulting alleles do not spend every imaginable placement of size but instead form a distinct pattern. On the right, as shown on this paper from Caribou, genome editing using Cas9 armed with guide RNA carrying a match to this protospacer and waited all this time to introduce yet more terminology. The bit in of the chromosome that gets cut is called the protospacer and the bit in the RNA that Cas9 carries that matches that bit is called the spacer. Again, I realize this is challenging, but here we are. So if you have a Cas9 armed with the guide RNA carrying a match to the protospacer and then use it on living cells, you get a 1 base-pair insertion as a dominant, and then 3 base-pair deletion and the 1 base-pair deletion and others in decreasing order. The pattern will differ from gene to gene and cell type to cell type, and this really provides a magnificent example of how the biology of the cell is the prison for which editors act. Remember that in the in vitro, the double-strand break is either a blunt 1 or a 1 base-pair standard, whereas look at what's happening in leading cells. So the pattern will differ from gene to gene and cell type to cell type. And this provides an example of how the biology of the cell is the prison for which editors act. Now such endless can form a living cells at genomic positions that are only a partial match to the guide RNA spacer, the so-called of target size. Next slide, please. And these, of course, deserve a very careful look. So what I will do is give you a preview of the structural biology by physics and by chemistry and we'll touch on the in-cell activity briefly, and Dr. Bauer will speak in a lot more depth to all about this. So as Martin Jinek and colleagues write, the target DNA specificity of the CRISPR associated genome editor Nucleus Cas9 is determined by complementarity to a 20-nucleotide segment in this guide RNA. However, Cas9 can bind and cleave, partially complementary of target sequences, which raises safety concerns for use in clinical applications. In this work, the Unique Lab identified off-target sites for Cas9 with the guide -- armed with guide RNA for the FANCF gene and the sequences are in this multicolored stretch on the upper right and determine the high-resolution atomic structure over the enzyme guide RNA complex balance of the target shown at the top and then to 2 off targets shown here. Next slide. For off-target #2, there is a clear structural explanation for why Cas9 binds it. The A2C mismatch you see on the left is accommodated by a wobble interaction, which we all learned in bio 1 in terms of tRNA anticodon interactions. So it's somewhat of a base pair. And this somewhat of a base pair happens between the other mismatch between this you and this gene. And I say sort of because as you can see in the thermodynamic analysis below, this in perfect pairing severely penalizes the enzyme. Look at the numbers, the off rate is different by 2 orders of magnitude. So the enzyme just loves falling off the off-target and the KV is nearly 100x higher from the off targets. So this is why most of targets, which requires which miss-pair driven binding are cleaved much less efficiently. Before I leave this slide, I want to slow down and state very clear, it is not the case that you can sprinkle in any number of mismatches anywhere in the target and the enzyme will cut there to some extent. In fact, rigorous data have shown that a combination of mismatches is guide RNA specific. And for each guide only some, but not other mismatches are tolerated. At a high level, this means, knowing whether a given person's genome has a partial match or a given guide RNA spacer is only the first step towards understanding whether or not that exact target will be cut by that enzyme in cells from that person. Next slide. There is more to the story. You will recall these beautiful data from the Doudna Lab, ARM Cas9 in a test tube with 5 different guide RNAs that will efficiently cut a plasma DNA using each 1 at a perfect match. That's not what happens in sets. Next slide. Instead, Cas9 efficiency varies dramatically from target to target even if each 1 is a perfect match. And this finding dates back to the earliest days of editing before Cas9. I mean we first sold this with [indiscernible] 20 years ago. But here is a 2012 [indiscernible]. In this experiment, Cas9 was armed with 1 of 14 guide RNAs, each perfectly matched to a sequence in the same small genetic region. And when you test each one of these cells and measure the efficiency, the result is pretty striking. Some guide RNAs just don't work. Most are mediocre and for some, for instance, guides number 10, 11, 12 and 14. They're pretty highly active. The cell imposes its own roles on what the enzyme can do. And it's going to get more interesting. Next slide, Shengdar Tsai and Keith Joung developed a way to identify our target sites that are actually cut and letting cells. The method is called GUIDE-seq in brief, you expose the cells to the editor, capture and [indiscernible] adapter into each break. Mapworthy adopter has landed. And when you do this exact analysis for all 14 guides, something striking emerges, yet again guides there this time in terms of specificity. But note how the correlation with the activities in poor work. For example, please compare guides 10 and 11. Both are equivalently potent. But guide 10 is a champ. It has 0 off-target sites. It only cuts the target. And in striking contrast, Guide 11 is, I guess, 1 divided by champ. It has nearly 300 off- target sites. Next slide. The art and craft of genome editor design involves finding the equivalent of Guide 10, maximally potent, maximally specific by screening for such guides in the relevant cell type. And I want to emphasize in-cell analysis for the following reason. There is an additional method to identify candidate off- target site. It was also invented by Shengdar Tsai, and it involves adjusting naked genomic DNA and finding where the enzyme pass. So when Shengdar and Keith [indiscernible] lab did this experiment for the same set of 14 guides, you find something that's frankly not surprising. The number of DNA targets that given Cas9 guide RNA can cut in the naked human genome is -- oh, sorry, I managed to an ingredious mistake, use the wrong word in the title. It should say is much greater than what it actually cuts in itself. Sorry about that. So the number of sites that Cas9 cuts in naked DNA is much greater. Apologies, I should have proved my own sites better than what it cuts them to cell. But I guess I'm glad I made this mistake because it lets me hammer in the point. Look at our Champion guide. It has no measurable off target, but it still cuts 176 targets in the naked genomic DNA, but not in cells. And look at the dirty guide, guide #11, cuts about 295 off targets and cells but it cuts more than 9,000 naked DNA. So this means yet again that the sell imposes its own rules on what the enzyme can do inside the cell. So that the number of sites in naked DNA is much, much bigger than the number of sites in the chromosome. So this -- bottom line what does this -- it means that in silico and on-naked DNA analysis are only the initial step in determining whether a given candidate off target site will be cut inside the cell and if yes, to what efficiency. Next slide. So ramping up for my last piece of primary research data on the science of editing. I want to showcase a recent finding that aligned with my own experience that is of this site dating back 15 years and of the field more broadly. The key takeaway from this is this how you handle the cells during genome-editing provides critical input to the outcome. In this recent study, the lab of Jennifer Doudna that here at the innovative genomics institute investigated what happens to primary human T cells following genome editing. In a research setting, they found and other studies agree with this that double-strand breaks induced by the nucleases, lead to chromosome loss in primary human T cells. The scientist then collaborated with the group of Carl June and Pen, and did the same analysis on T cells manufactured in an optimized clinical scale process using a modified protocol. And the finding was "undetectable chromosome loss of our bad group". I am not repeat -- not saying that any clinical manufacturing scheme will leave the genome of the target cell in pristine shape. But what I am saying that how you make the cells will have critical input into what the genome of a cell will look like. And that is also a key part of the art and craft of editing, finding those conditions where you sort of love these cells, I guess, when you gene edit them. So next slide, please. In conclusion, the presence in the human genome of a perfect sequence match or a partial match to guide RNA spacer that Cas9 can carry is a modest utility being diplomatic in determining the potency or the outcome spectrum of genome editing using that Cas9 guide RNA in the living human cell. Next slide. Context is critical in determining the outcome of genome editing. What does that mean context? What Cas9 did you use, in what form? What was the guide RNA? What was the spacer? What is the chemical composition of both? What sequence were they targeted to? How were they delivering to what amount? Into what kind of cells? How did you handle the cells before and after editing? So again, the art and craft of genome editing in both permuting endlessly, this is really labor-intensive work, permuting all of these variables until you find sort of the Goldilocks conditions of maximum potency and lease genetics. But my own experience and that of our entire field is at the end of the day, the only way to truly determine what the functional consequences are of editing on the cells in the near and long term. The only way to do that is actually transplant the cells into a subject on the clinical trial and see what happens. Now all the ex-vito studies in this regard are important, but the trial data objectively speaking more so. Next slide. In the last minute, let me close by offering this perspective. As I look at the progress of our field in the 2 decades that I've had the honor of being part of it. Progress by every objective criteria has exceeded our wildest expectations. In 2005, if you'd asked Maria Jasin, Dana Carroll, Matt Porteus, my colleagues at Sangamo or pretty much anybody else, are we going to get to a world in 2023 when there will be 27,000 references on Cas9. It will have genome editors, which knock out 100% of the target with no measurable target sites. I don't know that we would have believed you. And yet here we are. It is certain that a decade from now, the field will grow further in ways that we can predict only to a certain extent. Last slide. But that said, as I think about the maturity of this technology, like where are we in the overall trajectory. Next slide. My personal perspective is we have completed our period of exponential growth in terms of, if you will, genome-editing quality score. I personally think that we are in a more linear stage for health. Having established methods for editor design, deployment and derisking. And this to me means that genome editing is ready for prime time, which, of course, is why we're here today. Again, I'm honored for this opportunity to speak with you today, and I will turn the floor over to Dan Bauer.

Thank you, Dr. Urnov for both the historical and technical context there, very important for us to understand. And so now we'll hold off questions for Dr. Urnov until we have the presentation by Dr. Bauer. Daniel Bauer is a Principal Investigator and Staff Physician at Dana-Farber Boston Children’s Cancer and Blood Disorder Center in Boston, Massachusetts. Dr. Bauer, if you could turn on your camera and go off mute, please.

Good morning. I'm delighted to present today regarding comprehensive evaluation of genome editing associated genetic modifications. Next slide, please. So my disclosure is also reviewed by FDA include that I'm a co-inventor of patents related to therapeutic genome editing for blood disorders. And I hold a license patent that's related to BLA-125787 from Vertex Pharmaceuticals, and it's possible that they could receive future related royalties. Next slide, please. Today, I will discuss the therapeutic genome editing can produce genetic modifications both away from and at the genomic target site that is off target and on target edits. Off-target edits may be influenced by human genetic diversity, on-target edits may include short indels and structural variants and the edit distribution reflects the clonal composition of the hematopoietic graft. Next slide. First, I'll discuss that off-target edits may be influenced by human genetic diversity. Next slide. Here, I'm considering off-target effects as genomic modifications away from the intended target locus. As introduced by Professor Urnov, based on its biochemical properties, Cas9 may bind and cleave genomic sites with sequence similarity to the target locus. Current methods to nominate candidate off-target sites are mainly based on 2 approaches: first, in silico approaches based on sequence homology and second, cell-based and/or in vitro assays that empirically assess modification of genomic DNA. Next slide. For the BCL11A +58 enhancer targeting Guide RNA, originally called #1617 and now referred to by the sponsor as SPY-101 both of which names indicate the same spacer sequence. The published off-target analysis using combined in silico and empiric approaches, nominated 24 and 223 off-target sites in publications from 2019 and 2021, respectively. Validation by deep sequencing of the candidate sites in edited cells identified no off-target sites with significant editing and prespecified detection thresholds of 0.1% or 0.2%. Next slide. However, in silico methods traditionally have been based merely on the human reference genome and cell-based and in vitro empiric methods usually interrogate a limited set of human donor genomes. Next slide. Therefore, we wondered about off-target sites that are not found in the human reference genome, but may be found in specific populations or individual patients. Next slide. This question motivated our research group working with computational biology colleagues listed below to develop a publicly available in silico tool called CRISPR Me, which takes his input a guide RNA spacer sequence, plus flexible sets of genetic variants such as from the thousand genomes project, human genome diversity project or any other source of flexible homology thresholds to nominate variant aware off-target sites and to associate them with genomic variant and guide RNA related annotations. Next slide. When we tested the guide RNA 1617, that is the guide RNA used in the editing therapy that is the topic of today's discussion, we found that the top fit candidate off-target site was related to a single nucleotide polymorphism, SNP called RS114518452. On the Y-axis is the cutting frequency determination score, which is based on the number and position of mismatches of a target sequence with respect to guide RNA. For the top hit site, on the far left, shown in red, there was a very low score suggesting a negligible likelihood of cleavage for the reference allele site. While in blue, with an arrow pointing to it is shown the nonreference allele site that had a predicted likelihood of privilege similar to that of the on-target site. Next slide. In this case, the variant changes the C to G on the bottom strand, which produces an NGG PAM sequence shown in Bold, which enables the binding of Cas9. The off-target site then just has 3 PAM distal mismatches shown in lower case for which a high likelihood of cleavage is predicted. This variant is present at different frequencies in different human populations with 4.5% minor allele frequency in African ancestry populations. This suggests that about 10% of the target population of African ancestry sickle cell disease patients would be expected to carry a risk variant for this off-target effect. Next slide. To test the variant specific off-target potential, it is essential to conduct the test in cells carrying the risk allele. We identified a CD34 positive hematopoietic stem and progenitor cell donor heterozygous for the SNP and performed gene editing. Above deep sequencing showed off-target short indel gene edits exclusively from the nonreference g-allele and never on the reference c-allele. The low shows off-target editing was never observed on the reference allele, either in this heterozygous donor or in homozygous donors carrying only the reference allele. In contrast, the nonreference allele showed 5% to 10% short indel off-target gene edits. Next slide. Since the BCL11A target sequence is on the P arm of chromosome 2, and the SNP is on the Q arm of chromosome 2, we hypothesized that simultaneous cleavage of both positions could lead to pericentric inversions of approximately 150 megabases. To test this, we designed droplet digital PCR assays to specifically detect and quantify each of the para-centric inversion junctions. Next slide. We validated that indeed allele-specific pericentric inversions were produced by gene editing at about 1 in 600 allele frequency in the heterozygous donor but were undetectable in cells lacking the risk allele. It's important to note that the biological significance of these off-target indels and pericentric inversions is uncertain and may be negligible. Next slide. The second point I will discuss is that on-target edits may include short indels and structural variants. Next slide. After Cas9 cleaves a target site, endogenous DNA repair mechanisms, repair the cleavage, when this leads to a genomic modification, this is what we call an on-target gene edit. The edits of the on-target locus may include short indels which were the easiest edit to identify since they can be amplified and sequenced by conventional short-range PCR and short-read sequencing. However, as shown in panels B through G, there are a range of other possible DNA repair outcomes at the on-target site, which collectively are known as structural variants including long deletions, translocations, insertions, inversions, copy neutral loss of heterozygosity and chromothripsis or chromosome shattering and repair. Standard short amplicon sequencing cannot capture these structural variant types of on-target gene edits. Again, it's important to note the biological significance of any individual structural variant is often uncertain and may be negligible. Next slide. There are a range of alternative methods besides short Amplicon PCR that can capture these structural variant types of on-target gene edits. This includes long-read sequencing, which is shown on top, indicates numerous repair alleles with deletions of hundreds to thousands of base pairs may be frequent on-target edits. On the bottom pie chart has shown that sometimes after gene editing, up to 40% of the alleles are comprised of intermediate or long deletions that may escape conventional short amplicon PCR detection. Next slide. Another set of methods is based on single primer amplification. We're binding of an expected primer on 1 side of a cleavage, the so-called bait side, can capture edits with unexpected sequence on the opposite side, the so-called pray side. One such method PEM-seq is shown below. Next slide. Droplet Digital PCR approaches by placing a probe far enough from a cleavage site to be unaffected by short indels can comprehensively capture structural variance as missing alleles. In this experiment, about 15% of the alleles after gene editing were some kind of structural variant. Next slide. The point here is that numerous assays exist to detect on-target structural variant gene edits and that these can be frequent occurrences, although multiple assays may be needed to fully characterize these gene edits. Next slide. The final point is that in ex vivo hematopoietic gene editing, the edit distribution within ingrafting cells reflects the clonal composition of the hematopoietic graft. Next slide. Although therapeutic gene editing of hematopoietic cells is relatively new integrating vector gene therapy has been studied for more than 20 years as a treatment approach for a variety of inherited blood disorders. Unlike genetic therapies targeting other tissues, it is straightforward to measure the distribution of genetic modifications in the blood system of treated patients. Similar to therapeutic gene editing, hematopoietic cells are collected from a patient, ex vivo gene modification is performed in this case by treatment with an integrating vector, here, individual stem cells are shown as distinct colors. So there's a red cell, a purple cell, green cell and so forth. These cells are marked by unique vector insertion sites. Then blood or bone marrow samples are routinely collected from the patient over time after cell infusion. The distribution of the vector insertion sites in these samples reflects the contribution to blood cells of hematopoietic stem cells marked by a given insertion site. In this way, measuring vector insertion sites can indicate the evenness of clonal diversity. In the illustrated example, an alert is raised when there's evidence of a decline in diversity suggesting clonal dominance. In this way, monitoring funnel composition may inform the approach of safety by detecting clonal dominance and clonal dynamics, and efficacy by detecting a level of potentially therapeutic gene edits. Gene-editing therapy has a strong analogy to integrating vector gene therapy, although gene edits may not be as diverse as vector integrations since different cons may share the same edits. Next slide. So is it possible to evaluate the clonal diversity and dynamics after gene editing. Here is a recently published study suggesting the answer is yes. This study was a CRISPR/Cas9 mediated gene editing of the BCL11A enhancer for pediatric beta 0, beta 0 transfusion-dependent beta thalassemia. Two patients were treated with Cas9 plus guide RNA 1617 with ex vivo gene editing. The edit distribution was tracked in cell products and in serial patient samples from the blood. Next slide. Using this powerful approach, the investigators made some important observations. First, they found that the frequency of gene edit alleles based on microhemology mediated end joining repair, MMEJ was substantially lower in engrafting cells in the blood as compared to the input cell product, while the frequency of edits showing nonhomologous end joining repair, MHEJ was reciprocally increased. This shows that the edit distribution may differ substantially between cell products and engrafting cells. Next slide. In addition, the investigators track the top 20 short indel edit alleles over time in the blood and reassuringly found a stable edit distribution suggesting polyclonal hematopoiesis. This study illustrates how tracking edits enables monitoring of clonal dynamics in treated patients. Next slide. In conclusion, therapeutic genome editing can produce genetic modifications, both away from and at the genomic target site that is off target and on target edits. Off-target edits may be influenced by human genetic diversity. In general, genomic diversity is most pronounced in African ancestry populations. For the 1617 guide RNA targeting the BCL11A +58 enhancer, there's a likely off-target site due to the RS114518452 variant with about 5% minor allele frequency in African ancestry populations, including a risk of a rearrangement that is a pericentric inversion between the on-target and off-target site. This off-target can only be detected in cells sharing the risk allele. A risk assessment could include uncertainty about the biological relevance of indels or rearrangements at the off-target site. Patients could be screened and/or patient samples could be monitored to gather information about the frequency and consequence of such events. Next slide. on-target edits may include short indels and structural variants. Shorty Amplicon PCR with short-read sequencing will miss structural variants, assays exist to characterize and quantify structural variants although more than 1 assay may be needed for comprehensive measurement of these on-target edits. A risk assessment could include uncertainty about the biological relevance of structural variants. Next slide. The edit distribution reflects the clonal composition of the hematopoietic graft. The distribution of edits in the cell product may not mimic the distribution of edits and engrafting cells over time which could impact safety and/or efficacy. Gene edits that do not impact cell fitness that is passengers nonetheless, mark engrafting stem cells and their progeny, clones to offer opportunity to track clonal dynamics. Gene edits that do impact cell fitness, if any, exists that is drivers would be expected to cause clonal loss, more expansion, which might be detected by tracking the edit distribution. Tracking genetic distribution over time is akin to vector integration site analysis in integrating vector gene therapy studies. Thanks for the opportunity to participate in today's discussion.

Thank you, Dr. Bauer. And so now we have a period of question and answers. So questions from the committee. I do want to encourage you while Dr. Urnov will be available in the afternoon to answer some questions during our discussion period if needed, Dr. Bauer will not be. So now is our opportunity to ask our questions of these 2 guest speakers. If you can raise your hand if you have questions, committee members, this would be the time to do so. Dr. Ott, can you turn on your camera and go off.

Yes. I wanted to ask both speakers for a comment on the question of length of expression of the Cas9 enzyme in cells and the relationship or the chance of off-target effects, especially when it comes to the applied method, I believe, which is an RNP guide RNA electroporation. Would welcome any comments on this. Thank you.

Dr. Bauer, do you want to go first? Should I start?

I'm happy to. So that's been -- that's a great question and has been well studied as a main modifier of the risk of off-target potential that the longer the duration of exposure to the editor, the more -- there's risk of off targets. And 1 could imagine once the on-target effect has been achieved, there's no more potential benefit of exposure to that editing reagent and that any continued exposure might only incur more off-target risk. And that's been shown many times in the field that delivery methods that have long-term or permanent expression have much greater off-target risk than a short pulse. And the RNP, ribonuclea protein delivery method is generally the shortest pulse that can be achieved with Cas9 gene editing and is expected to have thereby the shortest degree of off-target risk.

So the numbers that you have shown, are they done with long term or with short-term expression of enzymes or a mixture of both?

No, the experiments that I showed were all with RNP delivery -- in a therapeutically relevant delivery context. So I think that kind of distinguishes the 2-step approach of nomination followed by validation for off-target effects. So in the nomination step, often, one is very broad, one might have relaxed thresholds to try to find many possible sites knowing that it's going to be a much larger list than the real edited sites will be and then the validation can occur in a clinically relevant delivery context, in relevant cellular context to look for those edits. And so for example, that SNP associated edit that we observed was in a CD34 cell donor cellular context with RNP delivery. So close similar to what would be expected for the therapeutic delivery.

If I may step for just a second, everything Dr. Bauer said is absolutely true. The additional sort of almost hydrodynamic thing to consider is when you make 1 million cells in a small pivot, whether you make 250 million cells in a baggie, the parameters through which the RNP then enters the cell and then stays on the cell and leaves the cell are actually different, which is why a major part of what we do in our field as we go from the research branch to developing a product for potential and human use you spend a lot of time basically doing exactly what you just alluded to by Dr.Ott. But My colleague at the [indiscernible] will some calls that the area under the curve. The basic idea is you want to have a short pulse that looks like this rather than a pulse that looks like this. But critically, that even for the same RNPM for the same sales, the shape of that will depend on the scale at which we do the experiment.

Great. Thank you very much for that complete answer. Who I have next is Dr. Lisa Lee, please if you could go on camera get off mute.

Thank you for these extremely helpful presentations. It's really well done. I want to take us from the baggie of cells to a higher level view of patients and even higher level of families. And I wonder, Dr. Bauer, if you could talk a little bit about, if you were talking to a family about this kind of treatment, how would you characterize the consequences off-target edits both cellularly and clinically. If I were concerned about, well, what does this mean if they are off-target, if things get cleaved at the wrong place. What does this mean for a patient?

Yes. I think it's a great question, and I would emphasize to patients that there's often uncertainty about the functional significance of off-target edits that only a small part of the human genome actually codes for genes. Most of the human genome is noncoding. Its functional importance could be regulatory, but it's likely that many places in the human genome can tolerate an off-target edit and not have a functional consequence. The challenge is we just don't know for sure. And the only way to know that is careful follow-up, I would say. What I would emphasize to patients is it was obvious to them the known risks of the disease that this is a terrible disease and that, that has to play in. And then the risks of the therapy, which are known, which include things like the busulfan conditioning that's used or whatnot. So I would say this my guess is it's a relatively small risk in the scheme of this risk benefit. But it's new, it's unknown, but it's easily measurable, and that's one of the goals, I would say, of doing this under very careful circumstances is to try to learn what that risk is so that we can continually improve those therapies.

I mean theoretically is it catastrophic?

The -- in theory, as Dr. Urnov said, these cell products have lots of cells in them, hundreds of millions of cells and any 1 cell that goes [indiscernible] could cause leukemia. Now has that ever been shown at an off-target effect of gene and cause leukemia? No? So theoretically, could it? Yes. Is there any evidence to suggest that it does? I would say no. But we -- this hasn't really been done very much we need to be humble and open to learning from these brave patients who are participating.

Thank you, Dr. Bauer. Dr. Joseph Wu, if you could go on camera and unmute yourself, please.

Yes. So I want to thank Dr. Bauer and Dr. Urnov for 2 great presentations. My question is with regard to -- I guess this is for Dr. Bauer with regard to the off-target papers that show -- for example, the -- as I understand it, the paper that you have high off-target assessment, BCL11A and the guide RNA for 1617, that 1 was Nature Medicine and then they another one in France in New England Journal. It says the number of adolescent [indiscernible] and it was four and then the follow-up slides, you have the CRISPR Me software that you have. I think it's an assessment tool, and it's a prediction model -- a prediction model, not an actual cell that you validated. So I just wonder, what is the scale and the field that people have done to do the actual editing the cell samples and to see what our target is on the scale of 10? Or has somebody been a scale which you take 100 patients, 200 patients hematopoietic cells tested and just see what the prevalence is and what the consequences are. What is the scale? How much has been tested as a sample size?

Yes. That's a good question. As Dr. Urnov pointed out, there's great variability from a guide RNA to a guide RNA. So you could have what we call like a clean guide RNA where we can't find any off-targets, you could -- what we call dirty guide RNA, where you could find thousands of off targets. So if those experiments were done, the findings might be dramatically different depending on the guide RNA. So it's hard to answer when you say what's the scale that's been done, I guess it depends if you mean with just 1 given guide RNA, like the guide RNA.

Yes. So Guide RNA 1617 be tested on 100, 200 different patients, donor hematopoietic cells, just to see what happens in the bigger population rather than an equal for on the New England journal paper.

Yes. I mean I think the sponsor has done a few more donors. So it's probably greater than 4 at this point. They could answer. In our studies, we've done on the order of fewer than 10, 5 to 10 different donors. But the problem is when you -- when you validate off targets, you generally find what you're looking for in the sense that you're doing Amplicon sequencing of sites that were nominated in the first step. So if you did hundreds of donors, you would need to do it in a way where you were looking at the relevant sites in those donors. So for the reference genome related sites, these off-target results are very reproducible. If you do it in 1 donor or another donor, you get very similar effect. So I think it will be unlikely at a reference genome off targets that you'd find a substantial difference if you tested it in many more donors. We haven't seen that. I don't think anyone in the field have seen that. I think where the risk really comes from is when there's genetic variants and those donors might carry different genetic variants, then the number of mismatches for that guide RNA may differ substantially and the likelihood of cleavage may be different. So just to clarify, the comment on the CRISPR Me tool, you're right, it's a prediction tool. But then what we did was we found a sample, a CD34 cell sample that carried that risk variant and we validated. In fact, there was off-target editing. And I think that's kind of the key point that when 1 tests donors, unless 1 knows the variance that they carry, it's hard to interpret what are the off-target sites that they may be at risk for.

So based on what you just mentioned, when do you want to test on many, many more samples instead of just on less than 10, if you're pushing for this product or any product in general. Yes.

I think, like I said, testing on many, many samples -- unless you did it in a very focused way, I don't know that it would be of high value because if you're just looking at the reference genome sites, I don't think you'd find new information. If there's genetic variant sites, you could learn something from those samples. But depending on the allele frequency of that variant, it could be 1 in 1,000. It could be 1 in 10,000. So doing 100 samples, you still might not find it. What the tool allows you to do is understand both the allele frequency of the variant and the likelihood of cutting or the other genomic annotations and then prioritize what are the most key variance to consider. There's other ways that one can minimize the risk of off-targets, and that's the genome editing procedure itself. And what we talked about that a few minutes ago that limiting the duration of exposure can do that using certain cast variants or guide RNA modifications or other methods can limit the risk of that off target. But I think saying we should do 100, then you could say we could do 1,000 and you could say we move to 10,000. I'm a little concerned that without some statistical rigor that study might be kind of ambiguous in terms of what's its power and what's its goal.

Dr. Wu, I'd just add 1 sentence, if I may. First, let me just emphasize the extent to which the overall thrust of your question is completely sensible. I want to compare and contrast CRISPR/Cas with, let's say, a standard small molecule for which we can study the pharmacogenomics. In introductory biochemistry will teacher or students about tamoxifen and how it's metabolized by CYP2D6, and there are alleles of CYP2D6 that cause differential metabolism of that to the actual active drug, which is endoxifen. And if a physician knows what the patient's genotypes for CYP2D, they can route -- guide the patient's care. The challenge with editing is we do the experiment just discussed, we sequence 10,000 people, and we identify 7 additional off-target sites. In most cases, perhaps not all, it's going to be incredibly difficult to look at that hyper -- potential target site and say that, that off-target site is -- gives us actionable data. And again, this is because most of these, if not the overwhelming majority are intragenic. So I want to be clear. I don't want to throw my hands up being like agnostic orals to know there's nothing we can do. But I think we should also be mindful of the objective limitations of what we can and cannot do in terms of derisking the editor. And as Dr. Bauer said, we would need the level of statistical rigor and functional analysis, what would need to make sense of this larger scale sequencing, we might not be there yet. But again, I don't want to understate the fact that your overall line of question is completely sensitive.

That's a great point of how we take that theoretical information and use it on a per patient basis, it's very challenging. Dr. Komor, can you go on camera and unmute yourself, please?

Yes. Yes, I just [indiscernible] right on the nose. Each person has several million genetic variants in their genome. So it's kind of a question of what's reasonable. But I had 2 quick questions. I just want to clarify, is this new off-target that you identified due to the genetic variant, there seem to be some like yes or no, in terms of like did Vertex identify that as a putative off-target their in silico analysis because of the threshold that they used. Was that 1 of the off targets that they had identified.

I mean I just read the public materials a few days ago, and it looked like it may have been on their nominating list. But I think the key question is not if they nominated it. I think the key question is we only saw that off-target when we look in cells that carry that variant. So unless that test was done, I think that would be uncertainty. And based on our results, I think it would be extremely likely that in cells carrying that variant, there would be cleavage at that site.

Okay. Yes. I guess in the briefing, it does mention that the additional off-targets they saw, they did test them in patient samples that had the variant, but it's unclear which one. But then I also -- I just wanted to ask about the edit distribution, that third bullet point that you covered, so like the on-target indel profile, I'm assuming. I mean, in theory, like the sequence surrounding the double-stranded break site should be the same for all of these patients. And so do you typically see for this guide RNA those indel sequences are quite reproducible across different patients. Or do you have to worry about human genetic variation on that point as well?

I mean there's no common genetic variance that would disrupt the binding of that guide RNA, but -- so I think it would be a vanishingly rare event where someone carried a variant that would impact that, but it's a possibility for any sequence specific therapeutic and any gene editing. But yes, like any guide RNA, the edit profile is quite reproducible and characteristic, and there's a certain set of indels that are seen on a given frequency. Now it's not the same actually in the cell product and engrafting cells in that other clinical trial and the experiments we've done in animal models likely due to differences in the editing in true engrafting metabolic stem cells versus progenitor cells in that edited cell product population. But certainly, those can be measured over time and would indicate clonal dynamics and clonal diversity.

But you would expect that to be similar across various patients? Or is that reasonable?

I think that's reasonable. We have never seen patient differences in that. But it depends on the clonality of engraftment. If many, many cells engraft and it's highly polyclonal then you might expect patients would have similar distribution. As it becomes more oligoclonal, there could be stochastic differences or other differences in terms of which cells engraft and which cells give rise to that. And it's known that hematopoiesis, the clonal contributions of different hematopoietic stem cells can vary over time with different factors influencing that. And so I don't know that if we can totally predict what would happen in patients.

And Dr. Komor, maybe your question about what the sponsor had identified might be a question for the FDA -- after the FDA presentation as well. Let's see. I think at this point, there are no more raised hands. Anyone else have any questions that they would like to ask of our 2 guest speakers. And I'd like to remind you that Dr. Bauer will not be available in the afternoon. So now is the opportunity. Dr. Tisdale, if you could turn on your camera and go off mute.

Yes. Thank you, Dan. I get this question in before you go. And I think the 1 thing that I'm interested in contextualizing is sort of your view on the risk of off-target effects and how they should -- how the should be monitored. I mean given that this tool is following Watson-Crick base pairing, can you say anything about the overall risk and how you see it and thus, how it should be monitored?

Yes. Like you said, the tool nominates off-targets taking us input genetic variance, and it can use any external annotation. So any off-target prediction tool could be implemented along with the tool. And as those prediction methods improve, our ability to predict variant associated off targets will improve. I think the risk is modest that there's no biological significance that we know of, off editing at this on this off-target site. I think the indels in the interionic non-coding sequences are unlikely to be functional, but we don't know that for sure. I think the pericentric inversions that we saw something like 1 in 600 allele frequency are a little more notable, but still may not be of biological significance. I think there's really no methods that I would say that are reliable to predict the function of off targets. I think the main benefit of doing gene editing in the blood system is that it's easy to follow blood samples over time and that a broad characterization of on-target edits should be able to find the pericentric inversion as a rearrangement between the on-target and off-target site, and it could easily be followed over time. And if it's nonfunctional, it could provide reassuring data on this point. But I don't think there's any preclinical analysis that could be convincing to say a given off-target effect is certainly safe. So I think that's a main benefit of editing in blood disorders is that we can follow patients.

Fyodor, I see you on the edge of your chair. Does that mean you'd like to add something?

I mean I think any professional genome editor, like Dan or myself or Alexis, who looks every -- the entire world in the INSS. Look, we are completely certain that our nuclease is 100% of the time behave pristinely. I mean we have a 30-year history of our field, John, you wrote much of the textbook of it where things can happen clinically that we couldn't predict. The one thing I will stand by is the thing I closed with, which is -- as I look at the trajectory of where we were when genome editing entered the clinic 2008, 2009 and where we are today, our ability to do a deep analysis at the sequence level, at the functional outcomes level is really in a different part of -- like we're in a different dimension of how deeply we can look. And so to me, as I said again, I'm just going to stand by what I closed my talk on, my technologically speaking is the technology is in fact ready for prime time. And by that, I mean, we're kind of reaching asymptotic places in terms of how we can derisk it nonclinically. Like I don't know what else to do at this point in terms of like understanding the benefit risk, which, again, I'm grateful for the opportunity to be part of this today.

Great. So it looks like we have addressed the questions from the committee. I do want to thank the 2 guest speakers today, those were very, very thorough and informative presentations. I think that, that led to some good question and discussion. And I think for the rest of the day, it sets the stage to think about 2 things, which is when is enough theoretical data sufficient to support a patient-specific risk assessment and also, to your point, Dr. Urnov, which is, where are we in that curve of risk mitigation? And have we actually started getting to that asymptote or is there more work to be done? Those are 2 very important questions. And while we will start the discussion today, I think that this will continue to evolve over time. So -- but thank you so much for setting the stage for the conversation today. I look forward to the rest of the day. At this point, we have time for a 10-minute break and we will reconvene at a little less than 10 minutes at 10:35 a.m. So thank you all and see you then. [Break]

Welcome back. At this point in the day, we're going to hear from the sponsor. And so with that, I would ask that there's going to be a series of speakers from the sponsor that each speaker introduce a subsequent speaker, and I will introduce the first speaker. So first to speak will be Dr. Stephanie Krogmeier, who is the Vice President of Global Regulatory Affairs at Vertex Pharmaceuticals. Dr. Krogmeier, if you would like to start.

Good morning. My name is Stephanie Krogmeier, and I'm the Head of Global Regulatory Affairs for cell and genetic therapies at Vertex Pharmaceuticals. We are excited to be here today and would like to thank the FDA, the panelists and the patients in our clinical trials as well as their families for making this meeting possible. Exa-cel was developed for the treatment of sickle cell disease in patients 12 years and older with recurrent vaso-occlusive crises. In other words, exa-cel was developed for severe sickle cell disease. In parallel, Vertex is evaluating the same drug exa-cel for the treatment of transfusion-dependent beta-thalassemia. This BLA is also under review by the FDA, but is not being discussed today. I'll begin by discussing sickle cell disease. Severe sickle cell disease is a serious, rare, debilitating and life-shortening genetic disorder affecting hemoglobin function. Approximately 20,000 people in the U.S. have severe sickle cell disease, defined as 2 or more vaso-occlusive crisis per year in each of the 2 previous years and who are candidates for transplant therapy. Of those patients, approximately 90% of people with sickle cell disease in the U.S. are of African descent. The clinical hallmark of sickle cell disease is recurrent, painful VOCs. These events not only require care at a hospital, outpatient clinic or ER, but culminate in acute and chronic organ complications leading to significant morbidity and mortality. The current treatments for sickle cell disease are only partially effective and do not eliminate VOCs. Allogeneic stem cell transplantation is the only curative option, but has substantial limitations. Thus, there is a high unmet need for transformative therapy, and that is why we are here today. Exa-cel is a nonviral, onetime autologous CRISPR-edited cellular therapy. The development of exa-cel is grounded in human genetics showing that fetoglobulin can substitute for sickle globin in erythrocytes and eliminate VOCs. Specifically, the permanent, irreversible and precise edit made by exa-cel results in the reduction of BCL11A gene transcription, which upon erythroid differentiation leads to the increase in HbF I just described. Consistent with this mechanism and site of action, comprehensive nonclinical studies demonstrate no off-target editing, which will be discussed in detail later in the presentation. Turning now to the sickle cell disease development program. The exa-cel development program consists of Study 121, a pivotal Phase I/II/III study and Study 131, a long-term safety and efficacy follow-up study. Given this is a rare disease with an intended population of only 20,000 people combined with the expected treatment effect, we designed the study in collaboration with the FDA to be approximately 45 patients. Study 121 has completed enrollment in dosing of all patients, 46 in total, including 12 adolescents. The patient journey for Study 121 is shown here, and there are 3 things I will point out. Sickle cell disease patients undergo CD34 mobilization and cell collection utilizing single-agent plerixafor. The editing process is nonviral and occurs ex vivo for an electroporation of Cas9 and the highly-specific guide RNA. And finally, the patient is prepped for transplant by undergoing myeloblative conditioning with busulfan to ablate their existing bone marrow prior to excel infusion. In the presentation today, you will hear from Dr. Hobbs on the efficacy, the data were highly positive and that both the primary and key secondary end points. You will also hear from Dr. Altshuler on the comprehensive nonclinical safety package with a specific focus on the off-target assessment, which did not identify any evidence of off target editing by exa-cel. Finally, Dr. Simard will describe the safety profile of exa-cel, which was generally safe and well tolerated. In summary, the results from the exa-cel program in severe sickle cell disease are unprecedented. Exa-cels demonstrate transformative efficacy, a strong safety profile and a highly positive benefit risk for patients with severe sickle cell disease. With that background, here's the agenda for the remainder of the presentation. Unmet need will be presented by Dr. Thompson, Chief of Hematology at Children’s Hospital of Philadelphia and Pediatric Hematologists who has cared for patients with sickle cell disease for the past 30 years. Next, efficacy will be reviewed by Dr. Hobbs, who is the Head of Hematology Clinical development at Vertex and has spent his career treating people living with sickle cell disease. Then nonclinical safety will be discussed by Dr. Altshuler, Chief Scientific Officer at Vertex. Prior to Vertex, he was a founding member of the Broad Institute at Harvard MIT with a deep background in population and human genetics. Later, clinical safety of exa-cel will be shared by Dr. Simard, Head of Clinical Safety for Cell and Gene Therapies at Vertex who has been with the program since the first patient was dosed. Lastly, clinical perspective will be presented by Dr. Frangoul, Director of Pediatric stem cell transplant program at the Sarah Cannon Research Institute of TriStar Centennial Children's Hospital in Nashville, Tennessee and the lead investigator in the sickle cell disease exa-cel clinical trials. Dr. Thompson and Dr. Frangoul are presenting on behalf of Vertex and have been compensated for their time. We have -- we also have additional experts from Vertex here today who are available during the Q&A session. Thank you, and I will now turn the [indiscernible] over to Dr. Thompson.

Thank you. I'm Alexis Thompson, and I'm the Division Chief of Hematology at the Children's Hospital of Philadelphia. For the past 30 years, I've cared for patients with sickle cell and have regularly witnessed the debilitating consequences of this life-threatening disease. I'm pleased to be here today to discuss the current treatment landscape and why I believe that patients with sickle cell greatly need a curative treatment. Let me share some background on the disease. Sickle cell is considered a rare condition in the United States, affecting approximately 100,000 Americans. Among these, about 20,000 have what would be considered severe disease defined by recurrent VOCs, and are, therefore, candidates for transplant therapy. Sickle cell disease occurs at disproportionately high rates among individuals of African ancestry and also at lower rates among individuals of Middle Eastern, Mediterranean, Indian or Asian descent. People with sickle cell often live in low income areas and communities with high unmet medical need, further adding to substantial health care disparities. Sickle cell is caused by mutation in the beta-globin gene, which encodes a key component of hemoglobin. This mutation leads to production of an abnormal form of hemoglobin called sickle hemoglobin. In the deoxygenated stage, sickle hemoglobin polymerizes and produces deformed or sickle-shaped red blood cells that are prone to hemolysis leading to chronic anemia. Individual sickle cell disease commonly experience episodes of severe acute pain known as vaso-occlusive episodes or crisis that can last a few hours to sometimes many days. Over time, with repeated sickling events, sickle cell results in progressive injury, potentially impacting multiple organs in the body, which can progress to organ failure and a shortened lifespan. Frequent painful episodes and chronic pain significantly diminish the quality of life, not only for the patients but also for their caregivers and their families. In addition, sickle cell has profound psychosocial consequences for the patients with higher rates of anxiety, depression and absenteeism from work and school. In addition to high morbidity, VOCs are the most common cause of hospitalizations for individuals with sickle cell disease, resulting in approximately 100,000 admissions per year. VOCs that require hospitalizations are associated with increased risk of mortality. While the overall lifespan for patients with sickle cell has certainly improved over time, it is still reduced by 20 to 30 years compared to the general population with a median life expectancy of only 45 years in recent reports. Unfortunately, there is no broadly available treatment option that will eliminate VOCs. Allogeneic stem cell transplant, the only potentially curative option is only available to approximately 18% of patients who will have a suitable donor. Allo transplants are associated with significant risk, including transplant-related mortality, graft failure, graft versus host disease and other significant complications. Turning to fetal hemoglobin. Fetal hemoglobin is an established powerful modulator of clinical and hematologic features of sickle cell disease and has been robustly studied. Elevated levels of hemoglobin F results in improved morbidity and mortality in sickle cell disease. And this is demonstrated by 2 examples from natural history. The first are neonates or infants with sickle cell who by and large are asymptomatic when they produce primarily hemoglobin F, which is non-sickling, and sickle cell disease patients who have co-inherited hereditary persistence of fetal hemoglobin. Fetal hemoglobin levels of 20% or greater have become the clinical target for patient with sickle cell to protect against disease complications. So a durable therapy that consistently raises fetal hemoglobin higher than 20% would provide an important treatment option. In summary, sickle cell disease is a rare debilitating and life-shortening disease. Patients will suffer painful vaso-occlusive events and other recurring issues that cause chronic complications across multiple organs and significantly impact their lives and lifespan. Allogeneic hematopoietic stem cell transplants are potentially curative, but they are not widely available for the majority of patients. In the current landscape of disease-modifying therapies, none of the approved agents are curative nor will they fully eliminate vaso-occlusive episodes. Hemoglobin is an established and highly relevant clinical marker in sickle cell. So a new treatment that raises fetal hemoglobin in a durable or sustained manner would provide an important therapeutic benefit. The bottom line is patients and families need curative medicines for this devastating disease. Thank you. I'll now turn the presentation over to Dr. Hobbs.

Thank you, Dr. Thompson. I'm William Hobbs, Head of Hematology Clinical Development at Vertex. I'm a hematologist and have spent over 20 years working with people living with sickle cell disease, including in patient care and for the last 10 years in developing new treatment options for the severe progressive disease. And it's an honor and a privilege to be here today to share the clinical data showing the transformational and durable clinical benefit of exa-cel in adolescents and adults with sickle cell disease. Exa-cel resulted in transformational clinical benefit, and I'll provide an overview of the efficacy data, which showed that the study met its primary and key secondary end point. The primary endpoint being the proportion of patients with no VOCs for at least 12 consecutive months, which is referred to as VF12. The key secondary endpoint was the proportion of patients with no inpatient hospitalizations for VOCs for at least 12 consecutive months, which is referred to as HF12. The efficacy of exa-cel was consistent across the patient population, including both adolescents and adults. And the clinical benefit of exa-cel was durable, including for approximately 4 years of follow-up. Key characteristics of patients in the study were representative of patients with severe sickle cell disease expected to be treated with exa-cel. The primary efficacy set or PES, includes all patients with at least 16 months of follow-up who were analyzed for the primary anti-secondary endpoints. The full analysis set, or FAS, includes all patients who received exa-cel. Adolescents represented a significant proportion of the study population, making up approximately 30% of the dose patients and 20% of patients evaluated for the primary and key secondary endpoints. Patients experienced a mean of approximately 4 VOCs per year in each of the 2 years prior to exa-cel with a mean of almost 3 inpatient hospitalizations per year resulting in approximately 2 to 3 weeks in the hospital per year. The study met the VF12 primary endpoint, demonstrating remarkable clinical benefit. 29 of 30 patients, nearly 97% achieved at least 12 consecutive months without a VOC, with a mean VOC-free duration of over 22 months, almost 2 years and ranging up to 46 months or almost 4 years. To further illustrate the treatment effect in more granular detail, this figure shows each of the 44 patients who received exa-cel. Each black diamond indicates a VOC event, and you can see the remarkable absence of VOC events after exa-cel. The light gray bars to the left indicate the 2-year baseline period prior to exa-cel, demonstrating the high frequency of VOC events before exa-cel treatment. The purple bars to the right show the duration VOC-free after exa-cel, this evaluation period for VOC events began after a 60-day washout of transfused red blood cells, which are given for post-transplant support and identified as the red and dark gray bars for each patient. There were only 2 patients in the PES who had VOCs after the endpoint evaluation period. One patient had a single event and is the patient towards the top of the figure. This patient achieved both VF12 and HF12 and then had a single event after approximately 20 months VOC free. And I'd like to highlight a few features of this event because it illustrates the protective benefit of exa-cel. This event occurred in the setting of a documented parvovirus infection. Parvovirus infections are known to cause severe and potentially life-threatening events in patients with sickle cell disease due to parvoviral induced acute severe anemia that typically requires hospitalization, often in an intensive care unit and almost universally requires transfusion support. In contrast, this patient recovered quickly after an uncomplicated short hospital stay without any red blood cell transfusions. This case highlights the protective effect of exa-cel in preventing severe complications even from known acute precipitants of what could otherwise be potentially life-threatening events. There was only 1 patient who did not achieve VF12, and this is the patient on the figure who had several VOC events after exa-cel. None of these events required hospitalization and the patient achieved HF12, which I will show you in a moment. At the bottom of the figure are the patients who are not yet evaluable for the primary endpoint because they had not yet been followed for 16 months as of the data cut. One of these patients has had several VOC events and will not achieve VF12, but does remain eligible to achieve HF12, all of the other patients remain eligible to achieve both VF12 and HF12. I want to focus for a minute on adolescent patients. This is the same data that I just showed you, but now focusing on the adolescent patients who are grouped together at the bottom of the figure. Exa-cel demonstrated consistent clinical benefit between adults and adolescents. And this was as expected, given the same disease pathophysiology, the same mechanism of action of exa-cel and the same protective effect of HBS. The 12 adolescent patients who received exa-cel representing approximately 30% of all patients have VOC-free treatment effects similar to adults, and all of the adolescent patients in the PES or 100% of them achieved VF12. Turning here to the key secondary endpoint of avoiding hospitalization. All 30 patients, 100% of them achieved the key secondary efficacy endpoint, HF12, which is defined as patients free from inpatient hospitalization for VOCs for at least 12 consecutive months. This endpoint is clinically important because it informs the absence of the subset of VOCs that are associated with higher acute mortality risk. These data are represented here in the same format that I just showed you for VOC data, but now each Black Diamond represents a hospitalization for a VOC. The gray bars to the left show the frequent hospitalizations for VOCs the patients had over the 2 years prior to exa-cel and the purple bars to the right show hospitalization events after exa-cel. The clinical benefit of exa-cel is clear. There was only 1 patient in the PES who had a hospitalization for a VOC after exa-cel. This is the same patient event I previously described associated with the parvovirus infection. For the patients not yet in the PES at the bottom of the figure, there were 2 other patients who experienced a hospitalization early after exa-cel with both maintaining the potential to achieve HF12. Exa-cel resulted in rapid, robust and durable reactivation of fetal hemoglobin. As shown on the left, fetal hemoglobin levels increased to over 20%, and were maintained at approximately 40% over time. As Dr. Thompson described, increasing fetal hemoglobin to over 20% protects against disease complications, including eliminating VOCs and this was clearly achieved. As shown on the right, adolescents increased fetal hemoglobin levels similar to adults with all adolescents achieving fetal hemoglobin levels over 20%, which were also maintained at approximately 40% over time. Again, illustrating the similar treatment response of adolescents and adults. To further demonstrate the durability of exa-cel, shown here is patients allelic editing in bone marrow at the top and peripheral blood on the bottom, which remains stable and durable throughout follow-up in every patient. This demonstrates the stable engraftment of edited long-term hematopoietic stem cells with editing remaining durable through follow-up, including beyond 2 years. In summary, exa-cel demonstrated transformational clinical benefit in patients with sickle cell disease. 97% of patients achieved the primary endpoint of VF12 and 100% achieved the key secondary endpoint of being free from inpatient hospitalizations for VOCs. This efficacy was consistent across all endpoints and all subgroups. And in particular, adolescent patients had similar efficacy responses as adults. Again, this is as expected given the same disease pathophysiology, the same mechanism of action of exa-cel and the same protective effect of fetal hemoglobin. Efficacy was durable. Patients were VOC free for an average of over 22 months, including up to almost 4 years. High protective levels of fetal hemoglobin were rapidly achieved and were durable over time. Allelic editing was stable for up to approximately 4 years of follow-up. In totality, the data support the remarkable clinical benefit of exa-cel in patients with sickle cell disease. I'll now invite Dr. Altshuler to present the nonclinical safety.

My name is David Altshuler and I'm the Chief Scientific Officer at Vertex. I will be discussing nonclinical safety with a focus on the strategies used to minimize the potential for off-target editing by exa-cel. We designed and executed a comprehensive nonclinical safety package in support of the exa-cel program. The package included analysis of on-target editing of chromosomal integrity, potential for off-target editing and studies of tumorigenicity, engraftment, persistence and biodistribution. The nonclinical studies did not identify any exa-cel specific risk. I will focus this presentation on the potential for off-target editing. 10 years after the discovery of CRISPR gene editing, we now understand that the specificity of CRISPR is determined by the uniqueness of the on-target site and of the guide RNA. In cells exposed to CRISPR, the guide RNA guides the CRISPR enzyme to specific genomic locations based on sequence homology, that is where the guide RNA matches the DNA of the host genome. For CRISPR to edit a specific site, the DNA sequence must match both the guide RNA and the short adjacent sequence known as the protospacer adjacent motif or PAM. If the on-target site is unique in the genome as depicted on the left with a yellow dot, and if the guide is highly specific, then editing will occur only at the on-target site. However, if one were to choose an on-target DNA sequence that is present at multiple genomic locations is depicted on the right, in red, and 1 design to guide that binds promiscuously at many places in the genome that off-target editing can occur. Based on this understanding, 3 strategies to minimize the risk of off-target editing are first, to limit exposure to CRISPR; second, to select an on-target site that is unique in the genome. And third, to optimize the guide RNA, not only for efficacy, but also for specificity. From the start of the exa-cel program 8 years ago, we were focused on minimizing and assessing the risk of off-target editing. The design of exa-cel was shaped by 3 strategies to minimize off-target risk. First, we use an ex vivo approach and transiently express CRISPR only in cells of the hematopoietic lineage. Second, to select an on-target site that is unique in the genome; and third, to optimize the guide RNA, not only for efficacy, but also for specificity. From the start of the exa-cel program 8 years ago, we were focused on minimizing and assessing the risk of off-target editing. The design of exa-cel was shaped by 3 strategies to minimize off-target risk. First, we use an ex vivo approach and transiently express CRISPR only in cells of the hematopoietic lineage. Second, we selected the on-target site in an intron of BCL11a that has a unique sequence with no other match in the human genome. Third, we screened hundreds of candidate guide RNAs to select an optimal guide RNA that is no other match elsewhere in the human genome. Now having designed exa-cel to minimize potential for off-target editing, we then systematically evaluated the risk of off-target editing using multiple orthogonal methods to detect potential off-target edits including sites nominated based on human genetic diversity and performing risk assessments as appropriate. And the conclusion is that the design of exa-cel minimize potential for off-target risk and multiple systematic evaluations did not identify evidence of off-target editing by exa-cel. I'll start by describing the framework used for off-target evaluation of exa-cel. As depicted in the box on the left, our approach involves 3 steps. First, we nominated candidate off-target sites using 2 orthogonal methods, computational homology search and a laboratory method known as GUIDE-seq. As will be discussed in the next section, the nominating process included analysis of human genetic diversity relevant to the exa-cel patient population. Both nomination methods are known to be sensitive in their ability to detect sites at which off-target editing may occur, but both methods have high rates of false positives. And for this reason, to determine if any off-target editing occurs at any nominated site, the second step was to compare the DNA sequences of edited as compared to unedited cells using high coverage, hybrid capture next-generation sequencing. The third step was to perform a risk assessment in 2 settings. First, if any sites were confirmed as having an off-target edit and second, for any site nominated based on a rare genetic variant that was not directly evaluated in our hybrid capture experiments. I'll now review in a bit more detail each step in this process. One nomination method was a systematic computational homology search of the human genome sequence. In the box on the right, you can see the DNA sequence of the exa-cel guide -- the exa-cel on the target site and in the NGG PAM. Below that is a sequence of a potential off-target site that has 3 mismatches and an alternative PAM sequence highlighted in red. In the first study, we searched the genome and nominated 5,007 candidate sites based on criteria of up to 5 mismatches or a bulge or an alternative PAM sequence. While sites with a bulge or alternative PAM are very unlikely to cut, we included them for completeness. In the second study, we narrowed the mismatch criteria to include only those sites with up to 3 mismatches, because this enabled sequencing more deeply at the candidate sites with the highest likelihood of having any off-target editing. The third study added 50 additional candidate sites nominated based on human genome sequence diversity. Now on the next slide, to help quantitate the risk of off-target editing, I will review literature on how the number of mismatches between a guide RNA and the cells genome sequence can impact the likelihood of off-target editing. This table includes data from a paper by Haeussler et al that measures the likelihood that any given site would be subject to off-target editing by CRISPR/Cas9 as a function of the number of mismatches between the guide RNA and that genomic sequence. This paper analyzed many different guides and many different off-target sites. They found that sites with 1 or 2 mismatches to guide RNA have a reasonably high chance of detectable off-target editing. By the time there are 3 mismatches, less than 2% of such sites with 3 mismatches have any detectable off-target editing. And by the time there are 5 mismatches, only 1 in 20,000 such sites had off-target editing. Now how does this data apply to exa-cel? Well, in the human genome sequence, there are no sites with 0 mismatches or 1 mismatch or 2 mismatches as compared to the exa-cel guide RNA. In fact, there are only 6 sites in the entire human genome with 3 mismatches to a guide RNA. So all the other sites that we nominated and tested have more than 3 mismatches and/or contain a bulge or an alternative PAM. And sites with these features have an even lower likelihood of off-target editing. Now the last 2 slides are about computational homology search. We also nominated candidate sites using a second orthogonal laboratory-based method known as GUIDE-seq. GUIDE-seq is a well-established empirical nomination method that has performed directly in living cells. And we perform GUIDE-seq in CD34 cells from both healthy volunteers and from patients with sickle cell disease and transfusion-dependent thalassemia using the process used by exa-cel. Now GUIDE-seq has high sensitivity. But to validate this in our experiments, in each experiment, we use the on-target site as an internal positive control to document that editing occurred and could be detected. But GUIDE-seq also has a high rate of false positives. And this is because normal cells have double-strand breaks even in the absence of genome editing. Given that both computational homology search and GUIDE-seq have a high rate of false positives, it was necessary to perform a second independent test to determine of off-target editing actually occurs at any of the nominated sites. To test each candidate site for off-target editing, we use a sensitive and accurate method known as high coverage hybrid capture sequencing. Specifically, we compared the genomes of edited and unedited cells at each of the candidate off-target sites. We use very high sequencing coverage depth ranging from 2,500 fold in the first study to 19,000 fold in the third study. We use such high sequencing coverage depth to enable detection of off-target editing as few as 2 in 1,000 DNA copies in edited as compared to unedited cells. Finally, in each hybrid capture experiment, we again use the on-target BCL11a site as an internal positive control, confirming that editing occurred and the hybrid capture sequencing could detect it. The third step in our framework was to perform risk assessment. The reason to perform risk assessment is that the presence of an off-target edit, if one were to be found, does not in and of itself create risk to the patient. The risk of a potential off-target edit would be of an increased risk of malignancy or impacted the function of a gene known to play a role in cells edited by exa-cel. For this reason, we performed risk assessments on sites meeting either of 2 criteria: first, if hybrid capture sequencing are found any confirmed off-target edit, we would have performed a risk assessment; second, some of our candidate sites were nominated based on genetic diversity and if a specific variant allele was not present in any of the samples tested with hybrid capture, we would perform a risk assessment. The pre-specified questions considered in our risk assessment were: does the off-target site overlap a gene known to play a role in hematologic malignancy; does the off-target site overlap an exon; and does the off-target site overlap a gene known to play a functional role and be expressed in blood cells. In a few minutes, I'll discuss the results of these studies. But first, I next discuss the approach used to include genetic diversity in the off-target analysis. Because the intended patient population for exa-cel is diverse, our off-target analysis includes genetic diversity. We nominated candidate sites based on a variant-aware homology search. This identified additional sites that met the criteria for potential off-target site only in the presence of a genetic variant. Specifically, we identified all variant sites in the 1,000 Genomes Project database with a frequency greater than 1% in samples from populations living in each of 5 continents, that is donors residing in or with ancestry from Africa, East Asia, South Asia, Europe and the Americas. And in the 1,000 Genomes Project, there are more than 21 million genetic variant sites with a frequency greater than 1% in 1 or more of the populations. We included these 21 million variant sites in our off-target variant-aware homology search, and this led to the nomination of 50 additional candidate sites. As a second approach to include genetic diversity, the 14 donors in whose cells we performed hybrid capture sequencing had self-reported ancestry that was diverse and this included 4 donors with African-American ancestry, 3 of whom had sickle cell disease. To evaluate the adequacy of this approach to incorporating genetic diversity, it's helpful to review 2 aspects of our current understanding of the human genome sequence and how it varies across populations. First, any 2 copies in the human genome sequence are 99.9% identical. That is, they differ on average at only 1 of 1,000 DNA letters. Of the 1 of 1,000 or so DNA letters that vary in any individual, the overwhelming majority are due to genetic variants that turn out to be common and shared across populations. The reason for this is that all 8 billion people living on the planet today are descended from a small founder population that lived in Africa tens of thousands of years ago. Our shared ancestry means that we are very similar to one another at the level of DNA sequence. Because most human genetic variation is both common and shared across populations, it's possible to build a comprehensive database of common human genetic variation. The second aspect of the human genome that I want to mention is that only 1% spans protein-coding exons. This means that the millions of genetic variants in each of us, only a tiny fraction overlap functional sequences. To serve human genetic variation, we use the 1,000 Genomes Project an NIH-funded, gold standard, global reference database of human genetic variation. And we use the 1,000 Genomes Project database, because all samples were consented for public data release, including community consultation. The sample set is large and diverse, including 2,504 individuals from 26 different global populations. And of the 2,504 samples, 661 resided in and/or have recent ancestry from West Africa, East Africa, African-American or Afro-Caribbean populations. This slide briefly compares the 1,000 Genomes Project database to another well-known database called the Human Genome Diversity Project that is discussed in the FDA briefing book. In addition to the 1,000 Genomes Project having informed consent for public data release, the 1,000 Genomes Project contains more individuals than the HGDP, 2,504 as compared to 929; more samples residing in or with recent African ancestry, 661 as compared to 104; and contains 61 samples from individuals with African ancestry residing in the United States. These data document that the 1,000 Genomes Project database is an appropriate resource for studies of human variation relevant to the exa-cel target population. We also performed calculations to evaluate the power to detect variants with a frequency 1% or higher in 661 donors of the 1,000 Genomes Project from individuals residing in or with recent ancestry from sub-Saharan Africa. The answer is that 661 individuals provides greater than 99% power to discover variance with a frequency greater than 1% in these population samples. Moreover, both internal and external analyses have independently confirmed the completeness of the 1,000 Genomes Project database for variance of frequency 1% and above in these samples. Now I want to again emphasize that because most genetic variation in each individual is shared across populations and because patients potentially treated with exa-cel will have ancestry from many parts of the globe, all of the samples from the 1,000 Genomes Project contribute information relevant to our off-target assessment. Having described the framework for evaluating off-target editing and inclusion of genetic diversity, I will now review the results of the off-target analysis. In summary, 3 off-target studies were performed and these studies did not identify any evidence of off-target editing by exa-cel. The first assessment was performed in 4 healthy donors and more than 5,000 candidate sites nominated by both homology search and GUIDE-seq. The coverage depth was 2,500 fold median depth and no off-target editing was detected at any site in any individual. The second study was performed in 4 additional samples from healthy donors, and this study focused on 171 sites with 3 or fewer mismatches or a bulge or an alternative PAM and also included additional sites nominated from GUIDE-seq in 2 additional healthy donors. The sequence step was increased to a median of 15,000 fold depth, and no off-target editing was detected in any individual at any site. The third study was performed in 6 patient samples, 3 each with sickle cell disease and transfusion-dependent thalassemia. As discussed, we performed variant-aware homology search, nominating 50 additional sites based on genetic diversity. We included these sites. We included sites also nominated by GUIDE-seq in each of the patient samples. The sequence step was a median of 19,000 fold depth. No off-target editing was detected at any site in any individual. To the best of our knowledge, this is the most comprehensive evaluation of off-target potential performed to date. This slide provides an alternative visualization of the hybrid capture data for all nominated sites tested in 1 patient with sickle cell disease. The x-axis is chromosomal position with chromosome 1 on the left and the X and Y chromosomes on the right. The y-axis is the rate of editing at each in unedited -- sorry, in edited CD34 cells using our manufacturing process as compared to unedited CD34 cells using the same process. You can see the purple dot showing the high rate of editing at the on-target site. You can also see that no other site was editing detected above the threshold of detection. This slide provides the hybrid capture results for the 8 healthy donors from the Study 1 and 2 and in each case, you can see very consistent results, high rates of editing at the on-target site and no evidence of off-target editing. And this slide shows a very similar result for the 6 patient samples, 3 with sickle cell disease, 3 with transfusion-dependent thalassemia. Again, the results are consistent, higher rates of editing at the on-target site and no evidence of off-target editing at the nominated sites. We genotype each of the samples tested for the genetic variants that led to the nomination of the 50 additional sites, that is we genotyped each sample tested with hybrid capture for the sites, the 50 additional sites nominated based on genetic diversity. Of those 50 sites, 9 were nominated based on a common genetic variant with a global frequency greater than 10%. And at each of these 9 sites, 1 or more individuals characterized by hybrid capture carried the genetic variant. In the individual donors who carry the variant allele, no off-target editing was detected. For the remaining 41 of the 50 sites, the genetic variant that led to nomination had a frequency less than 10% globally. And at 3 of these 41 sites, the variant allele was observed in 1 or more patient samples studied by hybrid capture and no evidence of off-target editing was detected. But because some of the variant alleles were not seen in our hybrid capture samples, we performed a risk assessment of each as if editing had been seen. This is what we would have done if editing had been observed in hybrid capture experiments. We found that none of the sites overlapped with the gene involved in hematologic malignancy and none of the sites overlapped with the protein-coding exon. We also evaluated the candidate genomic site described earlier this year in a paper by Cancellieri et al. Cancellieri et al. developed a computational algorithm, as you heard from Dr. Bauer, for identifying candidate off-target sites based on genetic diversity, and they use BCL11a as a test case and highlighted the particular variance site as having the potential risk of off-target editing. Our initial homology search actually nominated this candidate based not on genetic diversity, but based on the presence of an alternative PAM sequence. So we tested this locus in all 14 hybrid capture off-target assessments and no off-target editing was observed. But we genotype each of the 14 donors to see if any carry the low-frequency site discussed by Cancellieri et al. and none of the 14 donors carried that allele. Now this is unsurprising given the variant has a frequency of 5% in both the 1,000 Genomes Project and the Human Genome Diversity Project in samples from Africa. Because none of our donor samples contain the variant, we perform the risk assessment to determine if potential off-target editing this site would be expected to create risk for patients in whom it might occur. The site occurs in a non-coding intron of a gene called CPS1. CPS1 has no known or hypothesized role in malignancy. CPS1 encodes a metabolic enzyme that is expressed specifically in the liver and small intestine and is not expressed in any blood cells. Thus, the risk assessment did not highlight any specific risk attributable to potential gene editing at the Cancellieri site. In summary, we designed exa-cel to minimize off-target risk by choosing an ex vivo editing procedure with transient expression of CRISPR/Cas9, selected an on-target site with a sequence that is unique in the human genome and carefully screen guides to select one that is highly precise and specific for the on-target site. We empirically assessed off-target editing using hybrid capture high coverage sequencing in cells edited with our protocol, including sites nominated based on sequence diversity. And we performed a risk assessment on each potential site that was nominated based on genetic variation, including an addition for the site highlighted by Cancellieri et al. And none of the candidate sites nominated based on genetic variation overlapped with a gene involved in hematologic malignancy nor a coding exon of any gene. In summary, a comprehensive nonclinical data package did not identify any evidence for off-target editing by exa-cel. And I will now turn to Dr. Simard, who will discuss clinical safety.

Thank you, Dr. Altshuler. Good morning. I'm Christopher Simard, Vice President of Global Patient Safety at Vertex. This morning, I'll be sharing a summary of the clinical safety data for exa-cel, which supports a favorable benefit risk in adults and adolescents with severe sickle cell disease. By way of an overview, adverse events and serious adverse events after exa-cel were consistent with that of myeloablative conditioning with busulfan and hematopoietic stem cell transplant. No patients experienced graft rejection or graft failure and all patients successfully achieved both neutrophil and platelet engraftment. Safety has also been similar across subgroups, including adults, and adolescents. No new or unique safety events have emerged during long-term follow-up, including no malignancies. And we'll lastly review key elements of our proposed post-approval pharmacovigilance plan, including product labeling and long-term follow-up. Beginning with the safety database. The safety database for exa-cel consists of 44 patients with sickle cell disease, which included 32 adults and 12 adolescents. Patients have been followed for an average of 20.1 months with 73.5 patient years cumulative follow-up. 30 patients or 68% have been followed for 18 months or longer with a maximum follow-up of approximately 4 years. Now let's look at the adverse event data. The safety profile following exa-cel can best be summarized as being consistent with that of myeloablative conditioning with busulfan and hematopoietic stem cell transplant. All patients in Study 121 experienced at least 1 adverse event, 30% had adverse events considered related to exa-cel, all of these were nonserious. One patient died, this was from COVID-19 infection, which led to respiratory failure 9 months after treatment. The event was attributed to COVID-19 infection and possibly related to busulfan. On this slide, we summarize the adverse event rates per patient months over time. Not unexpectedly following myeloblation, most adverse events, grade 3 and higher adverse events and serious adverse events occurred in the first 3 months and all decreased over time. Here we see the most common adverse events, including those Grade 3 and higher. These 2 were all consistent with the known safety of busulfan myeloablation and HSCT. And while we're just showing the most common adverse events here, additional details on adverse events, serious adverse events as well as safety in adolescents and adults, which was similar, has been included in the briefing materials. Turning to engraftment. All patients who received exa-cel, successfully achieved both neutrophil and platelet engraftment. Platelet engraftment time was somewhat longer than reported in the allo-HSCT literature. However, overall platelet recovery was robust in patients with longer times to platelet engraftment had similar efficacy and safety outcomes as other patients in the study. Before we conclude, I'd like to briefly summarize our post-approval pharmacovigilance plan. Within product labeling, we propose to include the risk of delayed platelet engraftment as well as risks associated with busulfan and myeloblative conditioning used as part of the exa-cel regimen. We also plan to monitor the safety of exa-cel over the long term, including clinical trial patients and patients treated post approval in a registry for 15 years. We have multiple surveillance mechanisms in place to closely monitor patients for long-term safety post approval. Beginning on the far left, we will follow all clinical trial patients for 15 years, including the safety and efficacy data shown. In addition, we are fortunate that data from over 90% of patients, who undergo bone marrow transplant in the U.S. is collected and available through the CIBMTR transplant registry. Importantly, all centers in the U.S. where exa-cel will be used participate in CIBMTR and will provide data on exa-cel treated patients to the registry, and we will have access to this data. We are further also planning a 250-patient Vertex registry-based study, which will leverage CIBMTR and European transplant registries where patients will be followed for 15 years. The study will collect all the data, which the registries collect as well as all SAEs and malignancies, which will be reported to us immediately or within 24 hours. I would like to point out that for patients in the pivotal studies and the long-term follow-up Study 131, we collect in-store bone marrow and blood samples before exa-cel treatment and periodic intervals after exa-cel treatment. Finally, in addition to what is summarized on the slide, I'd like to further highlight that as part of our manufacturing process, we collect and store samples of CD34-positive cells before and after editing in all clinical trial patients, and we plan to do the same for all patients, who will receive exa-cel in the post-approval setting and all of these samples would be available for DNA testing should the need arise. Through these extensive surveillance activities, we will closely monitor patients for potential safety signals over the long term. In conclusion, the safety data demonstrate that exa-cel has a favorable safety profile in patients with severe sickle cell disease. The clinical safety profile of exa-cel has been consistent with that of busulfan myeloablation in HSCT with delayed platelet engraftment, the only exa-cel specific risk. All patients that were able to successfully achieve and maintain both neutrophil and platelet engraftment after exa-cel. The data also demonstrates the safety profile of exa-cel was similar in adults and adolescents. To date, we've seen no long-term safety findings, including no malignancies in the entire exa-cel program and long-term monitoring will continue post approval. In totality, these data demonstrate that exa-cel has a favorable safety profile and support a positive benefit risk in adults and adolescents with severe sickle cell disease. Thank you. And I will now turn the lectern to Dr. Frangoul to share his clinical perspective.

Thank you. I'm Haydar Frangoul. I'm the Medical Director of Pediatric Hematology/Oncology and Cellular Therapy at the Sarah Cannon Research Institute in Nashville, Tennessee. I'm a hematologist and a stem cell transplant physician. So I see patients with sickle cell disease and their families from all over the region, who are referred to our center to discuss transplant options. I'm also the lead investigator in the study presented today, and I have seen firsthand the impact exa-cel has on my patients with sickle cell disease. It has been such a rewarding experience to take part in this program, and I'm excited to be here today to provide my clinical perspective and experience using exa-cel. As you've heard from Dr. Thompson, sickle cell disease is debilitating and shortens the patient's lifespan. Patients who experience severe recurrent vaso-occlusive crisis live with debilitating pain and chronic progressive complications across multiple organs. I see this diminish the quality of life for my patients and their families. So it is clear that patients need a curative therapy. I have been performing allogeneic transplant for sickle cell disease for more than 20 years. And I have seen the impact of a cure on patients and their families. It's truly life-changing. But we must remember that 80% to 85% of patients with sickle cell disease do not have an HLA-identical related donor. And there are many risks involved with transplant using alternative donor transplant that the patient must consider. Our related and haploidentical transplants have been associated with risk of graft rejection, transplant-related mortality and high rates of acute and chronic graft-versus-host disease especially in the unrelated setting. I would like to share some patient stories to illustrate this experience. The first patient we consented was a 33-year old mother of 4 children and have been in and out of the hospital roughly 7 times over 2 years. She was suffering with severe and painful sickle cell crisis where a time she couldn't walk or even hold up a spoon to feed herself. She described the pain as lightning striking her chest. And because of this, she couldn't keep a job and was struggling to care and enjoy time with her 4 active children. The patient was initially referred to us for a haploidentica bone marrow transplant, but she was worried about the risk of graft-versus-host disease and the need for prolonged immunosuppression and decided to enroll on the exa-cel trial. Following exa-cel, she has remained VOC-free and is now spending time with her family and working full time, something she was not able to do prior to receiving exa-cel. The second patient is a 13-year-old girl, who was diagnosed with sickle cell disease on newborn screening. She had her first hospital admission at 6 months of age. And despite hydroxyurea therapy, she was hospitalized many times per year, including an episode of severe acute chest syndrome. She could not attend school regularly because of her pain crisis. Following exa-cel treatment, she had not experienced any VOC. She has not been hospitalized once and she's attending school and enjoying her teenage years. The highlighted stories are not unique to those patients. I see the same effect on the patients with sickle cell disease they have treated with exa-cel. And many of the adult patients which they were given the opportunity to be treated at younger age, so they could have their lives and live it to the fullest. My patients, who participated in the trial went on to benefit in the same way as my post-allogeneic transplant patients do over time. They live their lives as patients, who do not have the disease without the severe painful vaso-occlusive crisis and hospitalizations. They go back to school or work. They participate in their normal activities, and they enjoy time with families and friends, all things that were previously challenging, because of their sickle cell disease. Because exa-cel is an autologous product, it avoid the major limitation of allogeneic transplants, because every patient is their own donor. Therefore, there is no risk of graft-versus-host disease or graft rejection and no need for long-term immunosuppression. And exa-cel eliminate those risks while providing transformational clinical benefit and potential functional cure for sickle cell disease. I am from the camp that says to treat at a younger age as possible. Over time, sickle cell disease can cause lasting organ impairment, such as kidney disease, stroke or bone damage, because some of the damage that occurs prior to transplant is irreversible. I explain it to my patient this way, sickle cell disease is like a hammer hitting a wall. If you hit the wall with a hammer, it leaves damage. With transplant and I can take away the hammer, but we cannot reverse the irreversible damage. We cannot fix the wall. So if someone comes in with a joint that has been completely destroyed by sickle cell disease, a transplant will stop another joint from being destroyed, but it will not repair the original joint. That is why intervening early is better. I want to take away the hammer before it permanently damages the organs. For sickle cell disease, we have no way to tell what an individual patient trajectory will be, but we consistently see that disease will get worse as children and adolescent approach adulthood. That's why some hematologists perform HLA typing on patients and their siblings early in childhood to identify potential match siblings even when no signs and symptoms of the disease are yet present. Dr. Hobbs showed us earlier that the exa-cel data in adolescent is consistent with the adult data, as we would expect, given the mechanism of disease and mechanism of exa-cel are the same regardless of age. And adolescent patients often tolerate the myeloablative conditioning and transplantation procedure better than adults, further supporting the benefit of treating early. Therefore, the extrapolation of adult data to adolescent is very appropriate, and I would be happy to have this therapy available for my adolescent patients. In conclusion, exa-cel data have demonstrated transformational and durable clinical benefit for patients with sickle cell disease. And I have seen this clearly in the patients I have treated in the study. All study patients received substantial clinical benefit, and these results were demonstrated consistently across adolescent and adult patients. Regarding safety, exa-cel was generally safe and well tolerated, consistent with that of busulfan myeloablation and hematopoietic stem cell transplant. As we did in the trials, experienced medical staff, who regularly care for patients receiving transplant will be able to monitor appropriately for safe use of this therapy. It has been an honor to participate in this trial and see exa-cel change my patient's lives. I hope to soon have it available as an approved treatment option for the patient suffering with sickle cell disease. Thank you, and I'll now turn the presentation back to the sponsor to take your questions.

Great. Thank you very much for those presentations. That was very informative. We'll now take questions from the committee for the sponsor. And I just wanted to remind people that there will be an FDA presentation after lunch. And so opportunity for questions there as well for the FDA and then, of course, discussion in the afternoon. So Dr. [indiscernible], can you go on camera and unmute yourself, please?

Yes. Thank you for the presentation. This is, of course, quite interesting. I have a number of questions. I'm just going to start off with 1 or 2 and then see how the questions go. One is that you've shown really robust and stable percent levels of edits and hemoglobin F. So these percentages stay stable over time. But you didn't show hemoglobin levels or markers of hemolysis. Were they similarly stable? And can you comment on whether they normalized?

I will ask Dr. Hobbs to address your question.

Bill Hobbs, Clinical Development at Vertex. Your question is twofold. One is about hemoglobin and one is about hemolysis. First, I'll start with hemolysis. And we looked at hemolysis in a couple of different ways. We focused on measures of intravascular hemolysis, because these measures like LDH and haptoglobin and LDH in particular, are associated with increased effects on mortality as well as other vascular complications. And what we observed was a decrease in LDH and an improvement in haptoglobin. Shown here is LDH levels, which normalized after 9 months in patients and remained normal. In -- for haptoglobin, we saw an increase in haptoglobin as would be expected with a resolution of hemolysis or an improvement in hemolysis with levels becoming detectable in patients and remaining detectable in patients over time. The second part of your question is regarding total hemoglobin, partly because of the anemia of the disease and not unexpected for sickle cell disease, we saw increase -- for the effect of fetal hemoglobin, we saw increases in total hemoglobin as a function of that fetal hemoglobin with levels achieving normal or near-normal levels in almost all patients at approximately 12 grams per deciliter in large part due to that PAM cellular distribution of fetal hemoglobin, which is shown on the right. And so I think across the data, which is also in the briefing book for additional review was an improvement in anemia and improvement in hemolysis across all study patients.

You also look at the reticulocyte in total bilirubin?

We did look at additional measures of hemolysis, which included reticulocytes. And reticulocyte counts also improved over time in patients, which is shown here with a decrease from baseline, although remaining still perhaps somewhat elevated compared to normal.

Great. I also have a question about hybrid capture method that you used. So one thing that puzzles me about the method was that you only had 60% on-target editing in your donor samples. And that doesn't seem to be reflective of the graphic you put into participants because they have much higher -- even in vivo levels of editing. So if the editing rate is higher in the participant samples, I would think that the off-target rates might also be similarly higher. Why is it only 60% on-target editing in these samples used for the hybrid capturing?

I'll ask Dr. Altshuler to address your question.

The hybrid capture experiments were done using the same manufacturing process and the same cells and the distribution of on-target editing was the same in the distribution of all patients treated in the hybrid capture experiments.

So your participant samples products were 60% edited and then gave 90% neutrophil editing during follow-up?

I'm not sure -- the 60% that you're referring to might be the one slide, but there's a variety of different on-target edits and the samples used, obviously, it's not the samples from the clinical trial, the set of samples we use nonclinically, but it went through the same process, sampled in the same way and had the same results.

I can ask Dr. [ Moore ] to address your manufacturing question.

[ Kim Moore ], CMC. I just want to add that each patient may receive more than 1 lot. And so that specific lot used in the hybrid capture may have contributed to part of the dose, but more than 1 lot can be used.

Great. Thank you. Dr. [indiscernible] can you go on camera and unmute yourself, please.

Yes. I have a question about the off-target effect of the on-target editing. In other words, I would like to learn more about the lack of BCL2A expression in erythrocytes or precursors and other lineages coming out of HSCs. I understand that the editing is done in a very specific enhancer but nothing is always complete. And I would like to know whether the lack of BCL2A has any effect in other lineages, for example, is the delayed platelet engraftment caused by this? And also, do you anticipate any other effects there other than the effect on the hemoglobin gene?

Dr. Altshuler?

I think the simple answer is we do not expect any other effects, but let me explain a little bit more detail. So I'd like to present here our analysis of on-target editing. So just to contextualize for everybody. The on-target site is in intron of the gene, as you can see here depicted between exon 2 and exon 3. And just to give you a sense of how large a non-coding region, this is the nearest, exon on the right is 26,000 base pairs away, and the nearest on the left is 50,000 base pairs away. And you can see the distribution of indels of the different genetic perturbations or edits from exa-cel in the graph below, where the on-target site is right in the middle. And you can see that 88 -- you can't really see, the bullet shows you. But in fact, if you quantify it, 88% of all indels less than 30 base pairs in length. And so I think the question then is -- but there are some that are larger, but they're all as you can see here, modest in size. If the question then is, what would happen outside of erythrocytes, I actually think there are 2 ways of answering that question. One is experimentally where we actually transplanted the cells into non -- I'm answering non-clinically. We transplanted the cells into animals and looked at the distribution of edits across different cell types, and it was unchanged for the different edited cells and the other was obviously the clinical data, which could be described. But I think that there's another piece of data that's very informative, which is others, not Vertex have done extensive characterization of this region. In fact, Dr. Bauer, published in very beautiful papers, where both in human cells and in animals, in mice, they actually created systematic modification of this non-coding region including edits much larger than the ones we see with exa-cel. And they then looked in the animals and also in the cells. And so no effect of editing this non-coding region and this erythroid-specific enhancer in any other setting. And I guess the last point is the genetic variant we're recreating, which is this whole program was motivated by a genetic variant discovered in a genome-wide association study that increased hemoglobin F and decrease the risk of the severity of symptoms with both the thalassemia and sickle cell disease. And then that variant has been studied in millions of human beings. It's a common variant to look for other phenotypic consequences of modulating the site and none were observed.

Thank you. Dr. [ Wolfe ], will you go on camera and unmute yourself, please.

Yes. So I had a follow-up question on the off-target event. It was identified in the Cancellieri paper by the Bauer Lab and others. What's the plan to follow up with regards to treated patients to look at editing events at this off-target site in the context of the therapy?

I will ask Dr. Hobbs to address your question.

Bill Hobbs, Clinical Development. This is a really important question that we've thought really long and hard about and not only in relationship to the Cancellieri variant that was identified, but really to any potential off-target risk. And our approach to this if you boil it down is really that to do close clinical monitoring and follow-up, which we recommend and do for all patients in the clinical study irrespective of the variant or not. And the rationale for the approach is that we know we have established a strongly positive benefit risk for this in a patient population with severe unmet need, who are -- who not only have a disease that is -- impacts quality of life, but also shortens their life as the disease relentlessly progresses. The approach for that Dr. Altshuler described for the nonclinical package, which I didn't identify any specific off-target risks and did a risk assessment of any additional variant that could potentially occur in a patient concluded that there was a low risk of functional consequence to a patient. And therefore, we had neither an off-target to follow nor a specific variant of concern for a clinical outcome. And we concluded from that, that the appropriate approach for all patients in the clinical study was close and careful clinical monitoring which is independent of whether they have the variant or not and assumes that any particular patient could have an off-target effect, which we could then pick up. In that process, we also collected laboratory samples from both before and after treatment that would allow us to go and then subsequently investigate should the need arise. And so this is also reflected in our pharmacovigilance plan that Dr. Simard described. And so our approach has been to do careful close clinical monitoring, which was also referred to in the session this morning as the appropriate approach for all patients, who receive a genetic therapy like exa-cel.

So there's no plan for molecular follow-up to look at editing at the off-target site. It seems like there is quite a bit that could be learned with regards to off-target editing rates in your treated patient population by looking retrospectively at editing at this site now that you've treated more than 45 patients.

I will ask Dr. Altshular to address your question.

It's an important question, and we've thought deeply about it. And the way we think about it is, first, take into consideration not only the off-target assessment we've done, but all the other assessments and the package of data and as Dr. Hobbs described, we don't believe from the totality of data that we've collected that additional nonclinical studies are going to be informative. And then we believe for clinical studies, the important thing is to follow all the patients, see if any events occur, and then we'll have the samples and the data to try and understand those events, and that's our approach.

Thank you. Dr. [ Shapiro ], can you turn on your camera and come off mute, please.

Yes. Thank you. I have some clinical questions. I think specifically for Dr. Thompson. Can you comment on fertility preservation protocols and what standard of care and issues specifically related to that in individuals of childbearing age or pre-puberty for individuals who might undergo this therapy?

I'm going to take that in 2 parts. First, I'm going to ask Dr. Hobbs to comment on the clinical perspective and then Dr. Thompson to comment on the patient perspective.

Bill Hobbs, Clinical Development. And thank you for the question because this is, I think, a really important one for patients and families as they think about going through a treatment such as exa-cel. In the clinical studies, for all patients, we offered fertility preservation, and that's largely because the reason for that is the busulfan myeloablative conditioning that patients get, which has a high frequency of potential infertility afterwards, not related to exa-cel itself. So we did offer that for all patients in the clinical study. I'll now turn it over to Dr. Thompson to discuss the additional clinical perspective on that for patients.

Thank you, Dr. Shapiro for asking that question. I think this is a critical issue that we need to deal with in terms of efficacy. I think it's safe to say prior to programs like this current program, this was not the standard of care, although one could have made the argument some time ago, given that myeloablative therapy even used in allogeneic stem cell transplant has been associated with infertility. It's been very reassuring that recent programs in this space have included that as part of the studies, including the payment for it. I do believe that many of us have an opportunity to advocate with insurers to be sure that they also consider this in the totality of cost for transplantation. It is absolute tragic for families happy to choose between a possible cure and their children having future children. And so we would strongly as a community support in and all efforts, including those by Be The Match, which will now help to support in a limited way, fertility preservation for individuals with sickle cell disease, who are undergoing chemotherapy-related conditioning that may impact their fertility.

Great. Actually I have a follow-up question. Is that okay? Or do I need to...

Yes if you can keep it brief. We have a few people with questions, yes.

Okay. In this regard, would you -- if both allogeneic stem cell transplant were available as well as exa-cel, would you prefer exa-cel over allogeneic treatment for individuals with sickle cell?

I will ask Dr. Frangoul to address your question.

This is Haydar Frangoul from Sarah Cannon. I think the decision to go with exa-cel versus an allogeneic transplant even when there is an HLA-identical sibling identified, is a decision that should be made by the physician as well as the family. There are so many things to consider, including recovery time, the need for immunosuppression, the collection of cells from the donor, which can put the donors at risk to donate bone marrow. So there are multiple variables, but the results we are seeing are equivalent to what we see with the HLA-identical sibling transplant. And I think that discussion should take place between the physician and the families.

Great. Actually, I'm going to insert myself here to ask a question myself, which is the efficacy seems to be very impressive in terms of data and its durability. Do we expect -- is there any reason to believe a change in off-target effects with repeat treatment? We're hoping -- it seems like there is a propensity to try to treat earlier with adolescents, but this is a lifelong disease. If you were to do a repeat treatment or if the patient were to receive another genetic editing therapy later in life, can we expect a difference in off-target effects?

I will ask Dr. Hobbs to address your question.

Bill Hobbs, Clinical Development at Vertex. Exa-cel was developed and is intended to be a onetime treatment, and we do not envision any need or approach that would include retreatment.

Great. And could you speak a little to the off-target effects if they were to receive a different genetic editing therapy subsequently in life?

Thank you for the question. I'll turn that over to Dr. Altshuler to continue that answer.

I'd like to just go back and answer your question to the transient nature of CRISPR editing with exa-cel. So the cells obviously are harvested. And then in the manufacturing process, they are briefly exposed to the CRISPR/Cas9 enzyme using a [ ribonucleated ] RNA protein complex. And then that is a shortly short duration of editing and that it's gone. So I guess if your question again, as Dr. Hobbs said, we do not imagine the need for nor intend there to be another treatment. But just as you're hypothetical, if it were -- the exposure to CRISPR/Cas9 is extremely brief and not in the body of the person, who has the cells because it all takes place in the manufacturing process and is then gone. And none of it residually is there and makes it into the patient.

Great. Thank you. And you expect no residual effects in the cells that once they've been edited. Dr. [ Brewer ], if you could go on camera and take yourself off mute.

Thank you for your presentations and congratulations on your promising clinical trial results. My question pertains to the labeling. Was any consideration given to adding off-targeted -- possible off-target effects to the label? While I recognize that your preclinical studies did not show evidence of that. I think given the nature of this meeting and the emerging field and the difficulty of potentially trying to identify these things ahead of time, might that be something to consider?

We are still in discussions with the agency on the label. We -- those discussions will certainly include the safety of exa-cel.

Great. Thank you. Dr. [indiscernible]?

So I have a question about clone diversity. There's a lot of talk about off-target effects and the effects of an off-target could have on subsequent hematopoiesis. But I think one buffer against a clone getting out of control is to have a diverse set of hematopoietic stem cells that have this edit. So I'm wondering if you have ways to estimate HSC number contributing over time. I know it's more difficult than, for example, an integrating vector where you can use integration sites to do that. But perhaps the diversity of edits could somehow give you a sense of how many corrected cells you're putting back in and whether that's a high number and a number high enough to hopefully prevent clonal events later?

Dr. Altshuler?

It's a great question. And I'll think during the break, if there's a quantitative answer to your question, but I can tell you that just in terms of you trying to estimate the number, but I will tell you a couple of things that are relevant to your question. One is in the New England Journal Paper in 2021,[indiscernible] we actually published a plot of the distribution of indels, which are not each, of course, clonal, because you got the same indel occur multiple times. We see a very broad distribution of different indels. And that figure in the New England Journal Paper shows 3 different lots and then they were each transplanted into mice, and there were many mice for each. And we both followed the number of clones and also the distribution of those clones across the animals, and it was -- there were many different indels in each animal in the cell lines and across many different animals, that diversity was maintained and similar. And then the other thing we've done is as part of process of qualification. We have characterized the 19 different donor lots just for the distribution of indels and again, see a very broad distribution of indels. So I'll think about your specific question, if I can quantify it, but I think the answer is that there is a broad distribution of cells that get engrafted and there's a broad distribution of different indels to the nature of what you were asking, I think that is the case, but I'll think about your very specific question, see if I can come up with a more quantitative answer for you.

Great. Thank you. We are going over time, but I think this is an important discussion. So we'll continue a little bit more before we go to lunch. Dr. [ London ]?

Yes. Thank you. I'm wondering about the samples from only 14 donors or patients that were tested for off-target editing, how was the sample size chosen? It seems small for detecting the kind of rare event that we're concerned about.

Dr. Altshuler?

If I could have the slide from the core presentation on genetic diversity. The way that, that we think about this in terms of the assessment of genetic diversity really comes back to -- and actually, if I could have the slide on the 2 different types of genetic diversity, please, the 1,000 Genome Project and the sample [ owners ]. So the way we think about this is, first, that, as described in the -- both our presentation and the previous presentations, we do understand that off-target events are directly related to homology between the guide and between the host genome. We know the sequence of the host genome, and we also know the sequence using the 1,000 Genomes Project of 2,504 people, including 21 -- more than 21 million genetic variants. We did the analysis. So I would think of it as the nomination of sites is not about 14 people. It's actually about 2,500 people from around the world, including 661 people with sub-Saharan Africa. Having used all those genetic variants to nominate the sites, then we went and looked at were any of those sites, either from the reference genome or the genetic diversity did they have off-target editing. And we did look at those in 14 individuals of diverse ancestry. But the 14 individuals are not the limit of detection for the variation in people, because we know the variation from the 1,000 Genomes Project of 2,500 people, and we looked at all those sites. And all of the sites in the reference genome, of course, we examined all the sites with a frequency greater than 10% in the human population from 1,000 Genomes were directly evaluated. And there were sites that were low frequency, like 1% in a group from 1 continent to another, and we didn't see them all. We acknowledge that. So we performed the risk assessment that we would have performed had we seen off-target editing. And that risk assessment did not identify any change overlapping with a gene involved in hematologic malignancy, using the [ myelo cPanel ] or an exon of any change. So we believe that the assessment is not an assessment of 14 people, it's an assessment of the genetic variation across 2,500 people that then was queried in appropriate samples.

Great. Thank you. Dr. [ Kumar ]?

I have a quick question. In the brief it said that no chromosomal abnormalities were detected, but I was just wondering how you -- like what was the assay for looking at those? And if that would have picked up any like larger insertions or inversions or translocations or truncations?

I will ask Dr. Altshuler to address your question.

We evaluated chromosomal abnormalities using 2 different orthogonal methods, one was karyotyping which is -- of edited cells, which is a standard approach. And the other was we used a combination of long-range PCR and split-read analysis to evaluate both the indel patterns at the site because it's -- one of the talks mentioned, I think it was Dr. Bauer, you can't simply use PCR to look at large indels, because there's an amplification bias against large sites. So we used a thing called split-read analysis and got actually very similar results for those. So we saw no chromosomal abnormalities in these studies. And I would just note a few other points just in how we think about it, which is that to the best of our understanding, creation of a chromosomal abnormality involves cutting at 2 sites. And one would be beyond target site. And as we said, systematic evaluation we described did not identify any on-target -- off-target editing by exa-cel that would be the substrate. And then just 2 other points that we at least think about are: one, that cells contain that DNA repair system that Dr. [ Arnab ] described, which exists to identify DNA damage and then either rest the cells and either repair the DNA damage or induce apoptosis. And so even if such sites are created doesn't necessarily mean they'll survive. And the last point, just because a lot of discussion in the field is about laboratory experiments that are transient rather than transplant experiments in order for such cells, if they did have DNA repair, if they did have any damage, and they did actually undergo -- survive the DNA repair response, it also to survive the engraftment process and make it to the patient. So those are just additional considerations.

We are getting pressed for time, Dr. Will, if you could [indiscernible] that would be great.

Yes, a very quick question. You may have covered this already, but what is the number of cells that hematopoietic cells that you fuse back to the patient and -- do you have a sense what the -- what percent of these cells have been successfully edited from patient to patient? And then you also showed a patient that did not have any -- that have recurrence at the BOC. Is it because the number of edited cells was lower compared to the other batches compared to other patients?

I will ask Dr. Hobbs to address your questions.

Will Hobbs, Clinical Development. The first part of your question is about the number of cells infused in patients and the protocol specified a minimum of 3 x 10 to the 6 per kilogram and a maximum of 20 x 10 to the 6 per kilogram. And in the clinical study, that range was infused into patients.

Great. Thank you very much. I think we've addressed all the questions for now. There will be opportunity if we need to -- if the committee feels like they have questions directly to the sponsor. We can arrange for that. But I think for now, we're set, and we'll be taking a break for lunch, and we will reconvene at 12:35, so in 30 minutes. And so enjoy your break, and I'll see you all then. [Break]

Meeting from the lunch break, we are now going to move forward with the open public hearing, and I have an announcement to read. Welcome to the open public hearing session. Please note that both Food and Drug Administration and the public believe in the transparent process for information gathering and decision-making. To ensure such transparency at the open public hearing session of the Advisory Committee meeting, FDA believes that it is important to understand the context of an individual's presentation. For this reason, FDA encourages you the open public hearing speaker at the beginning of your written or oral statement to advise the committee of any financial relationship that you may have with the sponsor, its product and if known, its direct competitors. For example, this financial information may include the sponsor's payment of expenses in connection with your participation in this meeting. Likewise, FDA encourages you at the beginning of your statement to advise the committee if you do not have any financial relationships. If you choose not to address this issue of financial relationships at the beginning of your statement, it will not preclude you from speaking. So with that, we'll move forward, and I hand this over to Cicely Reese, who will be handling the open public here.

Thank you, Dr. [indiscernible]. This is Cicely Reese speaking. Before I begin calling the registered speakers, I would like to add the following guidance. FDA encourages participation from all public stakeholders in its decision-making processes. Every Advisory Committee meeting includes an open public hearing session, during which interested persons may present relevant information or views. Participants during the open public hearing session are not FDA employees or members of this Advisory Committee. The FDA recognizes that the speakers may present a range of viewpoints. The statements made during the open public hearing session reflect the viewpoints of the individual speakers or their organizations and are not meant to indicate agency agreement with the statements made. In fairness to all open public hearing speakers here today, since this is a 1-hour session, we ask that you please remain within your 4-minute time frame. To assist speakers and adhering to 4 minutes each, we are placing timer in the lower left of the screen for each presentation. We greatly appreciate your cooperation. [Operator Instructions] We will now begin with open public hearing speaker number one.

Good afternoon. I do not have any [indiscernible] onto get paid financially to be at this meeting. My name is [ Victoria Gray ]. I'm a 38-year mother and wife. I am the first sickle cell patient to be treated with CRISPR gene therapy. Before this treatment, my entire childhood and most of my adult life was played with severe pain, fatigue, numerous hospital stays in the [indiscernible]. The pain will come on so certainly, it felt like I was being hit by a truck [indiscernible] lighting at the same time. In order to manage my pain, I had to take 3 different opioids. Oxycodone, [indiscernible] and [ Fentanyl ]. Even with this combination, I was still in a lot of pain. I received regular blood transfusions and hopes to increase my blood counts and improve my symptoms of pain and fatigue, but it was only a temporary solution. One hospital stay in particularly has been primarily in [indiscernible]. It was in October [indiscernible] that I had one of the worst sickle cell crisis in my life. It ended my college pursuit of being a nurse. With this crisis, I had to wait for 3 days straight. I couldn't use my legs or my arms. I was in so much pain that I couldn't even move my hips to sit on to bear pain. I couldn't lift the fork to feed myself or use my hands to wash my face. I depended on physical therapy team to help me regain the control of my body. This was all result of severe pain episode from sickle cell disease. I didn't get released from this hospital stay as of January 2011. I missed Thanksgiving, Christmas and all 4 of my children's birthdays. I became so weak from being beat down by this disease. I had to have someone coming to my home to help me with my normal day-to-day routines. It wasn't as a [indiscernible] to say that his behavioral change -- excuse me, because he thought that I was going to die. I knew I had to fight for my kids. When I met Dr. [indiscernible] when the -- he presented the opportunity for me to join gene therapy trial. I said yes, without hesitation, knowing that I will be the first person, but this was my opportunity to fight. After receiving this treatment, I no longer have pain, so I no longer have to take opioids. I no longer have hospital stays or received blood transfusion. I just participate with my kids and join them in their activities. When they play sports to the [indiscernible] and just be here and just to play with them and knowing that I no longer have to leave them to go to the hospital. And I work full time and contribute to my household and my community. I believe if you say yes to this treatment, that is going to change to last positively of many people who are suffering from diseases in [indiscernible], who now feel hopeless. But once this call, they can feel hope again just like I did.

Thank you so much for sharing your personal story. We'll now open the public hearing speaker #2.

All right, yes. Can you hear me okay?

Yes.

Good afternoon, everyone. Michael Abrams here from Public Citizens Health and Research Group. We have no financial conflicts of interest on this matter. The exa-cel gene editing therapy to reduce the frequency of vaso-occlusive crisis in patients with sickle cell has demonstrated apparent efficacy in at least 29 of 30 subjects who have received this therapy thus far. This therapy is we've heard involves stem cell extraction from patients, CRISPR editing aimed at reigniting the expression of fetal hemoglobin and autologous reinfusion of the reengineered stem cells back into the patient. Chemotherapy, of course, is required and used to prepare patients for this auto transplant. The FDA scientific review of exa-cel has concluded that these results, although limited to small single-arm studies, are overall strongly positive. This review also notes that if the therapy is approved, a 15-year follow-up study, yet pending in design has been proposed to fully evaluate safety outcomes, including the possibility that [indiscernible] gene-editing may lead to plausible adverse effects such as malignant cancers, blood diseases, organ damage, transplantation related illness and even the possibility of early death. The focus of this meeting is accordingly not so much on the efficacy of exa-cel, but on its safety, specifically there is considerable uncertainty about off-target gene editing that is unintended editing of other genes besides those which turn on the expression of fetal hemoglobin. For the FDA's review, the sponsors thus far assessed the probability of off-target gene editing in 2 ways. First, by using algorithmic or [ silico ] reviews of existing genome databases, and second, by using more direct cellular assays looking at cells, and how they've been modified with the exa-cel therapy. Unfortunately, at present, both of those evaluations have insufficient scope. The algorithmic analysis relies on a limited amount of sequencing data that may not capture all of the variants that are vulnerable to off-target editing. For example, the review knows specifically that only 61 whole genome maps of individuals of African descent from the Southwest U.S. were actually used to consider whether tens of millions of genetic variants maybe at risk for off-target editing. Moreover, the review notes that 1 recent silico study published in Nature Genetics, which we heard about this morning, did not identify the same variant of concern that were identified by the sponsor study described today, a discrepant finding that may underscore sampling concerns. But finally, the cellular assay data was limited to just 9 subjects, 3 healthy, 3 with thalassemia, 3 with sickle cell disease. As stated by the FDA in their packet, "It is unclear whether this limited sample size will provide for an adequate understanding of the potential risk of off-target editing." Sickle cell disease, for example, is known to have to alter chromatin structure and stem cell function, such alterations could plausibly affect the risk of off-target editing. Accordingly, Public Citizens Health Research Group presently strongly believes that more study is needed to determine if off-target gene editing is a concern for patients receiving the therapy. We thus encourage this Advisory Committee and the FDA to request additional comprehensive studies to be completed before exa-cel is approved for a wider spread use. Thank you very much.

Thank you. We greatly appreciate your comments. We would like to have open public hearing speaker #3.

Hi, my name is [ Jimmie ] over here. I participated in exa-cel about 36 months ago, and I've got nothing to disclose. For most of my adult existence, my life has revolved around one thing, sickle cell disease. It dominated every facet of my life. Hospital admissions were so regular that they even had a bed reserved for me. It was a circus bouncing from specialists to specialists, and constantly desecrating my body with endless amounts of prescription pills, all in the hopes of finding a [indiscernible] of what it feels like to be truly alive. So when the opportunity came to participate in the gene editing clinical trial, I lead that a chance with no concern of any future consequences. Now instead of going to hospital rooms, I'm out here, living life to the fullest, no more days wasted under the fluorescent lights of the ER. No more pain and subsequently no more pain meds. No more endless forms, no insurance battles and no waiting room that's seem designed to test your patience. I can breathe easier, both literally and figuratively. Prior to the therapy, I focused on the short term, life was in the state of touch and go. Long-term planning meant planning for a world without me being able to support my family. Now those long-term plans include me. My family can do more and achieve more because we're all able to work towards the same goals. My quality of life has sought to new heights, allowing me to achieve things I once thought weren't possible. Gene therapy has given me the ability to take full control of my life. I can chase the proverbial sunset, write novels and even dance in the rain without a care in the world. Most importantly, gene therapy has given me the ability to be a present father and not encumber my children with the burden of caretaking. In a world where the deck was stacked against me, gene therapy has been a winning hand. While I recognize gene editing won't be the solution for everyone, I strongly recommend [indiscernible] warriors to consider this onetime therapy as it has the potential not only to change the individual's life, but also impact generations to come.

Thank you, open public hearing #3. We really appreciate you sharing your personal story. We'll have open public hearing speaker #4. Excuse me, we'll have open public hearing speaker, #5. We'll try to come back to #4.

My name is [indiscernible]. I've lived my entire life with sickle cell. I had constant crises. I had a crisis every 2 weeks, every -- twice a week, constant hospitalizations. And I was approached with the gene therapy where they take my cells, altered my fetal hemoglobin and gave me my own cells back. And after the process. I haven't had any crisis, any hospitalizations. Sickle cell, I had a little brother that was 2 years younger than me. And he passed away from sickle cell for -- because of organ failure. And I wish that this gene editing was around longer, and I want others to have it as well and had the opportunity to [ serve ] that way. Everyone else can experience it, just like I wish my brother could have. My life has changed drastically. I have more energy. Like I said, I don't have any crisis, I'm not in hospital. I haven't been in the hospital in 6 months. I met my 6-month period, and I haven't had any problems with sickle cell. So I ask that this is offered to others. Thank you.

Thank you for your moving comments. We appreciate it. We'll now move on to open public hearing speaker #6.

Can you hear me?

Yes, we can hear you.

Okay. My name is [ Sarde Kelly Howard ], and I have sickle cell disease. Before the age of 1, I was hospitalized over 13x. Last year, I was hospitalized 100 times. Over the years, I have experienced stigma surrounding my disease. I have been doubted, dismissed, [indiscernible] for having pain. Throughout my life, all I could think was I wish there was a cure. I wish I didn't have to go through all this pain. I wish I didn't have to be in the hospital. Well, today, and here's to tell you that I am 3 months post off of having a [ BMT ]. Although it's still early, this transplant has improved my quality of life tremendously. It has relieved me of so much pain. It's freed me from continuous hospital stays and has given me some quality of life back. This process liberating me from a disease that I have been fighting all 33 years of my life. I am so grateful because I didn't know that I would be without the transplant. However, BMT is not accessible to all SCD warriors because they do not have a stem cell donor. Gene therapy is an additional option that can cure SCD as well. It's more accessible. It doesn't require full body radiation and has a shorter recovery time. I'm asking the Advisory Committee to prioritize research and development of both methods. These are life-altering treatments that are desperately needed. Awareness and access are extremely important to improve and save the lives of people battling SCD. This is my plea. I pray you take note and actions. Thank you, from a surviving sickle cell warrior.

Thank you for your comments. We greatly appreciate your comments. We'll now have open public hearing speaker #7.

Hello, everyone. My name is [ Evan Sander ], and I have no financial conflict of interest in this matter. I'm here today on behalf of my incredible wife, [ Eleni Antola ], who is a sickle cell warrior and also on behalf of the roughly 100,000 sickle cell warriors living in the United States who are battling sickle cell disease every day. I'm here to voice my support for gene therapy as a curative therapy for sickle cell disease. Eli and I met in 2018, and I quickly fell in love with her infectious laugh, her wisdom and her immense enthusiasm for adventure and everyday life. I also learned quickly about the very real challenges and obstacles that Eleni faced to someone living with sickle cell. When we met, Eleni was recovering from a severe stroke and was receiving 8 units of blood every 6 weeks via an exchange blood transfusion. These transfusions served as a treatment and helped her sickle cell stay at bay. However, by 2019, to keep up with the progression of the disease, she was having to receive a blood exchange every 4 weeks. As you can imagine, this was a huge challenge for her and her family. Even though our life had many hurdles because of sickle cell, we were still able to have many moments of joy and celebration. And by 2020, we decided to get married. By 2021, we were exploring our options to become parents. It was then that we found out through a brain MRI that Eleni had small vessel disease in her brain. We were told that she was likely to have another stroke. This incredibly difficult news served as the catalyst for us beginning to research available curative therapy options to cure Eleni of sickle cell. In our search, we met with multiple doctors and hospitals in Wisconsin, Virginia and Ohio. We learned about the 2 curative therapy options, which were gene therapy and bone marrow transplant. Eleni's first choice was gene therapy as she felt it was less scary, less risky and has a better chance of success. However, due to her history with stroke, gene therapy was not an option for her. We decided to pursue the bone marrow transplant as a possible cure for Eleni. Luckily, we found out rather quickly that Eleni's father was a bone marrow match and would be able to be her donor. In September 2021, Eleni successfully received her bone marrow transplant and was cured of her sickle cell. Eleni, her family and friends, and I recently celebrated her 2-year anniversary of her successful transplant and it marked a truly life-changing milestone in her battle as a sickle cell warrior. I just want to share a quick photo if I can. This is -- I don't think I'm able to. Okay. Today, Eleni is living her best life. She is able to have a full-time job. She is able to swim and exercise regularly. No longer has to battle regular pain crises. She doesn't have to receive monthly blood transfusions or live with the worry of having another stroke. I am here today sharing Eleni's story to highlight the immense importance for all sickle cell warriors that have access to life-changing curative therapies like bone marrow transplant and gene therapy. Thank you all for your time.

Thank you so much for your moving story. We really appreciate that. We'll now have open public hearing speaker #8.

We have no disclosures. I'm [ Adrian Chapero ], and I'm here representing 5 generations of mothers in my family to have a child born with sickle cell disease. I think of these mothers often, I think of their pain, I think of their children dying, I think of their reality of their lifetime. In 1865, there was the emancipation proclamation. In 1890, [indiscernible]. In 1815, the beginning of World War I and 1940, World War II and 1965, the civil rights movement was going on. And in my daughter's generation, the 1990s was the beginning of the Genome project. As a young child, I told my mother that someday, I was going to speak to the FDA. I can't remember why, but she always said to me, "God is good, but science is going to fix this. She was the first generation of mother to understand the cause of the disease, and I was the first to benefit from [indiscernible] testing. No [indiscernible] science, I was good. We were all taught to look after my brother. As my mother learned, we all learned. We learned about cold, viruses, weather, sleep, hydration. Visits to the doctor with just the 3 of us. We learned about life, division of parenting duties and isolation that comes from having a way here in your family. Everybody told my mom that she should send my brother away. He had a stroke at 3 and it left him mentally and physically disabled. She kept him with us. When everybody said he was going to die, she kept him living. So my daughter was diagnosed with sickle cell, I learned 2 things: [ cheap ] science and [ bad ] science, and sickle cell disease was not just a disease for Black Americans. Everybody said anyone in the family could meet this challenge, it was me. I was trained by the best. So now we have 2 more generations living through this well-documented lens of sickle cell disease trauma. Nothing was ever going to be normal, education, employment, enjoyment, nothing. I was determined. She was going to remain alive, and she was going to be healthy. Well, she was alive, but not healthy. The generation she spent month in either the ER, the ED, somewhere until we got treatments that made her have a better life. I know that I may not be the last mother with a child with sickle cell disease in my family. But with these treatments, I will be the last mother to watch my child suffer and die without hope. Science is fixing this and science is only going to get better. Please support this. Thank you.

Thank you very much for your very moving personal story. We'll now have open public hearing speaker, #9.

Thank you for having me today. I don't have any ties or disclosures to speaking today. I'm going to start off by saying, I am a 42-year-old male who lives with sickle cell disease. I was diagnosed with sickle cell at the age of 2. I was the only one out of 4 kids to have sickle cell. So as you can imagine, life for me was different. I was in and out of the hospital, pain crisis because of my complications from sickle cell. I had pneumonia as a kid. I had my gallbladder removed as a kid. And I dealt with excruciating pain crisis that would have me in and out of hospital for days to weeks at a time because the pain was so excruciated. It felt like I was being hit with a hammer or someone who had a vise-grip around my arms and was just squeezing and I couldn't get rid of the pain of my arm. Even with the prescribed medications I had at home like Percocet. So I had to go to the hospital and rely on the hospital. And that took a heavy financial burden on my family, my mother, she had to take off of work to care for me. Because I couldn't be in a hospital alone as a child, so she will miss days to weeks at a time at her work. And when her work didn't understand that, she had a chat with sickle cell and didn't know what sickle cell was, she will be relieved of her duties at times. That also put the pressure on my dad because while I was in a hospital and my mother was in a hospital with me, he will have to take care and provide and run the household for the other 3 children. And his job also didn't understand. So at times, because of the financial struggle we had because of my health, we will go without things or have to borrow money so that we can have food on the table, simple necessities like tissue. And so over a lifetime, that financial burden doesn't go away. It continues. Within a year, over $10,000 is spent on medical costs, medical care. And as I got older, I will still be in a hospital with the same excruciating pain. Pain that, if I was -- if it was in my legs, I couldn't walk from here to the bathroom or pour a glass of water from a pitcher. So it's very important that we support gene therapy. Because there's a lot of people like me who want to be relieved of this pain and stigma of going to the hospital. [ Those ] devices on my disease. Lastly, with inadequate care because I was a man, I didn't get the right and proper care. So it's important that we address the issues and concerns for sickle cell because our entire life, we come into this world fighting. We fight with hospital systems. We fight for our health. We fight with insurance companies for coverage. We fought with pharmaceutical companies to come up with medications that affect, that would help us so that we wouldn't have to go through this pain because 100,000 people live with this pain and just the fact that we only had 4 medications. And now that we have the pharmaceutical companies on our side and they see the importance and they're taking action and they understand how much this affects the community. I think it's important that we support gene therapy. So those 100,000 people can live a normal healthy life and work. And can have jobs and be providers for their family. So I thank you for this time, and I ask that you support this gene therapy.

Thank you so much. We appreciate hearing from you. We'll now have open public hearing speaker #10.

My name is [ Trinity Ebbs ] and I do not have any financial ties on this matter. I was born with sickle cell hemoglobin SS disease. At the age of 16, I received the CRISPR stem cell transplant. And since then, my life has been so much better than I imagine it could be. All my life, I suffered from chronic and severe pain crisis, along with other complications that came with sickle cell disease. Many times, the pain would put me in the hospital to receive IV fluids with strong pain medications and blood transfusions. When I was not in the hospital, I had to take pain medicine just about every day of my life. When I was in elementary school, getting up in the morning was hard for me. I was tardy just about every day with some of the time having to use a wheelchair to assist me around the school because it was too painful for me to walk. Frequently, after having so many consecutive missed days of school, I would have to be put on homebound schooling. Physical activity or a change in the weather could also bring the onset of pain crises. I could always tell when the rain or first cool front of the season was on its way 3 or 4 days before and even times with a prediction, not even being made by the neurologist. By the time I got to middle school, my condition became worse. It was [indiscernible] episodes with me still having many hospital visits, making it extremely hard for me to attend school. Eventually, when I became old enough, I became dependent on Hydroxyurea, which minimized some of my hospital visits, but long after I had to have surgery to have my spleen removed, which is common for patients with sickle cell. Shortly thereafter, my mom made the decision to remove me from public school and enrolled me in a self-paced online and private school because I go too far behind in my classes and was not learning anything from missing so many days of school. It's been 2 years now since my transplant, and I have not been hospitalized due to any sickle cell pain as minimal pains or taking pain medication has been reduced. I have no longer pain when the weather changes, I can be physically active, walking a mile without having a pain crisis. I can swim staying in the water for long periods of time without needing a [ wetsuit ] to keep me from getting cold. I am now currently finishing up my last 2 years of high school, attending in person learning for the first time since middle school with the ability to focus and learn with almost perfect attendance. Some of my classes consist of dual credit courses, and I plan to attend college after I graduate. My overall health has improved, 95%, and I'm able to spend time with my family, friends without having to miss out on special events all the time. I'm so glad I enrolled in the CRISPR study and would like other patients to have this opportunity to receive this treatment. The best part of the transplant is that you are able to choose your own cells, especially when you have no one else as a match. Thank you for this opportunity to speak.

Thank you so much for sharing your story. We truly appreciate it. We'll now have open public hearing speaker #11.

Good afternoon. I'm Lewis Hsu, and my colleague, Dr. Donnell Ivy and I would like to represent Sickle Cell Disease Association of America. We volunteer as Chief Medical Officer and Vice Chief Medical Officer, respectively. I declare no financial ties in this matter. Next, please. Sickle Cell Disease Association of America has a mission to advance the search for universal cure, and that's what this gene therapy is about. Sickle cell disease is a rare disease, but if you count up the families impacted, it is probably 0.5 million or more affected by a disease with a lot of suffering and day-to-day insults as you've heard, as well as high cost and high utilization of the emergency department and of the hospital. Next, please. And you've already heard what the community feels about gene therapy, enthusiasm and seeing the potential benefits for individuals living with sickle cell disease and their caregivers and Sickle Cell Disease Association of America, likewise, says, yes, for gene therapy as a yes, but next, please. So there are issues to deal with as we seek that there could be approval of this gene therapy approach -- there we go -- yes, that this would be something where you do pay attention to coverage for fertility preservation that there can be addressing lack of insurance coverage in many states for fertility preservation. There can be also potential not just to pain and to cancer risk, but also to behavioral and mental health and that the services provided for people with sickle cell disease who don't get gene therapy, don't get tossed to the side as we pursue gene therapy plus for those who have the gene therapy that there can be ongoing care, ongoing attention during the planned 15 years of follow-up to look for additional problems whether there could be something beyond secondary cancers, organ damage or other kinds of effect. I'm going to turn the rest of the time to my colleague, Dr. Donnell Ivy. Next slide.

I do not have any financial relationships to disclose. Thank you for this opportunity to provide testimony on behalf of the Sickle Cell Disease Association of America and on behalf of individuals with sickle cell disease. My name is Edward Ivy and in addition to serving as the Vice Chief Medical Officer for the Sickle Cell Disease Association of America, I also am an individual living with sickle cell disease. As we have heard this morning from experts, the potential gene editing treatments for sickle cell disease would be of tremendous benefit to individuals suffering from this painful condition. As with many other therapies to treat disease, the potential risk-benefit analysis for gene therapy must continue to be evaluated and strategies to adequately inform patients and their families of this risk benefit must be provided to the population and language that is easy to understand and help individuals to make informed decisions. As pointed out by several speakers this morning, sickle cell disease is a very serious disease. So the absence of therapy can also be present at individual -- as a risk factor for individuals. And this should be accounted for in any risk-benefit analysis that is made. However, in addition to the risk benefit from the gene editing therapies, we must also consider the societal factors that can affect the therapies for this population. It is estimated at over 100,000 Americans suffer from sickle cell disease. The majority of individuals with sickle cell disease are lower income and depend on government-sponsored health insurance for their care. Given the expected high cost of this onetime treatment, the risk of many patients who cannot afford this therapy will be left out of this potentially life-altering benefit must be considered. Although the role of the FDA to ensure access to the therapies from the cost perspective is limited, it's important that the FDA remains in conversation as the risk-benefit analysis is continued to be evaluated. This is particularly important on how the FDA develops language around the analysis of the risk benefit so that the determination of who receives the therapy remains between the sickle cell expert provider and the individuals receiving therapy. I see that my time is out. So thank you so much, and I encourage you to consider the risk benefit analysis for this patient, particularly around cost as this therapy moves forward.

Thank you for sharing your comments. We'll have an open public hearing speaker #12.

Greetings. I am Dr. [indiscernible]. I have no personal financial disclosures to make at this time. I am sickle cell disease patient warrior, research scientist and disease expert as well as community leader as the Executive Director of the Sickle Cell Community Consortium. I have experienced it all from stroke, multiple acute chest syndrome, bilateral hip replacement, the most recent of which was 2 months ago and hip still has not healed. And I have tried it all. Trials, every kind of experimental treatment, even bone marrow transplant where I have failed to find a consistent donor. But yet by the grace of God, I have made it to age 45. And at 45, I have made it to this transition of the second transition in sickle cell from a young adult living with sickle cell to an older adult. For a very long time, I was the oldest person that I knew living with sickle cell disease. I did not realize how that affected me and so I began to meet those much older than me. This past July, at our Annual Warriors Convention, where we brought together hundreds of sickle cell warriors, we recognized for the first time something that we named Golden Warriors. And as those Golden Warriors shared with us their age, all over 55, some all the way into their 70s, as they share with us their life, I realize that they represent hope. A hope that I had needed to see and hadn't seen. Despite all of my activity and work in this space, I needed to see that hope. And that is what this gene therapy represents. These warriors represent hope, these golden warriors. And so does the option of genetic therapy, these curative therapies. This idea that I could be my own donor and that there some of these trials, I could potentially see a day where I do not wake up in excruciating pain. The sickle cell consortium started at an FDA meeting, the FDA patient-focused drug development meeting back in 2013 or '14, and it has now come full circle back to this meeting at the FDA, where once again, hope is on the horizon. And we are looking towards this hope for a change of the lives that we are living of excruciating pain. We are often faced with a population growing older and yet significant unmet needs. These unmet needs have left us feeling in many cases, hopeless, but at the convention where there are dozens and dozens of young people there, that hope was renewed. We had many, many discussions about gene therapy and the sickle cell community is excited and ready to walk into these curative therapies. We often find ourselves going and facing mistreatment and finding ourselves having to choose between what sounds ridiculous to say but death and dignity. Do I choose my life or my dignity, poor treatment, unmet needs. Many of us do not choose the way that you will instantly think that we should. We are now here to ask for support. For not only dignity but hope, hope that we can have a better life and better future. I'm grateful to have made 45, and I look forward to another 20 or 30 years to provide hope to the next warrior. Thank you.

Thank you so much for sharing your personal story. We greatly appreciate it. We'll now have open public hearing speaker #13.

Good afternoon. I do not have any financial disclosures. My name is [ Maria Jacklin Scott ]. I'm a 32-year-old sickle cell warrior from New Jersey. First and foremost, I would like to express my gratitude for allowing me to speak to FDA Advisory Committee today. This is a special day where the community voice our journeys and how we need to see the investment of sickle cell therapies in a health care system. As I speak to you today, I woke up in pain as I have chronic pain every minute and every day and yet I keep hope for what is about to be approved, gene therapy. This gene therapy exa-cel has future implications that a warrior like myself has been looking for, for ever since we became aware of what sickle cell disease can do. This will be the first gene therapy approved after it was first discovered over a century ago. In addition, not many are aware of the depression and anxiety sickle cell creates for our families and ourselves. Alleviating the fear and worry of pain and suffering [indiscernible] gene therapy can prevent some of these mental anxieties. I was diagnosed with sickle cell disease at 6 months old in 1991, but parents are unaware they carry the [indiscernible], living with this diseasewas challenging for myself and my family. I came into this world wondering if I will live after being resuscitated from emergency C-section. After my first crisis resulted in splenectomy, the doctors told my parents, I may not live past 5. Moving beyond those predictions was the first milestone in battling this disease. I always had a fervent drive for education and learning more about the disease at a molecular level or [indiscernible] can cause infections and vaso-occlusive crisis and how patient reported outcomes are value measures of what is important to the patient. I'm applying these skills for my community as a research coordinator with success. However, these accomplishments did not come easy. In 2016, I received my first shoulder replacement. In 2018, I needed my right hip replaced. This continues to 2020 when my left required a joint replacement. Yet what is ingrained in my mind, in December 2016, I went to an annual OB/GYN appointment [indiscernible] because the physician directed me to be on birth control because I should not have children, "Your risk of dying is too high. My mental health declined, I became depressed so much that I couldn't work as a professor and my physical health declined. Despite the many times, sickle cell made me feel alone and won't have any chance of having a fruitful life. I had my beautiful daughter with my fiance in 2021, and yet that came with a price. My veins are incredibly hard to access due to being at the hospital. I was stuck for IV access 4 times before the anesthetiologist decided to put a central line in my neck. In addition, a year after my daughter, I was constantly in the hospital requiring my fourth joint replacement just after 2 months giving birth. Imagine your mom unable to [indiscernible] just in contact because my shoulder collapsed after rocking her into sleep. By providing my postpartum hospital data between March 2022 and March 2023. According to this graph, I had 8 hospital stays where my average length of stay was 10 days. That was 10 days I had to facetime my baby. 10 days, I missed her attempting to take her first steps, and 10 days working on [indiscernible]. To this committee, I want to highlight what sickle cell can takeaway but you can give hope after this approval for exa-cel for that future mother, father, and anyone who feels sickle cell hinders their future [indiscernible] medical and mental implications this disease can cause. This gene therapy is another chance for this community to live free from pain, hurt and for dreams to come true. Thank you once again for this opportunity to speak.

Thank you so much for your comments. We greatly appreciate it. So thank you so much. So we were going to give speaker #4 the opportunity to speak, but the speaker 4 has not had the ability to log in. So with that, we are grateful to each of you for sharing your thoughtful remarks today with this committee, and with the agency. And for taking the time to be with us today. We invite you to watch the rest of the day's proceedings on the YouTube link being provided earlier and also on the Committee's web page. Thank you so much, and we will now proceed to the next portion of our meeting, and I'll hand it back over to Dr. [indiscernible].

Thank you, Cicely, and thank you so much for all of the folks that took the time out today for the open public hearing. That their viewpoint is very much appreciated and really an important component of the day in terms of how we look at the discussion point. So thank you very much for everyone's comments and the sharing of their experiences. So at this point, we move on and close off the open public hearing. We have a break now, and we will start at 1:45 P.M., so we have a little bit of extra time. And then we will see everyone there for the FDA presentation and then subsequent discussion. [Break]

Welcome back. And now we're going to move forward with the FDA presentation. There'll be 2 speakers, and I will present -- I will introduce first Dr. Karl Kasamon, who is a reviewer in the Office of Clinical Evaluation, division of hematology, benign hematology branch, OTP, CBER, FDA. [Operator Instructions] Dr. Kasamon, we cannot hear you. I think it's working now.

How about now?

Yes.

I'm so sorry. Okay, thank you. So welcome back to this part of the cellular tissue and gene therapies advisory committee meeting regarding biologics license application #1 to 5787 on exagamglogene autotemcel, which is CRISPR/Cas9 notified autologous CD34 positive hematopoietic stem and progenitor cell, cellular therapy, which seeks the indication for the treatment of sickle cell disease in patients 12 years and older with recurrent vaso-occlusive crisis. Next slide, please. My name is Karl Kasamon, as was mentioned, and I'm a hematologist and a reviewer of the Office of therapeutic products within the FDA Center for Biologics Evaluation and Research, next slide. The goal of my presentation is to briefly review the clinical aspect of this BLA and set the stage for [ Dr. Singh ], a bioinformatics expert to lead you through a crucial discussion of potential off-target editing by exa-cel and strategies to manage this issue. I'd like to start by introducing sickle cell disease and its current therapy. Then talk about exa-cel, including the mechanism of action and how it is manufactured, after which we will review the studies providing evidence to support efficacy and safety. Next slide. Sickle cell disease is a group of hemoglobinopathies that includes sickle cell anemia, sickle beta plus and sickle beta zero thalassemia and sickle SC disease. Sickle SC disease will not be further included in this presentation as it was not studied in the clinical trials that will be discussed. Sickle cell disease largely affects persons of African, Southeast Asian and Mediterranean ancestry, including about 80,000 patients in the U.S. As shown in this graphic, sickle hemoglobin differs from adult hemoglobin by a point mutation that [indiscernible] for glutamine at the beta-globin gene. When deoxygenated sickle hemoglobin polymerizes creating rigid fibrils that deform red blood cells, making them sticky and leading to occlusion of blood vessels and hemolysis. Next slide. As shown in this slide, sickle cell disease causes a number of debilitating manifestations, which include recurring severely painful episodes called vaso-occlusive crises in addition to anemia, retinopathy, stroke, pulmonary hypertension and chronic ischemic damage to various organs such as brain, kidney, liver and bone. And to date, sickle cell disease continues to shorten the survival substantially, especially for adults. Next slide. Sickle cell management consists of supportive care, including analgesics and red blood cell transfusions during vaso-occlusive crises and in children, penicillin prophylaxis and transcranial doppler monitoring, approved drugs include hydroxyurea, L-Glutamine, voxelotor and crizanlizumab. While these have modestly improved outcomes of many patients with sickle cell disease, none of these is curative, and they require lifelong adherence. Furthermore, not all patients can tolerate these. The only available curative therapy is allogeneic hematopoietic stem cell transplant. However, fewer than 20% of patients with sickle cell disease have an appropriately matched donor. Consequently, treatment for severe sickle cell disease remains an unmet medical need. Next slide. At this time, we'll go over the mechanism of action of exa-cel and look at how it is manufactured. Next slide. To help explain exa-cel's mechanism of action, it's useful to review the role of BCL11A in the control of hemoglobin expression around the time of birth. Hemoglobin is an oxygen-carrying protein within red cells. And as shown in this figure, it's a [indiscernible] that is made up of 2 copies of 2 distinct peptides. Fetal hemoglobin consists of 2 alpha and 2 gamma-globin chains and adult hemoglobin consists of 2 alpha and 2 beta-globin chains. BCL11A, which is shown here in Orange is a zinc finger protein that's responsible for the transition from gamma globin to beta globin. The line graph at the bottom shows that starting late in fetal development, gamma globin expression becomes repressed by BCL11A, leading to a coordinated transition from fetal hemoglobin to adult hemoglobin. Next slide. Exa-cel is a cell-based gene therapy product that is composed of autologous CD34 positive hematopoietic stem cells, edited by means of the SPY101 guide RNA and a CRISPR endonuclease at erythroid lineage-specific enhancer region of the BCL11A gene. This diagram shows the mechanism of action of exa-cel. The exa-cel works by disrupting [ data 1 ] binding and downregulating BCL11A expression. Therefore, it uninhibits gamma globin expression and upregulates fetal hemoglobin production within red cells. It's important to consider why increasing fetal hemoglobin and decreasing sickle hemoglobin would be expected to be clinically desirable. It has been observed that fetal hemoglobin is therapeutic and individuals who have coinheritance of sickle hemoglobin and hereditary persistence of fetal hemoglobin. Therefore, upregulation of fetal hemoglobin by the action of exa-cel is predicted to lessen symptoms of sickle cell disease. Next slide. The manufacture of exa-cel, as shown in this diagram starts with collection of autologous hematopoietic stem cells using apheresis. Then CD34 positive cells are isolated, purified and genome edited with a CRISPR endonuclease and then SPY101 guide RNA. Following editing, the cells are incubated in a culture medium, washed and prior preserved. After completion of lot release testing and manufacture of the appropriate dose, exa-cel lots are shipped to qualified clinical centers for infusion. Next slide. The next section will focus on the clinical data. Next slide. The clinical data come from a single study 121 and the long-term rollover safety follow-up study, 131. Study 121 was launched in 2018 as a Phase I study with a client population of 17 subjects and evolved over time to become a Phase I and II and III study that is still ongoing as a multinational single-arm trial with a planned population of approximately 45 of whom 12 are adolescents under age 18. Following participation in Study 121, all subjects who have been dosed with exa-cel are eligible and encouraged to participate in Study 131 where they will continue to follow up for 15 years more following exa-cel infusion. Next slide. The primary efficacy endpoint was defined as a proportion of subjects achieving VF12, which is freedom from severe VOCs for a period of at least 12 months at any point on Study 121 after exa-cel infusion. The valuation of VF12 started only 60 days or more after any last red blood cell transfusion was given for post-transplant support or sickle cell disease management. Notable secondary efficacy endpoints included proportion of subjects achieving freedom from hospitalization for severe VOCs for a period of at least 12 months after exa-cel, which was called HF12 as well as several other endpoints that assess the durability of effect, the expression of fetal hemoglobin above 20% or reduction in the rates of VOCs, hospitalizations and transfusion requirements compared with baseline. Finally, safety endpoints assessed neutrophil and platelet engraftment reported on adverse events, abnormal laboratory values and mortality. Next slide. Study 121 enrolled adolescents and adults 12 to 35 years of age with a genotype that is shown here, who had severe sickle cell disease. Genotype severity was demonstrated by having had at least 2 documented clinical sequelae that are quite specific for sickle cell disease such as acute chest syndrome, splenic sequestration or prolonged priapism, that would require a visit to a health care facility or having had at least 2 severe vaso-occlusive painful crises in each of the 2 years preceding screening. To be considered a severe VOC required that a subject had a valuation for a pain event at a health care facility and received either red blood cell transfusion, intravenous NSAIDs or opioids. Key exclusion criteria included having a matched donor for allogeneic stem cell transplant, having had a history of a prior stem cell transplant at baseline fetal hemoglobin above 15%, or several clinical features that could make autologous transplant process unsafe. Next slide. I'd like to use this diagram to explain the schema of Study 121. Starting on the left and going across, we'll note that in Stage 1, screening, eligible subjects were advised of the option of fertility preservation and again, red blood cell transfusions for a minimum of 8 weeks before mobilization with a goal to lower their sickle hemoglobin to less than 30%, while keeping total hemoglobin no greater than 11 grams per deciliter. In Stage 2, mobilization, each subject is injected with plerixafor in order to mobilize the stem cells and allow them to be collected from the peripheral blood with apheresis, which would then permit the manufacture of exa-cel. In Stage 3, which was myeloablative conditioning and exa-cel infusion, first, busulfan was administered intravenously either daily or every 6 hours for 4 consecutive days. And after a washout of busulfan, exa-cel was given, IV. Finally, in Stage 4, subjects remain in the hospital until neutrophil engraftment was observed and then were followed on the study for up to 2 years after [ exa-cel ]. Next slide. 44 subjects have received exa-cel as of the time of data lock. And of these subjects, those 30 with at least 16 months of follow-up after exa-cel are considered evaluable for efficacy. This population is defined as a primary efficacy set, or PES, which will be the focus of the remainder of the efficacy discussion. At baseline, evaluable subjects had a median annualized rate of severe VOCs of 3.3 and spent a median of 12 days in the hospital for severe VOCs. The subjects required transfusion of a median of 3.3 annualized units of red cells for sickle cell disease. Next slide. I'd like to reiterate that the primary efficacy endpoint was VF12, which again was the absence of severe VOCs for a period of at least 12 months on Study 121 following exa-cel. This was achieved by 29 out of the 30 subjects who are followed for at least 16 months and thus were eligible for efficacy analysis. All 30, 100% of the evaluable subjects reached a key secondary efficacy endpoint age of 12, and thus avoided hospitalization for severe VOCs for a period of at least 12 months while on study after exa-cel. All 30 evaluable subjects had a sustained fetal hemoglobin level of 20% or more for a period of at least 12 consecutive lines, starting 60 days after any last red blood cell transfusion. While all 6 treated adolescent subjects followed for at least 16 months did achieve VF12, a seventh adolescent subject with 14.3 months of follow-up, experienced recurrent VOCs between month 11 and month 14 and therefore, cannot meet the definition of VF12 responder regardless of additional follow-up. Next slide. I'd like to walk you through this rather busy slide to illustrate some important efficacy end points. On the left, you'll notice the great time line of baseline severe VOCs. And on the right is a follow-up after exa-cel, among the 44 subjects who received exa-cel. Adolescents are in purple, and adults are shown in green. The dark blue diamonds are the severe VOCs. The 30 subjects who are shown above the orange line are those with at least 16 months of follow-up. The safety profile of exa-cel recipients in Study 121 was largely consistent with the toxicities typically seen with autologous transplant. Next slide. In conclusion, exa-cel administration to patients with severe sickle cell disease led to achievement of primary and secondary efficacy endpoint by a large majority of patients. The long-term outcome following CRISPR-based gene therapy in humans remains unknown. And questions still remain regarding off-target unintended genome editing. This important topic will be further discussed at this time by Dr. Komudi Singh of Bioinformatics. And therefore, I'd like to turn it over to her. Thank you.

Thank you, Dr. Kasamon. Good afternoon, everybody. My name is Komudi Singh. I'm a Bioinformatics Reviewer at the Office of Therapeutics Product at CBER. In this presentation, I will provide an overview of the applicant's off-target safety assessment of exa-cel using bioinformatics method. Next slide, please. I will first introduce the CRISPR/Cas9 technology, which will be a recap of the presentation provided by Professor Urnov earlier this morning, the risk associated with off-target editing, methods of off-target analysis. I will then present the applicant's off-target safety analysis of exa-cel, summarized potential issues leading to the discussion topic today. Next slide, please. The CRISPR/Cas9 systems are naturally occurring microbial defense system that has been engineered to introduce DNA breaks in animal and human cells. A double-strand DNA break caused by Cas9 endonuclease occurs upon base bearing between the guide RNA and the target sequence in the genome, in the presence of a short protospacer adjacent motif sequence, or PAM sequence for short, that is present on the noncomplementary strand of the genomic DNA. A precise editing by Cas9 endonuclease at an intended genomic location can be achieved by designing the guide RNA to align with the region of the genome targeted for editing in the presence of PAM sequence. The PAM sequence motif serves as a binding signal for Cas9 and is strictly required for a Cas9-mediated double-strand break. Shown on the right side of the slide is a CRISPR/Cas9 ribonucleoprotein complex, that shows a perfect base-pairing between the guide RNA and the target genomic sequence that would result in an on-target double strand break or an on-target edit. However, a growing body of evidence has shown that Cas9-mediated edits can also occur when there is an imperfect base pairing between the guide RNA and the genomic DNA in other locations, giving rise to unintended off-target editing as shown in the figure on the lower right portion of the slide. Next slide, please. If an unintended genome editing occurs at the region of the genome known to have regulatory elements, then a double-strand break in such locations can disrupt regulatory function. Similarly, off-target editing at genes coding region can lead to gene inactivation. If the gene happens to play an essential role in cell function, then such unintended edits can be deleterious. These edits can also increase the risk of cancer. Therefore, an adequate off-target analysis is needed to allow for safety assessment of genome editing products intended for therapeutic purposes. To provide context to the information I will be presenting today, my talk is going to revolve around the off-target safety assessment of exa-cel that applicant conducted, and the adequacy of these approaches. I will spend some time to introduce the off-target editing methods that have been developed for safety assessment of CRISPR/Cas9 genome editing products before presenting the applicant's off-target safety assessment of exa-cel. Next slide, please. The CRISPR/Cas9-based genome editing technology is rapidly expanding and so are the bioinformatic tools that are being developed to assess off-targets. These bioinformatic tools use sequencing information alone or with next-generation sequencing data to perform off-target analysis. These methods are broadly divided into 3 categories. Next slide, please. First, the in silico off-target analysis methods use computational algorithms that require user provider guide RNA sequence information and user provided mismatch criterion, while scanning the human genome reference sequences to nominate potential off-target low sign. The cartoon on the middle left section of the slide shows an example case of a perfect base-pairing between a guide RNA and the target genomic sequence, where in silico algorithm will nominate additional loci across the reference genome, by identifying regions with imperfect base-pairing occurring due to a mismatch, as shown in the cartoon depicted in the center of the slide, or when an imperfect base-pairing occurs because of a gap between a guide RNA and genomic sequence shown on the right section of the slide. These methods are straightforward to implement. However, the findings of these type of analysis are biased by user-provided mismatch criteria. Additionally, these methods do not account for cell-type specificity, arising from unique chromatin landscape within a cell. Next slide, please. The cellular methods of off-target analysis used the genomic sequence information of CRISPR/Cas9 genome edited cells. The cells, in this case, are edited in the presence of an oligonucleotide gap that marks the loci where double-strand breaks have occurred. The genomic material from these cells are isolated and subjected to high throughput sequencing and analysis. These methods can provide high-confidence off-target candidates. However, determining right experimental parameters need careful consideration. Additionally, this method can be hard to implement due to toxicity associated with oligonucleotide tasks in certain cell types. A third method for off-target analysis includes biochemical methods that use genomic material from the cells that are edited and assessed for off-target. Since the applicant did not use this method, we will not be discussing this in the presentation today. For the remainder of my talk, I will present the applicant's off-target safety assessment of exa-cel leading to the discussion question for today's Advisory Committee Meeting. Next slide, please. The applicant used 2 [ orthogonal ] methods to perform off-target safety assessment of exa-cel. In one of the approaches, they used in silico methods to nominate off-targets based on homology to the human genome reference sequence. We would like to know that the Cas9 endonuclease recognizes a native or cognate PAM sequence, NGG shown in the bold font on this slide, where N can be any nucleotide base. Additionally, Cas9 has also been shown to recognize different variations of PAM sequence, but exhibit lower activity at these alternate PAM sequences, and I will refer to them as suboptimal PAM sequences. The applicant used 3 in silico analysis tools for this analysis, and they included both cognate or native PAM sequences as well as suboptimal PAM sequence patterns in their search. In the second method, the applicant performed cellular GUIDE-seq off-target analysis on healthy donor and sickle cell disease donor cells. These cells were edited with Cas9 Spy 101 GUIDE RNA, and the genomic material was extracted for high-throughput sequencing and analyzed. I will now present the findings of the off-target safety assessment of exa-cel and discuss potential issues surrounding this analysis. Next slide, please. As mentioned in the previous slide, the applicant used 3 different in silico off-target analysis tools. They used 2 mismatch limits of 3 and 5 with scanning the human genome reference sequence and nominated off targets for Spy 101 GUIDE RNA. The mismatch criteria were inclusive of many mismatches and permissive of 1 gap. The applicant used a more lenient mismatch criteria of 5 when including cognate or native PAM sequence pattern in their search, and suboptimal PAM sequence patterns were tested with lower mismatch criteria. Increasing the mismatched criterium would result in inclusion of more loci as potential off targets. Consistent with this, the applicant identified 171 loci, when the homology-based search was implemented with 3 mismatches. And they identified 5,007 loci, when the search was implemented with 5 mismatches. The data presented by the applicant shows that the number of mismatches implemented can impact the number of off-target loci nominated. We would like to note that several of these in silico nominated sites are sequences in the genome that can base there with the guide RNA withstanding the applicant provided mismatch criterium and harbors any of the PAM sequence patterns that applicant use in their search. For such in silico nominated loci, confirmatory testing should be performed, ideally using more than one sample to allow for testing editing potentials at these sites in the presence of all potential PAM patterns used in the in silico nomination process. We will discuss this issue when presenting the applicant's confirmatory testing in the later part of the talk. Next slide, please. One of the issues with the in silico off-target analysis methods is that these tools, while scanning the reference genome sequence does not account for individual genetic variations that may result in off-target editing at a new locus harboring the variation. Shown below is a cartoon representation of several genomes harboring nucleotide variations across individuals contributing to heterogeneity. These individual nucleotide variations could be of concern, if it contributes to decreasing the mismatch between guide RNA and genomic DNA as shown in the cartoon below, or if it contributes to generation of a PAM site. Next slide, please. To account for heterogeneity, the applicant used 1000 Genomes Project database and included variants present at greater than 1% frequency in this database, which includes greater than 1% frequency in every subcontinental group represented in this database. Specifically, they applied a 1% frequency cut off, and I will present the analysis result in the next slide. Next slide, please. The database that the applicant used had 83 million single nucleotide variations. Of these, 21 million variants were present at frequency greater than 1%. The applicant implemented a variant-aware homology search that expanded the homology space to include sites that we either have decreased mismatch, all would include a PAM sequence in the presence of a variant. From this analysis, they identified 50 additional off-target loci that accounted for heterogeneity. Next slide, please. Since all the loci were reported -- that were reported were nominated using computational algorithms, the applicant performed confirmatory testing using hybrid capture sequencing. Briefly, this technique allows for enrichment of DNA fragments using biotinylated RNA fragments that act as baits or probes. In this case, the probes were designed to enrich DNA fragments from the loci that were nominated by the in silico off-target analysis. To ensure optimal capture of target DNA, the baits were tiled around the off-target loci. The genomic material from control and CRISPR-edited cells were incubated. The captured DNA was sequenced, aligned and after removal of duplicated sequences, reads carrying indels within 3 base pair of a potential cleavage sites were counted. Next slide, please. The applicant used genomic materials from 4 replicates of CRISPR or control-edited healthy cells. The target DNA sequences were captured for hybrid-capture sequencing. Sequences with suboptimal coverage, high GC content, high background indels and homopolymers were excluded from this analysis. As a result, 4,340 loci out of 5,007 were tested. The applicant performed confirmatory testing for these 4,340 loci in 4 samples, for which they did not provide any sample metadata information. They however provided sample metadata information for 4 independent samples that were used in confirmatory testing of 171 loci. They reported that one sample was from an individual of African-American ethnicity and the remaining samples were from 3 individuals of Hispanic ethnicity. Next slide, please. We would like to note that the applicant's off-target nomination strategy included scanning the genome with predefined mismatch criteria that were inclusive of different PAM sequence patterns, that we have presented in Slide 28. In this case, confirmatory testing should be performed at all these loci in the vicinity of all PAM patterns included in the nomination process. It is unclear if the 4 samples used in hybrid-capture sequencing, allowed for testing of all PAM sequence patterns used in the nomination classes. Based on this, we conclude that many of the off-target loci nominated were not experimentally tested. The applicant reported that no off-target editing was detected at any of the loci nominated in the in silico analysis as shown in the table on this slide. Next slide, please. For the additional 50 off-target loci, nominated by the variant-aware homology search, the applicant performed confirmatory hybrid-capture sequencing using genomic material from 1 sickle cell disease donor sample and 2 transfusion-dependent thalassemia donor samples. The applicant reported that no off-target editing was detected at any of the 50 loci nominated from the variant-aware search. We want to point out that these 50 loci were nominated as potential off-targets because of the presence of variance at this site. Therefore, the presence of variants in the sample is necessary for confirmatory testing. The applicant reported the presence of 13 variants in at least one of the samples that were used for confirmatory testing. Hence, the absence of editing shown by hybrid capture does not completely roll out off-target editing at the remaining 37 loci nominated from this analysis. Additionally, we would like to know that out of the 50 loci, 20 nominated to the 18 genic locations. These genic locations were mostly intronic region with 1 locus close to an intron/exon border. Since intronic regions are known to have regulatory functions, adequate risk assessment of potential disruption of these sequences will be needed. Next slide, please. Several factors need consideration by performing in silico analysis accounting for heterogeneity. Implementation of off-target analysis accounting for heterogeneity requires using variant information from sequencing database. A database used in this type of analysis would be adequate if it contains adequate amount of samples from which sequencing data is generated, the samples should be from individuals representative of the drug product target population, a good quality of sequencing data to ensure optimal variant identification and a suitable allele frequency cutoff to subset variance for this analysis. All these factors would ensure adequate variant sampling that can be used to account for heterogeneity. As mentioned before, the applicant used the 1000 Genomes Project database that had sequencing data from 2,504 individuals across different continents. Of this, 661 sequencing data were from individuals, representing the target population of exa-cel. Among the 661, there's only data from 61 individuals in the United States. The limited number of sequencing data may not adequately represent a drug product target population across the United States. As mentioned in the previous slide, the applicant reported 50 additional off-target loci from this analysis. Next slide, please. We would like to refer back to the talk by Dr. Bauer earlier today, where he presented some data on the heterogeneity assessment of guide RNA that targets the same locus on BC11A gene as exa-cel. The Cancellieri study and the applicant's exa-cel analysis reported different number of variants contributing to potential off-target loci. Before I go deeper into the Cancellieri study, I want to remind that the Cas9 endonuclease recognizes the native PAM sequence NGG, shown in the bold font on this slide, where N can be any nucleotide base. It has also been shown to recognize different variations in PAM sequences, some of which are listed on this slide. One of the variants reported in the Cancellieri study was a variant in the CPS1 intronic region, the change the TGA PAM sequence present on the reference sequence highlighted in the orange color box to [indiscernible] TGG PAM sequence highlighted in the blue color box on this slide. Cancellieri study reported a higher off-target editing score at TGG PAM locus compared to TGC PAM locus present in the reference genome. The CPS1 locus was nominated by applicants in their in silico homology-based off-target assessment, in which the applicant had included alternate PAM sequences in their search. However, applicant-performed confirmative testing in samples that harbored TGC PAM sequence only. Hence, editing potential at this locus with TGG PAM was not empirically tested by the applicant. The potential off-target editing at this locus cannot be ruled out until sufficient information is provided. This lack of empirical testing applies to other loci that were nominated by the applicant in their trial in silico off-target analysis studies. Next slide, please. We would like to note that while the applicant reported the CPS1 locus in their homology-based analysis, they did not report the variant in the heterogeneity analysis, even though this variant is present at a greater than 1% frequency in the 1000 Genomes Project database. An off-target locus that is potentially impacted by a variant is a critical finding that needs to be reported and fully assessed for editing potential using appropriate samples. With the available data, we cannot perform adequate risk assessment at this locus in the presence of this variant. The applicant, however, reported other variants in their heterogeneity analysis from other loci, and we show some of them in Table 1. The variant locus and the associated gene information is present in Table 6 of the briefing document. These loci were likely reported in the applicants in silico analysis as it fulfilled the mismatch criteria they applied in their prior in silico study. We have provided the mismatched criteria that the applicant used in Table 2. Hence, it is not clear why the CPS1 variant was not reported in the applicants heterogeneity study. Because of the applicant implemented criteria and curations to the database, it is unclear how many other variants were not reported in the applicants heterogeneity analysis, and how many potential variants may have overlapped with the Cancellieri study. Since the Cancellieri study included variants from different database when compared to the database used by the applicant, some variants may be excluded due to different variants reported in specific database. Additionally, different variant allele frequency cutoff used in these 2 studies may also result in exclusion of variants from the applicant study. For instance, in Table 3, we present a variant that was reported in the Cancellieri study to contribute to an off-target locus. However, this variant would not meet the applicant's 1% allele frequency criteria they applied in the heterogeneity assessment. Next slide, please. To summarize the 2 studies accounting for heterogeneity, the differences in the findings published in Cancellieri study and those reported by the applicant may stem from different factors we have listed in this table. First, the applicant implemented a variant-aware homology search, while the authors of Cancellieri study developed and implemented a tool to account for heterogeneity. The applicant used 1000 Genomes Project database that included sequencing information from 2,504 individuals across continents. The authors of Cancellieri study used 2 different databases, the Human Genome Diversity Project dataset comprising of sequencing data from 929 individuals and the Genome Aggregation Database that has sequencing data from a much bigger sample. The applicant reported 50 potential off-target loci that were contributed by 1 or 2 variants, and the Cancellieri study mainly reported a detailed assessment of a variant that resulted in creation of PAM site and a potential off-target locus. Next slide, please. To summarize the in silico off-target safety assessment of exa-cel, we are concerned about the different number and subset of nucleotide variants -- variations that were identified in the 2 studies that contributed to off-target loci. These differences may arise potentially because of limited number of sequencing information present in the databases potential differences in performance of in silico algorithms used in these studies. It is not clear if the small sample size of database would allow for sufficient sampling of variants. Additionally, we would like to point out that the confirmatory testing of off-target loci requires that the cells or genomic material used in this test harbors the variant contributing to an off-target loci. Since appropriate cell samples harboring variants were not used in the confirmatory testing, majority of off-target loci arising from variants were not empirically tested. On the same lines, a subset of in silico nominated off-target loci were also not empirically tested. The lack of clarity on these indicated aspects of off-target analysis accounting for heterogeneity and lack of confirmatory testing using appropriate samples may support a need for additional studies to further assess the safety of exa-cel. Next slide, please. I will now present the applicant's cellular method of off-target safety assessment of exa-cel. Specifically, they implemented GUIDE-seq to identify off-targets in Spy 101 GUIDE RNA-edited CD34 positive HSPCs. In these experiments, the cells were edited with the Cas9 ribonucleoprotein complex, in the presence of a double-strand oligonucleotide tag, or dsODN for short. The oligonucleotide tag will mark all the daily breaks occurring during genome editing. The genomic DNA from these samples were sequenced by high-throughput sequencing and assessed using the GUIDE-seq pipeline. The applicant performed this experiment using 3 healthy donor and 3 sickle cell disease donor cells. Next slide, please. GUIDE-seq analysis of 3 healthy donor cells has identified several off-target loci in each sample, as shown in the table. Also shown in this table are GUIDE-seq data from analysis of samples derived from 3 transfusion-dependent thalassemia donors. We would like to point out that 2 different dsODN concentrations were used, which could interfere with identification of a consistent subset of off-target loci. However, the sponsor stated that they were able to detect adequate number of on-target reads shown in the fifth column of this table, and hence consider these parameters to be optimal. The applicant then used hybrid-capture sequencing on 4 independent healthy donors and reported that no off-target editing was detected at these locis. Next slide, please. In the next experiment, the applicant performed GUIDE-seq on 3 sickle cell disease donor-derived cells. They reported optimal cell viability as shown in column 3 of the table, high on-target editing frequency shown in column 4 and sufficient number of on-target reads in each sample as shown in column 5. From this analysis, they reported several off-target loci in each sample tested as shown in column 6 of this table. Next slide, please. They manually assessed a subset of the off-target loci identified in the GUIDE-seq experiment. For confirmatory testing, they used hybrid-capture sequencing on the same 3 samples, but reported lower on-target editing rates in these samples prepared for hybrid capture, as shown in the column 4 of the table on the left side of the slide. The applicant stated that high sequencing depth would allow for detection of editing at off-target loci. From this analysis, they reported that no editing was observed at the off-target loci identified in the GUIDE-seq experiment. They identified 3 locis within indels that matched to a DNA break hotspot. Consistent with this observation, they provided manual assessment of a subset of these loci that also reported a DNA break hotspot at the same location. And these DNA breaks were independent of CRISPR/Cas9 editing. Next, they postulated that the off-target loci identified in these samples are likely false positive. To address this, they used false positive filtering and reported that all of the off-target loci identified in the experimental samples were removed. Next slide, please. To summarize the cellular off-target safety assessment of exa-cel, the applicant performed 2 GUIDE-seq experiments, one using 3 healthy donor-derived CD34-positive HSPCs and another using 3 sickle cell donor-derived cells. From these experiments, they identified several potential off-target loci. But they reported no off-target editing was observed in their confirmatory testing. None of the off-target loci identified in the GUIDE-seq overlapped with the 171 in silico-nominated loci. We would like to note that sickle cell disease has been shown to impact HSPC function and lineage and induce stress response. These changes are likely to impact the cells chromatin landscape that is known to impact off-target editing. It is not clear if off-target analysis using healthy donor cells would adequately inform off-target editing risk in exa-cel. We are also concerned about the adequacy of using small number of samples in cellular off-target analysis. Next slide, please. In summary, the applicant performed off-target safety assessment accounting for heterogeneity using the 1000 Genomes Project database. However, the small number of sequencing data present in the database and lack of confirmatory testing of all off-target loci in samples harboring the variants is concerning. We are also concerned about the adequacy of small sample size in cellular off-target analysis of exa-cel. And if the use of small number of healthy donor and sickle cell donor cells would adequately inform us of potential off-target editing risk of exa-cel. We would like the advisory committee members to weigh in on these issues and provide recommendations. We would like to thank the Advisory Committee members for their time and for participating in this Advisory Committee Meeting today. This concludes the FDA's presentation on the clinical assessment of exa-cel and the applicant's off-target safety assessment of exa-cel in this BLA. Thank you.

Thank you very much, Dr. Kasamon and Dr. Singh for thorough presentations that will help inform this conversation. So we now have time for questions from the committee members directed towards the FDA speakers. We'll then follow that up with committee discussion, where there will be discussion between the members of the committee. If we feel at that time, which will start at around 3:00 p.m., that we have pointed questions for the sponsor for clarification, we can do that then. But at this point, this Q&A is for the FDA speakers. So if members want to raise their hands for those that have questions for the FDA speakers.

Dr. Scot, please go on camera and take yourself off mute.

Dr. Singh, thank you very much for the detailed overview of the applicant's off-target analysis. One comment you made about their computational assessments of off-targets and a subsequent analysis was, you had a concern about their analysis of off-target sites with suboptimal PAMs. I was wondering if you could expand on that just a little bit, to clarify what your concerns are there with regards to the reference genome.

This is Komudi Singh, Bioinformatics Reviewer from FDA. The applicant had -- in their description of their in silico nomination process, they had used 2 different mismatched criterion and different variations of PAM sequences in their off-target in silico nomination process. If that is the case, what the analysis would do, would scan the genome to identify additional loci, which should have mismatches to the guide RNA, which is within the limits of the mismatch criterion applied. In that case, the applicant had performed searches across the genome with suboptimal PAM sequences with a 3 mismatch criteria -- with up to 3 mismatch criteria. And a more linear mismatch of 5 when they were using a cognate or native PAM sequence. Our concerns are not with the criteria they have used in their in silico nomination process, but our concerns are with the confirmatory testing that they subsequently followed this nomination process with. And those confirmatory testings were done on 4 samples, and it is not clear if they had used a certain number of PAM sequences in their searches, did the samples have these off-target loci with the indicated PAM sequences in their confirmative testing.

Dr. Singh, so it sounds like you're unsure of the exact analysis that the sponsors did on the confirmatory aspect of the in silico, correct?

Yes. So we are not sure about what variations of sequences were empirically tested in their confirmatory testing. Correct.

Okay. So that is a question that we can ask them directly, not right now, but at the beginning of the discussion that we'll hold. And Dr. Wolfe, I apologize for calling you, Dr. Scot, just quickly reading off the names here. Dr. London, if you could go on camera and take yourself off mute.

Yes. Yes. Thank you very much, Dr. Singh. I am wondering -- I appreciate your concern for the small sample size. And I was just wondering if the FDA has guidance on methodology that could be used to determine how many more samples should be analyzed. I mean, does the FDA have guidance about how many more patient samples would need to be studied in order to identify enough variance.

We are not prescriptive about the methodologies that the sponsors select in their analysis. And right now, we do not have any guidance to indicate the number of samples or recommend the number of samples to be tested. In fact, one of the issues that we would like the advisory members to weigh in on is, if given the issue and given the lack of clarity on how many samples should be needed, we would like to hear Advisory Committee members discuss this issue and provide us with recommendations.

Dr. Wu, could you go on camera and take yourself off mute.

Yes. So I think I asked this question at the very, very beginning on the applicant. So do you get a sense why it's so difficult to just take the patients that they have done the hematopoietic cell transplant and just analyze the samples that they have, because they've had these samples for several years. Why is it so difficult? Why it just show the data 3 sickle cell patients instead of the 30-or-so patients that they've already done?

I would defer to the applicant to address that question.

Okay. I mean, I asked again, but I asked the same question, but I think they just didn't answer it. Yes.

Okay. So if the sponsor who's listening can be prepared at the start of our discussion. I think there's 2 questions. And Dr. Singh, correct me -- Dr. Singh and Dr. Wu, correct me if I did not capture it correctly. The second question was from Dr. Wu, which is, is there a reason why we cannot do cellular-based analysis of samples from the patients that have already been treated, right? And the first question that Dr. Singh was asking, which is the methodology to looking at the confirmatory studies of in silico and whether the PAM variations were accounted for there, okay? So we will get to that when we start our opening discussion. Any other questions from the Committee members for the FDA speakers? Dr. Shapiro, if you could go off -- go on camera and come off mute.

Well, perhaps this is what Dr. Wu was asking, but I was asking if any of the patients treated were positive for the rs114518452 variant. I think that's essentially what he's asking as well.

I don't know, Dr. Wu, would you like to come on camera and confirm whether that's the same question?

Yes. So that's one question, but that's based on in silico modeling, right? So I think for any of these samples, you still -- besides in silico, you still want it to be hardcore experiment, taking the cells, analyzing them and see what happens, if there's any surprises outside of what the in silico model predictions are. I mean, this is a perfect opportunity to look into that, right? Instead of just focusing on 3 samples, SCD patients that you see in silico modeling. Yes.

Right. So that's kind of subset of your question, more broader question, Dr. Wu.

To Mr. Shapiro's question, I think that the applicant needs to address that as well. We don't have that data at FDA.

Okay. Great. Good to know. So both of those are questions for the applicant that we can have when we open up the discussion, the short period of questions and them. Dr. Lee, I saw that you had raised your hand and then lowered it. Please raise it again if you want to ask your question. In the interim, Dr. Ott, could you go ahead and ask your question.

Yes. I had the same question about the guidelines, what FDA is expecting from applicants in terms of target effects that are there. And then I also would like to just confirm that the in silico prediction was not overlapping at all with the experimental off-target data that were achieved. I just wanted to confirm this with Dr. Singh.

The applicant's report have performed a comparison of off-target loci nominated from the in silico analysis where they had up to 3 mismatches and reported 171 loci, and they had reported all the locis identified from GUIDE-seq data and they reported none of those locis overlap. That is correct.

Dr. Singh, maybe you could tell me -- could you speak to why there may be results that are not overlapping like that? Could you bring that to the forefront for the committee and the public?