1

DEPARTMENT OF HEALTH AND HUMAN SERVICES

FOOD AND DRUG ADMINISTRATION

CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE

MEETING #37

Thursday, March 18, 2004

8:30 a.m.

Hilton Hotel

Silver Spring, Maryland

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PARTICIPANTS

Mahendra S. Rao, M.D., Ph.D., Chair

Gail Dapolito, Executive Secretary

MEMBERS

Jonathan S. Allan, D.V.M.

Bruce R. Blazar, M.D.

David M. Harlan, M.D.

Katherine A. High, M.D.

Joanne Kurtzberg, M.D.

Alison F. Lawton

James J. Mul, Ph.D.

Thomas H. Murray, Ph.D.

Anastasios A. Tsiatis, Ph.D

CONSULTANTS

Jeffrey S. Borer, M.D.

Susanna Cunningham, Ph.D.

Jeremy N. Ruskin, M.D.

Michael E. Schneider, M.D.

Michael Simons, M.D.

INDUSTRY REPRESENTATIVE

John F. Neylan, M.D.

GUEST HEALTH CANADA REPRESENTATIVE

Norman Viner, M.D.

GUEST SPEAKERS

Stephen Epstein, M.D.

Silviu Itescu, M.D.

Robert J. Lederman, M.D.

Philippe Menasch, M.D.

Emerson C. Perin, M.D., F.A.C.C.

Doris A. Taylor, Ph.D.

NIH PARTICIPANTS

Richard O. Cannon, M.D.

Stephen M. Rose, Ph.D.

FDA PARTICIPANTS

Jesse L. Goodman, M.D., M.P.H.

Philip Noguchi, M.D.

Dwaine Rieves, M.D

Stephen Grant, M.D.

Richard McFarland, Ph.D., M.D.

Donald Nick Jensen, D.V.M., M.S.E.E.

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C O N T E N T S

PAGE

Call to Order

Mahendra Rao, M.D., Ph.D., Chair 5

Conflict of Interest Statement

Gail Dapolito, Executive Secretary 5

Introduction of Committee 9

FDA Opening Remarks

Presentation of Certificate of Appreciation

to Retiring Member

Jesse Goodman, M.D., M.P.H. 14

Philip Noguchi, M.D. 16

Open Committee Discussion

Cellular Therapies for Cardiac Disease

FDA Introduction and Perspectives

Dwaine Rieves, M.D. 18

Guest Presentations

Overview Cardiomyopathy and Ischemic Heart

Disease

Emerson Perin, M.D., Ph.D. 35

Q&A 65

Clinical Experience of Autologous Myoblast

Transplantation

Philippe Menasch, M.D. 85

Q&A 115

Bone Marrow Cell Therapy for Angiogenesis:

Present and Future

Steven Epstein, M.D. 128

Q&A 148

Cellular Therapies for Cardiac Disease

Richard McFarland, Ph.D., M.D. 159

Guest Presentations

Myoblasts: The First Generation Cells for

Cardiac Repair: What Have We Learned?

Doris Taylor, Ph.D. 169

Q&A 202

Preclinical Models - Hematopoietic and

Mesenchymal Cell Therapies for Cardiac Diseases

Silviu Itescu, M.D. 219

Q&A 245

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C O N T E N T S (Continued)

From Mouse to Man: Is it a Logical Step for

Cardiac Repair?

Doris Taylor, Ph.D. 257

Q&A 275

Cardiac Catheters for Delivery of Cell Suspension

Donald Nick Jensen, D.V.M., M.S.E.E. 292

Transcatheter Myocardial Cell Delivery: Questions

and Considerations from the Trenches

Robert Lederman, M.D. 307

Q&A 333

Open Public Hearing 343

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1 P R O C E E D I N G S

2 Call to Order

3 DR. RAO: Good morning. Welcome to the

4 37th meeting of the Biological Response Modifiers

5 Advisory Committee.

6 Today\'s topic, as you all know, is related

7 to use of cells in cardiovascular disorders, and we

8 have a pretty full schedule for the next couple of

9 days, but before we can start the meeting, we have

10 to have a few sort of committee stuff that needs to

11 be gotten through, so I will turn the mike over to

12 Gail, so that she can make the mandatory

13 announcements.

14 Conflict of Interest Statement

15 MS. DAPOLITO: Good morning.

16 The following announcement addresses

17 conflict of interest issues associated with this

18 meeting of the Biological Response Modifiers

19 Advisory Committee on March 18 and 19, 2004.

20 Pursuant to the authority granted under

21 the Committee Charter, the Associate Commissioner

22 for External Relations, FDA, appointed Drs. Jeffrey

23 Borer and Susanna Cunningham as temporary voting

24 members.

25 In addition, the Director of FDA\'s Center

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1 for Biologics Evaluation and Research, appointed

2 Drs. Jeremy Ruskin, Michael Schneider, and Michael

3 Simons as temporary voting members.

4 Based on the agenda, it was determined

5 that there are no specific products considered for

6 approval at this meeting. The committee

7 participants were screened for their financial

8 interests. To determine if any conflicts of

9 interest existed, the agency reviewed the agenda

10 and all relevant financial interests reported by

11 the meeting participants.

12 The Food and Drug Administration prepared

13 general matters waivers for participants who

14 required a waiver under 18 U.S.C. 208. Because

15 general topics impact on many entities, it is not

16 prudent to recite all potential conflicts of

17 interest as they apply to each member.

18 FDA acknowledges that there may be

19 potential conflicts of interest, but because of the

20 general nature of the discussions before the

21 committee, these potential conflicts are mitigated.

22 We note for the record that Dr. John

23 Neylan is participating in this meeting as a

24 non-voting industry representative acting on behalf

25 of regulated industry. Dr. Neylan\'s appointment is

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1 not subject to 18 U.S.C. 208. He is employed by

2 Wyeth Research and thus has a financial interest in

3 his employer.

4 With regards to FDA\'s invited guest

5 speakers and guests, the agency determined that

6 their services are essential. The following

7 disclosures will assist the public in objectively

8 evaluating presentations and/or comments made by

9 the participants.

10 Dr. Stephen Epstein is the Executive

11 Director, Cardiovascular Research Institute,

12 Washington Hospital Center. He receives research

13 support, is a consultant to and has financial

14 interests with, firms that could be affected by the

15 committee discussions.

16 Dr. Philippe Menasch is employed at the

17 George Pompidou Hospital in Paris, France. He has

18 an association with a firm that could be affected

19 by the committee discussions.

20 Dr. Emerson Perin is employed by the Texas

21 Heart Institute. He receives consultant fees from,

22 and is a scientific advisor to, firms that could be

23 affected by the committee discussions.

24 Dr. Doris Taylor is employed by the

25 University of Minnesota, Center for Cardiovascular

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1 Repair. She receives consultant fees from a firm

2 that could be affected by the committee

3 discussions.

4 Dr. Norman Viner is employed by the

5 Biologics and Radiopharmaceuticals Evaluation

6 Centre, Biologics and Genetic Therapies

7 Directorate, Health Canada, in Ottawa, Canada.

8 FDA participants are aware of the need to

9 exclude themselves from the discussions involving

10 specific products or firms for which they have not

11 been screened for conflicts of interest. Their

12 exclusion will be noted for the public record.

13 With respect to all other meeting

14 participants, we ask in the interest of fairness

15 that you state your name, affiliation, and address

16 any current or financial involvement with any firm

17 whose product you wish to comment upon.

18 Waivers are available by written request

19 under the Freedom of Information Act.

20 Thank you, Dr. Rao.

21 DR. RAO: Now you know why I always have

22 Gail read that statement.

23 Before we start any committee work, I

24 would like to welcome two new members to the

25 committee, Dr. Murray and Dr. James Mul. We

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1 generally introduce everyone on the committee

2 first, and we generally go in alphabetical order,

3 but this time I will try and start with the new

4 members, so that they can tell us a little bit

5 about themselves before we have the others

6 introduce themselves.

7 Introduction of Committee

8 DR. MULE: I am Dr. Jim Mul. I am

9 currently the Associate Center Director for the H.

10 Lee Moffitt Cancer Center in Tampa. I oversee all

11 translational research at the Center including all

12 cell-based therapies for the treatment of cancer as

13 it applies to the clinical treatment of patients

14 with advance tumors.

15 Prior to being in Tampa since September of

16 last year, I was at the University of Michigan

17 Cancer Center for 10 years, and prior to that, the

18 NCI for another 10 years, and I am delighted to be

19 here.

20 DR. MURRAY: Good morning. I am Tom

21 Murray. I am President of the Hastings Center,

22 which is celebrating its 35th years as the world\'s

23 first research institute devoted to ethics in

24 medicine and the life sciences.

25 I spent 15 years as professor at medical

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1 schools including 12 at Case Western Reserve

2 University School of Medicine. My interests are

3 fairly broad. I write a lot about ethics and

4 ethics in the life science and science policy.

5 Thank you. I am delighted also to be

6 here.

7 DR. RAO: If we can go down the table, Dr.

8 Tsiatis.

9 DR. TSIATIS: Hi. I am Butch Tsiatis. I

10 am from the Department of Statistics at North

11 Carolina State University.

12 DR. BORER: My name is Jeff Borer. I am a

13 cardiologist. I work at Weill Medical College of

14 Cornell University in New York City. I run a

15 division and an institute at Cornell and, relevant

16 to this meeting, I am the Chairman of the

17 Cardiorenal Drugs Advisory Committee of the FDA.

18 DR. CUNNINGHAM: Good morning. My name is

19 Susanna Cunningham. I am a professor in the School

20 of Nursing at the University of Washington in

21 Seattle, and I am the consumer representative for

22 the Cardiovascular Renal Advisory Committee.

23 DR. SCHNEIDER: I am Michael Schneider. I

24 co-direct the Center for Cardiovascular Development

25 at Baylor College of Medicine, and our interests

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1 are in the molecular genetics of cardiac muscle

2 formation, cardiac growth, cardiac cell apoptosis

3 and its relation to heart failure, and, relevant to

4 this meeting, cardiac progenitor cells of different

5 kinds.

6 DR. SIMONS: Hi. I am Michael Simons. I

7 am Chief of Cardiology at Dartmouth Medical School.

8 I work in the area of vascular biology, gene and

9 cell therapy.

10 DR. RUSKIN: Good morning. I am Jeremy

11 Ruskin. I am a cardiologist and

12 electrophysiologist, and I direct the Cardiac

13 Arrhythmia Service at Massachusetts General

14 Hospital.

15 DR. NEYLAN: Good morning. I am John

16 Neylan. I am a nephrologist and an organ

17 transplanter by training. Currently, I am Vice

18 President of Clinical Research and Development at

19 Wyeth, and I serve as a industry representative to

20 the committee.

21 DR. KURTZBERG: Hi. I am Joanne

22 Kurtzberg. I am a pediatric oncologist. I direct

23 the Pediatric Bone Marrow and Stem Cell Transplant

24 Program at Duke University and the Carolinas Cord

25 Blood Bank at Duke.

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1 DR. ALLAN: Hi. I am Jon Allan. I am a

2 virologist at the Southwest Foundation for

3 Biomedical Research. My area is nonhuman primate

4 models for AIDS pathogenesis.

5 DR. CANNON: Good morning. I am Richard

6 Cannon. I am at the National Heart, Lung, and

7 Blood Institute. I am Clinical Director of NHLBI,

8 and I am representing NHLBI at this meeting.

9 DR. ROSE: Good morning. I am Stephen

10 Rose. I am Deputy Director for the Recombinant DNA

11 Program in the Office of Biotechnology Activities

12 in the NIH.

13 DR. JENSEN: Good morning. My name is

14 Nick Jensen. I am a reviewer in the Center for

15 Devices and Radiological Health. I am a

16 veterinarian and an engineer.

17 DR. McFARLAND: Good morning. I am

18 Richard McFarland. I am a reviewer in the

19 Pharm/Tox Branch in the Center for Biologics in the

20 Office of Cellular, Tissue and Gene Therapies.

21 DR. RIEVES: Good morning. My name is

22 Dwaine Rieves. I am a medical officer in FDA\'s

23 Center for Biologics Evaluation and Research.

24 DR. GOODMAN: Good morning. I am Jesse

25 Goodman. I am the Center Director of the Center

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1 for Biologics. I would just like to join in

2 welcoming especially the new members. My

3 background is as an infectious disease physician in

4 academic medicine for many years.

5 DR. NOGUCHI: I am Phil Noguchi, Acting

6 Director of the Office of Cellular, Tissue and Gene

7 Therapies in CBER.

8 DR. RAO: Thank you, everyone.

9 We are very fortunate in having some

10 really leaders in the field come and present some

11 of the data which will be the basis of where we can

12 address some of the questions that have been raised

13 by the FDA.

14 I am going to ask them to just briefly

15 introduce themselves, as well.

16 DR. EPSTEIN: I am Steve Epstein, a

17 cardiologist. I am head of the Cardiovascular

18 Research Institute at the Washington Hospital

19 Center. We are involved in vascular biology, gene,

20 and cell therapy.

21 DR. MENASCHE: I am Philippe Menasch. I

22 am cardiac surgeon at the Hospital European George

23 Pompidou in Paris, France.

24 DR. PERIN: Good morning. I am Emerson

25 Perin. I am an interventional cardiologist and

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1 Director of Interventional Cardiology at Texas

2 Heart Institute in Houston.

3 DR. TAYLOR: Hi. I am Doris Taylor. I am

4 a scientist. I just moved from Duke University to

5 the University of Minnesota to head the Center for

6 Cardiovascular Repair.

7 DR. ITESCU: Hi. I am Silviu Itescu. I

8 am Director of Transplantation Immunology at

9 Columbia Presbyterian, New York.

10 DR. RAO: I would also like to welcome Dr.

11 Viner who is from Health Canada. Health Canada has

12 been following a lot of what the FDA has been doing

13 and it is nice to have them there.

14 I would like to invite Dr. Goodman to make

15 a statement.

16 FDA Opening Remarks

17 Presentation of Certificate of Appreciation

18 to Retiring Member

19 DR. GOODMAN: My main purpose is to thank

20 Joanne Kurtzberg for I guess about four years of

21 service to the BRMAC. We really appreciate that

22 tremendously. She has also interacted with CBER

23 before that.

24 One of the reasons I really wanted to come

25 by this morning. Joanne is rotating off this

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1 committee. I know from interactions both within

2 this committee and outside, and from all the

3 leadership and staff within CBER, just what a

4 tremendous advisor and asset Joanne has been for

5 FDA and for your various fields here.

6 Of course, she has mostly contributed very

7 extensively in her areas of hematopoietic stem

8 cells, et cetera, but she has also been a very

9 important thinker and discussant and contributor on

10 the whole range of other cellular therapies and

11 even gene therapy.

12 Please join me in thanking Joanne for her

13 service over these years. Also, we like to say,

14 particularly CBER, that we are a family and that

15 nobody ever leaves it, and that we, just like a

16 family, we will keep asking for favors in the

17 future and probably causing grief in return.

18 Thanks so much, Joanne. We have a plaque

19 for her, of course.

20 \[Applause.\]

21 DR. GOODMAN: I guess I will just turn it

22 over to Phil to just give a brief introduction for

23 the meeting, but just to say that, as I mentioned a

24 little while back about the islet cell therapies,

25 we, at FDA, are extremely excited about cellular

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1 therapies and their potential, and I think nowhere

2 is some of that potential clearer, but also perhaps

3 more difficult to evaluate and help move forward

4 than in the area of cardiovascular disease whether

5 it is for ischemic disease or heart muscle disease

6 or trauma, et cetera, some of the uses where there

7 have been some very promising reports.

8 So, we think this is a very timely

9 meeting. It is very important to get input about

10 how to go forward with efficient development of

11 those products, how to address some of the clinical

12 and safety issues, and how to hopefully make this

13 field positioned to realize its successes in the

14 most efficient manner and also help FDA get that

15 right to the extent that we all can based on

16 incomplete information.

17 Again, we really look forward to this. I

18 apologize, my usual schedule means I will be in and

19 out, but I really appreciate it.

20 Phil.

21 DR. NOGUCHI: Thank you, Jesse, and, of

22 course, Dr. Kurtzberg, our sincere thanks for the

23 many years of service. Jesse is absolutely right,

24 don\'t be surprised if the next meeting, you get a

25 funny call early in the morning.

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1 This is one of our, in a way, continuing

2 series of dealing with things that seem really

3 wonderful and amazing when they come up, where

4 there is a lot of hope and there is perhaps a

5 little bit of hype, but what we have always found

6 over the years, and here I would like to just

7 acknowledge Dr. Rose in the Office of Biotechnology

8 Activities and the Recombinant DNA Committee, what

9 we have learned from them is that one of the best

10 ways that we have of really dealing with things

11 controversial and where there is both hope and

12 there is some trepidation about whether or not this

13 is actually going to work or not, is to bring

14 everyone together, put them in the same room.

15 Our continuing\--and this really goes back

16 at least 25 years through the RAC and many years

17 for the BRMAC\--is that when you get reasonable

18 people together who may have differing opinions

19 about things, but are presented the facts and the

20 realities, as well as the unknowns, we all

21 basically pretty much come out with the same

22 conclusion, and then we can make significant

23 progress in making these therapies not just

24 experimental, but a reality.

25 With that, what I would really like to

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1 do, because we have such a full schedule, is now

2 turn it over to Dr. Rieves for the introduction.

3 DR. RAO: As Dr. Rieves comes up to the

4 mike, I just want to remind people of a few simple

5 rules. Remember that when you want to ask a

6 question, make sure that you are recognized. Use

7 the button. You will see that the light comes on.

8 When you are done, just hit the button again to

9 switch it off, because otherwise, there is sort of

10 a feedback loop and noise. Make sure you identify

11 yourself when you ask questions.

12 Cellular Therapies for Cardiac Disease

13 FDA Introduction and Perspectives

14 DR. RIEVES: Good morning. My name is

15 Dwaine Rieves. I am a medical officer within FDA\'s

16 Center for Biologics Evaluation and Research. This

17 morning I am going to present a brief overview of

18 FDA\'s perspective on cellular products used in the

19 treatment of cardiac diseases.

20 As will be covered in a subsequent

21 presentation, certain cellular products, when

22 either perfused into the heart or directly injected

23 into heart muscle, are thought to be capable of

24 regenerating heart tissue and/or augmenting heart

25 function.

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1 Consequently, these products may have

2 special utility in the treatment of heart failure

3 and certain other cardiac diseases. Today and

4 tomorrow, we will discuss issues in the early

5 clinical development of these products.

6 \[Slide.\]

7 This talk is divided into three major

8 sections. First, I will cite the purpose in

9 convening this advisory committee. Secondly, I

10 will provide a regulatory background on FDA\'s

11 understanding and activities within the realm of

12 clinical development of these products. Finally, I

13 will introduce the major questions we have proposed

14 for discussion.

15 \[Slide.\]

16 Unlike many advisory committees where the

17 topics center around assessment of data associated

18 with a specific product or data related to a

19 specific regulatory concern, our purpose in

20 convening this committee is not to obtain

21 definitive regulatory advice, instead, FDA has

22 convened this committee to listen to, and learn

23 from, the voiced thoughts and perspectives with the

24 understanding that this information will enhance

25 our ability to promote the safe clinical

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1 development of these products.

2 As you are aware, the clinical development

3 of cellular products is in its infancy and many

4 questions surround the very early stages of product

5 development. Consequently, our purpose today and

6 tomorrow is to stimulate a solid scientific

7 discussion of the major facets associated with the

8 very early clinical development of these products.

9 As noted here, we will focus upon three

10 major areas: manufacturing aspects of the cellular

11 product, preclinical testing of the products, and

12 finally, items related to the early clinical

13 studies.

14 \[Slide.\]

15 What are the cellular products we will be

16 discussing? These products may be broadly grouped

17 into two categories.

18 Firstly, those manufactured without

19 ex-vivo culture methodology, that is, the cells are

20 harvested from humans, processed, and then

21 delivered to a recipient without maintaining the

22 cells in culture for a period of time.

23 In general, these cells consist of bone

24 marrow mononuclear cells and certain peripheral

25 blood mononuclear cells, hematopoietic progenitor

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1 cells that are variously referred to as stem cells,

2 cells thought to be capable of assuming phenotypic

3 characteristics of non-hematopoietic cells.

4 The second category consists of cells

5 that, following harvesting, are maintained in ex

6 vivo culture for a period of time before final

7 processing and administration.

8 In general, these cells consist of those

9 derived from skeletal muscle tissue, cells

10 frequently referred to as myoblasts, and certain

11 bone marrow stromal cells, cells also referred to

12 as mesenchymal cells. Whether these cultured cells

13 should be regarded as forms of stem cells is more

14 questionable than that for the hematopoietic

15 progenitor cells.

16 Lastly, as the slide notes, most of the

17 cellular products we will be discussing today and

18 tomorrow are of autologous origin.

19 \[Slide.\]

20 The many questions surrounding the

21 scientific basis for cellular product development

22 illustrate the very nascent nature of the field.

23 As we are probably all aware, there is almost no

24 precedent for the clinical development of products

25 intended to regenerate and/or augment disease

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1 tissue.

2 The scientific data surrounding this field

3 are relatively new, such that the data are limited

4 in depth and the extent of replication. Hence we

5 come to the table of clinical development with many

6 hypothetical considerations and some, but

7 relatively limited background supportive data.

8 \[Slide.\]

9 Given these limitations, our discussions

10 today and tomorrow assume a scientific focus in

11 which certain insights and perspectives are

12 presented, and you, the committee members, will be

13 asked to share your thoughts. Three points are

14 cited here.

15 First, we acknowledge that these thoughts

16 are all tentative and susceptible to revision based

17 on accumulating data.

18 Secondly, we are not requesting any

19 definitive assessment of data, and we note that the

20 data presented here today are within the public

21 arena, and have not undergone FDA vetting.

22 Finally, I reiterate an earlier comment,

23 that no specific cellular product discussed here is

24 under review with respect to regulatory

25 decisionmaking.

23

1 \[Slide.\]

2 This slide illustrates the

3 interconnectedness of clinical research and

4 regulatory paradigms. The connecting link between

5 the two fields is the science. Clinical research

6 generates the scientific background for clinical

7 development of cellular products and the scientific

8 background forms the major basis for our regulatory

9 paradigms.

10 \[Slide.\]

11 FDA is charged with many responsibilities,

12 but as cited here, two are especially relevant to

13 this discussion. Specifically, FDA\'s mission is to

14 promote and protect the public health by optimizing

15 pre-market product development and ensuring

16 sufficient post-marketing product monitoring.

17 The key word in these two statements is

18 \"product.\" A notation that whereas we frequently

19 hear the terms transplant, graft, and procedure, we

20 need to think in terms of a cellular product, a

21 product that is manufactured, labeled, and

22 potentially marketed.

23 \[Slide.\]

24 A little over 10 years ago, FDA clarified

25 the regulatory basis for oversight of clinical

24

1 development programs for cellular products. In

2 general, this regulatory framework is the same as

3 that for the drugs and biologic products we

4 commonly recognize as marketed products.

5 Hence, the commonly cited biologic

6 product, drug, and device regulations applied to

7 the clinical development of these cellular

8 products, and the clinical studies must be

9 conducted under the purview of submission of a

10 investigational new drug application.

11 The last bullet on this slide reminds us

12 that clinical development programs may be divided

13 into early and late stages, with the late stages

14 focused upon the ascertainment of data definitive

15 to safety and efficacy, and the early stage, what

16 we are talking about today and tomorrow, focused

17 upon the ascertainment of exploratory safety and

18 bioactivity data.

19 That is, we hope to examine the nature and

20 extent of background data necessary to introduce

21 the cellular products into small, sample size,

22 Phase I clinical studies.

23 \[Slide.\]

24 As previously noted, the keystone

25 consideration in early clinical development is

25

1 safety. Specifically, we need to ensure that the

2 tripod of product development is solid. That tripod

3 consists of manufacturing control and testing

4 information, sufficient preclinical testing

5 information, especially information that may inform

6 the design of a clinical study, and finally, the

7 clinical study itself.

8 The next few slides will cite each of

9 these three components.

10 \[Slide.\]

11 Cellular products must be manufactured in

12 some manner, that is, the cells must be harvested

13 and processed prior to administration to a

14 recipient. Manufacturing aspects may be divided

15 among four major areas, three being shown on this

16 slide.

17 The top bullet notes that documents should

18 describe the cell source and reagents used in the

19 manufacturing process, such as growth factors,

20 sera, salt solutions and additives. We need to be

21 confident that all the reagents used in the

22 manufacturing are of clinical or pharmaceutical

23 grade, or that if they are not pharmaceutical

24 grade, they are sufficient for human use.

25 One may envision many potential concerns

26

1 with these materials, such as the use of sera that

2 may contain infections agents, or the use of only

3 partially purified reagents that contain harmful

4 excipients.

5 Secondly, documents should describe the

6 procedures used in manufacturing, specifically

7 describing how cells are aseptically harvested,

8 isolated, and potentially selected.

9 For example, a distinct population of

10 cells may be selected based upon the presence of

11 certain cell surface markers, such as the CD34

12 antigen with the selection process involving

13 incubation with an antibody to CD34.

14 As we know, many investigational

15 antibodies have been developed to target cell

16 surface antigens, and we need to be confident that

17 these selection techniques are performed in a

18 reproducible and safe manner.

19 Additionally, documents should describe

20 the storage and tracking of the cellular products,

21 this being of special concern because certain

22 cellular products may be patient-specific products.

23 For example, measures must be in place to

24 ensure that for autologous products, the cellular

25 product is returned to the correct donor. Of

27

1 course, the cellular product needs to be labeled as

2 one for investigational use only.

3 The bullet at the bottom of this slide

4 emphasizes the importance of testing the cellular

5 product, an especially important concern since

6 cellular products cannot be sterilized in the same

7 manner as one might sterilize a drug product or a

8 device. Notable aspects of testing include tests

9 for sterility, endotoxin, viability, enumeration,

10 or cell counting.

11 \[Slide.\]

12 The fourth component of manufacturing

13 information is product characterization as

14 highlighted here. When one speaks of product

15 characterization, we are generally talking about

16 cellular phenotype and/or functional

17 characterization and the characteristics of the

18 product\'s final formulation.

19 For example, a product containing solely

20 CD34 positive cells in saline with no preservatives

21 or media. Product characterization is especially

22 important from a clinical perspective, because

23 failure to consistently manufacture a product makes

24 the clinical data virtually uninterpretable.

25 As noted here, the major aspects of

28

1 product characterization consist of a description

2 of identity, purity, and potency of the final

3 cellular product.

4 \[Slide.\]

5 Pre-clinical testing is the second major

6 component of product development, and the major

7 aspects of this testing are cited here. The top

8 bullet notes that consistent with the science, the

9 extent and depth of preclinical testing necessary

10 to support a clinical study is an evolving paradigm

11 and is a major topic for discussion at this

12 meeting. However, we generally take the stance

13 that this preclinical testing paradigm should be

14 consistent with that used for other biological

15 products.

16 The last bullet notes another important

17 aspect of preclinical testing, the testing of the

18 product administration procedure.

19 This is especially important because many

20 cellular products involve injection directly into

21 heart muscle either through the epicardial surface

22 or the endocardial surface. These techniques

23 represent inherent safety concerns that may be best

24 evaluated in animals prior to their use in humans.

25 As noted, all available catheters, whether

29

1 marketed or not, are regarded as investigational

2 with respect to administration of cellular

3 products.

4 \[Slide.\]

5 This slide highlights three aspects of

6 preclinical testing that will be the focus of the

7 preclinical questions tomorrow.

8 Firstly, the choice of the relevant

9 species is central to designing preclinical studies

10 with the major choices being between large animals,

11 such as pigs, versus small animals, such as mice,

12 as well as the choice between immunocompetent

13 animals where, for autologous products, the

14 cellular products would be the animal cells, not

15 human cells, or immunocompromised animals, where

16 the actual human cellular product may be tested.

17 Secondly, designing preclinical studies

18 raise questions of the choice of model, that is, a

19 disease model, such as ischemic heart disease

20 induced in the pig versus a healthy animal.

21 Lastly, preclinical concerns relate to

22 testing of the administration procedure itself,

23 such items as the impact of the catheter materials

24 upon cells, the potential for occlusion of

25 catheters by the cellular product, and the safety

30

1 concerns associated with manipulation of the

2 catheters in the heart.

3 \[Slide.\]

4 The third component of the clinical

5 development program for cellular products is the

6 clinical study. There are many aspects of clinical

7 study design that could be discussed, but at this

8 meeting, we are focusing upon two, the first shown

9 here, that is, adverse event detection.

10 This slide highlights two aspects of

11 clinical study design that are frequently

12 engineered to optimize adverse event detection, the

13 evaluation plan with attention to the duration of

14 clinical follow-up, the frequency of evaluations,

15 and the extent or nature of these evaluations.

16 Secondly, the clinical study safety

17 monitoring plan may be optimized through the use of

18 close scrutiny of each study subject based upon the

19 sequential, not simultaneous, enrollment and

20 treatment of the subjects, as well as the

21 prespecifications of the types and numbers of

22 adverse events that should prompt interruption of

23 the study, that is, the study stopping rules.

24 Tomorrow, the committee will be asked to

25 discuss potential adverse events in these early

31

1 clinical studies, both the nature of the events and

2 ways to optimize the safety of the studies.

3 \[Slide.\]

4 This slide illustrates an additional

5 clinical study design item that we will bring to

6 the committee, that is, a discussion of the

7 analysis of adverse events.

8 Exploratory clinical studies are, by their

9 nature, small sample size studies in which it is

10 often difficult or impossible to distinguish

11 treatment-related events from adverse events that

12 might occur in the natural history of the disease,

13 potential study design mechanisms that might help,

14 but certainly not resolve this issue are cited in

15 the bullets, design features that incorporate

16 randomization of subjects among groups, such that

17 comparisons may be made, the use of controls,

18 especially placebo controls, to make comparisons,

19 the use of masking or blinding to help lessen the

20 bias associated with concomitant therapies or

21 clinical care.

22 Tomorrow, the committee will be asked to

23 discuss mechanisms that might aid in adverse event

24 attribution.

25 \[Slide.\]

32

1 In this presentation, we have covered

2 three major topics. Firstly, we have noted that

3 the focus of this meeting is upon a discussion of

4 the scientific aspects of early cellular product

5 development.

6 Secondly, we have noted the regulatory

7 precedent for the cellular products.

8 Finally, we come to the questions.

9 \[Slide.\]

10 This slide highlights the four major areas

11 of tomorrow\'s questions. Specifically, questions

12 related to manufacturing, we will request a

13 discussion of the extent of safety testing and

14 characterization that should be performed prior to

15 the release of a cellular product for

16 administration to humans.

17 The second and third discussion areas are

18 especially critical and may consume the bulk of our

19 time, that is, the extent and nature of preclinical

20 testing necessary to support the introduction of a

21 cellular product into humans, testing that involves

22 questions related to the product itself, as well as

23 the delivery mechanism, the catheter.

24 Finally, we will pose clinical questions

25 centered around adverse event detection and

33

1 analysis with a discussion of the pros and cons

2 associated with the use of controls in these

3 studies.

4 \[Slide.\]

5 Our agenda is summarized on this slide.

6 As you can see, today, we have a series of invited

7 presentations by FDA staff and leading

8 investigators in the field, as well as the

9 opportunity for public presentations.

10 Tomorrow, we will have another opportunity

11 for public presentations followed by a discussion

12 of the questions.

13 \[Slide.\]

14 In closing, listed here are some documents

15 that are especially pertinent to our discussions.

16 All these documents are available at www.fda.gov

17 under the CBER sites, specifically the guidance

18 section.

19 The first document is entitled \"Draft

20 Guidance for CMC Reviewers: Human Somatic Cell

21 Therapy Investigational New Drug Applications.\"

22 This document describes the types of information

23 FDA reviewers will examine following the submission

24 of an IND. Consequently, it provides a very clear

25 description of the types of manufacturing

34

1 information that needs to be submitted with an IND

2 application.

3 The second document is from the

4 International Conference on Harmonization of

5 Regulatory Practices, and it is entitled \"

6 Preclinical Safety Evaluation of

7 Biotechnology-derived Pharmaceutics,\" the S6

8 document.

9 This document is cited because it contains

10 a paradigm that one may apply to cellular products.

11 Finally, the last bullet cites one of the

12 most useful guidances to sponsors and

13 investigators, the ICH Guideline on Good Clinical

14 Practice.

15 This guideline provides detailed

16 information on how to design and conduct a clinical

17 study, information presented in a simple to read,

18 yet relatively comprehensive format.

19 This concludes my presentation and I thank

20 you for your attention.

21 \[Applause.\]

22 DR. RAO: Before we continue with the rest

23 of the presentations, I would like to just welcome

24 Dr. Harlan and ask him to introduce himself.

25 DR. HARLAN: I apologize for being late,

35

1 but I am David Harlan, NIDDK. I study

2 transplantation of islets and immunotherapies.

3 DR. RAO: Our first speaker will be Dr.

4 Perin, whom you already were introduced to.

5 Guest Presentations

6 Overview Cardiomyopathy and Ischemic Heart Disease

7 DR. PERIN: I want to thank you for the

8 invitation to be here to present to you today,

9 especially Dr. Grant, who has helped me put this

10 together in a way.

11 So, what I want to do here this morning,

12 the task that has been laid before me is that of in

13 a way setting the stage or giving you a general

14 idea of the kinds of patients that we are treating.

15 Obviously, this is fundamental if we are

16 thinking about doing clinical trials. It is very

17 important to understand the nature of the disease

18 in which these kind of therapies will frequently be

19 applied.

20 What I plan to do is talk about the

21 following topics. First, we will start from the

22 beginning, define what heart failure is, look at

23 the scope of heart failure, talk a little bit about

24 the pathophysiology, look at some prognostic

25 markers, talk about the treatment to some extent

36

1 and that is important in terms of monitoring, and

2 then really work our way towards end stage heart

3 failure because that is where I think the focus of

4 most of the future clinical trials will likely be

5 initially, and finally, talk about adverse events,

6 which I think is a major concern, and the

7 monitoring of there adverse events.

8 Now, I know many of you are not

9 cardiologists, so hopefully, I can go from a level

10 where we are not getting too complicated, but not

11 too simple.

12 Starting with the definition of what heart

13 failure is. Firstly, heart failure is a clinical

14 syndrome very simply defined by certain symptoms

15 and certain signs that come together. These

16 symptoms are fatigue, shortness of breath, and

17 congestion, and these are translated on a physical

18 exam by being able to hear a third heart sound, the

19 patient manifesting peripheral edema, and jugular

20 venous distention.

21 If we start looking at this problem and

22 have a broad overview of this, first, I want to

23 show you a graph from the HOPE trial. This is a

24 trial that was conducted in thousands of patients,

25 as you can see here, over 9,000 patients. It was a

37

1 study primarily of ramipril and vitamin E in

2 patients with hypertension over a long period of

3 time, involved a five-year follow-up.

4 But it is just very interesting, as we

5 start out looking at heart failure, to look at this

6 patient population, and here we have over 500 days,

7 so here is about a year out, and if we look at this

8 population, who is not primarily designated as

9 particularly sick or harboring heart failure, that

10 identified the patients that did have heart failure

11 and we look at their survival, you will see the

12 mortality.

13 It separates from the beginning, and when

14 we get out to about a year, you have got a 10

15 percent mortality in the group that has heart

16 failure compared to less than 4 percent mortality

17 in the general population. So, you can see that

18 the problem that we are dealing with seems to be

19 very serious.

20 If we go here and let\'s just look at the

21 placebo arms of some very large heart failure

22 trials, these are trials pretty much aimed at

23 evaluating different forms of therapy now in heart

24 failure patients, and looking at different severity

25 of heart failure patients, for example, in the

38

1 V-HeFT trial, inclusion criteria might be an

2 ejection fraction less than 40 percent.

3 If we look at PRAISE, which evaluated

4 amlodipine in more severe heart failure, an

5 ejection fraction was less than 30 percent,

6 comparing this with Class III and Class IV

7 patients, very sick patients.

8 So, you can see here if we look at just

9 the placebo arms of all these trials, a very

10 striking mortality as we go along. If we look at 1

11 year here, this will vary from 10 percent down to

12 around 30 percent.

13 If we go out to 2 years in the very sick

14 patients, we see that half of the patients are

15 dead. So, heart failure, depending on the

16 presentation, carries a very ominous prognosis.

17 It is a very broad problem, 5 million

18 Americans are living with heart failure now,

19 550,000 new cases are diagnosed each year.

20 From 1979 to 2000, heart failure deaths

21 increased by 148 percent. Now, what is

22 interesting, over this period of time, we have

23 actually gotten a lot better at treating heart

24 failure, and we do treat it. I will get into this a

25 little later, and I will show you the modern treat

39

1 of heart failure and how much better we are doing,

2 but at the same time that we are treating heart

3 failure better, we are also treating the patients

4 that have coronary disease, which is a very

5 dominant problem in this country and around the

6 world, we are treating those patients better, too,

7 so what happens is we are getting more patients

8 with heart disease that normally would have died

9 earlier, to live longer, and as we are able to

10 bypass and stent and do all these revascularization

11 procedures and come up with better treatments, we

12 are getting people that go further down the road,

13 that otherwise would have succumbed a long time

14 ago.

15 So, despite our improvements in treatment

16 of coronary disease, we are dealing with an

17 increasing amount of heart failure deaths.

18 In individuals diagnosed with heart

19 failure, cardiac death occurs at 6 to 9 times the

20 rate in the general population. If you are more

21 than 40 years old, you have a 1 in 5 chance of

22 developing heart failure, and 22 percent of men and

23 46 percent of women that have heart attacks will be

24 disabled within 6 years with heart failure.

25 So, as you can imagine, the high

40

1 prevalence and multiple complications have an

2 implication in terms of health costs. If we look

3 at the costs, and these numbers vary, and it

4 depends on what you are looking at and what year

5 you are looking at, but this is a very significant

6 financial burden on the country, over 5 percent of

7 the total health care costs.

8 You can see that most of the cost involved

9 is really involved in inpatient care, and as I will

10 show you hopefully, that really translates to the

11 sickest portions of these patients, that as you get

12 sicker with heart failure, you start coming into

13 the hospital more, and that is what runs up the

14 cost of treating these patients. It is interesting

15 that transplant is just a little sliver out of the

16 pie here.

17 So, let\'s look at the causes of heart

18 failure, and I am not going to get into all the

19 little minor details, but let\'s look at the major

20 causes of what brings on heart failure.

21 Seventy-five percent of people that go on

22 to develop heart failure had hypertension

23 previously. Valvular heart disease is a big

24 contributor and also heart failure engenders

25 valvular heart disease, mitral regurgitation

41

1 further contributes to the problem.

2 Coronary artery disease, you are all

3 familiar with this, the number one problem in this

4 country, and this is really what we are going to

5 focus majorly on in terms of causing heart failure

6 and the specific kind of heart failure that this

7 engenders.

8 In cardiomyopathy, there is many different

9 kinds of things that get a heart to perform poorly,

10 all the way from an idiopathic cardiomyopathy to

11 such things as iron overload, et cetera, which are

12 not as common.

13 Now, what I want to talk about here is

14 really systolic heart failure. There is something

15 called diastolic heart failure, and that really has

16 a lot to do with compliance problems of the

17 ventricle, and in these patients, we are going to

18 see a normal ejection fraction.

19 So, this is really a different animal and

20 it is really not what we are focusing on, so what I

21 am going to be talking about today is systolic

22 heart failure, and as I will show you, with the

23 hallmark being a low left ventricular ejection

24 fraction.

25 This is just to give you a practical

42

1 example. This is an angiogram from one of the

2 patients that we treated with stem cell therapy in

3 Brazil, who all had an ejection fraction that

4 averaged about 20 percent. This patient has an

5 ejection fraction of 10 percent.

6 You can see the coronaries are calcified.

7 This is a catheter in the left ventricle. This

8 heart is supposed to be pumping this contrast we

9 just put into the aorta. As you can see, it is not

10 doing that very well at all. Only 10 percent of

11 what is in here gets out with each beat.

12 So, you can tell this is a dilated big

13 heart that just doesn\'t contract well. That is the

14 picture of severe heart failure right there, and

15 this is what I want to talk about.

16 Now, when we talk about heart failure, I

17 think everybody is aware of the classification.

18 There is Class I, II, III, IV, which are commonly

19 used, but it is important to acknowledge this.

20 Class I involves no limitation of physical

21 activity, Class II slight limitations, Class III

22 marked limitations, you can\'t walk up a flight of

23 stairs without getting short of breath, and Class

24 IV, you have symptoms at rest.

25 If we look at this, if we put Class III

43

1 and Class IV together, you see the division is

2 about a third for each of these pieces of the pie

3 here.

4 Now, if somebody comes in with Class IV

5 heart failure, they are very short of breath at

6 rest, you can give them some diuretics and they

7 will feel better. They are not Class IV anymore,

8 they are Class III.

9 So, it is interesting, there has been a

10 want in development of a little different way of

11 looking at heart failure, and a staging or

12 classification put out by joint AHA and ACC shows

13 four different stages, and really looks at heart

14 failure more like a disease like cancer.

15 So, where we can identify patients that

16 are at high risk of developing it, we can screen

17 patients, and then we can start treating patients

18 before they really manifest symptoms of the

19 disease.

20 Again, this is a progressive disease and

21 we are going to end up with people that are

22 refractory even to all kinds of treatment. I am

23 going to go over this a little bit more in detail a

24 little later.

25 So, in defining what heart failure is, I

44

1 hope I have given you a general idea of the scope

2 of the problem, just talk a little bit about what

3 causes it because it is important to understand

4 that to be able to know how we treat it and how we

5 monitor these patients.

6 Usually, we are talking about ischemic

7 heart disease and we are dealing with a myocardial

8 insult, which is usually a heart attack, so that

9 heart attack causes damage to the heart muscle, and

10 that is going to result in dysfunction of that

11 heart muscle.

12 Well, the body is going to try to

13 compensate this dysfunction and especially in two

14 major ways. One is neurohumoral activation, so we

15 will talk a little bit about this in more detail,

16 but essentially, these compensatory mechanisms are

17 going to make the heart change its shape and its

18 size. It is something we call remodeling. It

19 involves hypertrophy of the myocytes and then it

20 involves fibrosis and dilatation.

21 So, these mechanisms that the body helps,

22 to try to help to reverse what is going on,

23 actually wind up causing toxicity, hemodynamic

24 alterations that all lead to remodeling, and

25 remodeling really is the hallmark.

45

1 You saw that big heart. Well, remodeling

2 is how you get from a normal small heart, which you

3 have, to a big boggy heart that doesn\'t contract.

4 That is the problem of heart failure.

5 This was very simply put by Doug Mann in a

6 nice editorial a few years ago. Basically, here is

7 the heart over time, as we have an index event, and

8 basically, remodeling occurs, the heart gets

9 bigger, the ejection fraction goes down as time

10 goes by and symptoms occur as time progresses, as

11 well.

12 So, I have told you we have a myocardial

13 insult. This leads to LV dysfunction and

14 remodeling, and this really instigates a

15 neurohumoral response. In return, this is going to

16 have an impact on remodeling again.

17 So, what are these neurohumoral things

18 that happen? Well, first of all, most importantly,

19 is the renin- angiotensin-aldosterone system, and

20 there are several points in which the body

21 upregulates the system and ultimately, it acts on

22 the AT-1 receptor, which will cause

23 vasoconstriction, proteinuria, again LV remodeling.

24 As you can identify, here are several

25 sites in which medications, the mainstay of some of

46

1 the therapy for heart failure works, namely ACE

2 inhibitors that work at this point, ARBs that work

3 at this point, beta blockers have a role in

4 inhibiting renin, as well. So, some of the

5 mainstay of therapy is actually directed at one of

6 these mechanisms of compensation.

7 On the other side, we have sympathetic

8 activation. We have increased sympathetic activity

9 that again leads to myocardial toxicity and

10 arrhythmias, and then on the other side, with the

11 sympathetic outflow, we get vasoconstriction. This

12 impacts negatively on the kidney, sodium retention,

13 more vasoconstriction, and progression of the

14 disease.

15 Just to get a slightly little bit more

16 complicated, just to mention that it is really not

17 all that simple, there are other things involved,

18 and we have cytokines, TNF-alpha, IL-6,

19 inflammation that actually progresses with the

20 progression of heart failure.

21 Endothelin is a potent vasoconstrictor.

22 All these things lead to apoptosis and unfavorable

23 effects upon the myocyte, but then lead to LV

24 remodeling, which I have told you is one of the

25 mainstays of reasons for heart failure.

47

1 Now, natruretic peptides are important,

2 as well. It\'s another compensatory mechanism that

3 the body has. I am sure you are familiar with

4 these BNP, it\'s a B-type natruretic protein that

5 actually comes from the ventricle, the A types

6 comes from the atrium. We will just focus on the B

7 type.

8 What this does, basically, in response to

9 elevated pressure inside the heart, we secrete BNP.

10 This suppresses the renin-angiotensin-aldosterone

11 system and suppresses endothelin. It helps with

12 peripheral vascular resistances, decreases

13 vasodilatation, and it increases natruresis.

14 So, if we go on to understand now that

15 there is an interplay between LV dysfunction and

16 remodeling, and that basically, this will lead to

17 low ejection fraction, and that is what we see in

18 the patients.

19 On the other hand, as a result of this, we

20 will start getting a constellation of symptoms, and

21 it is the combination of having a low ejection

22 fraction and symptoms that defines heart failure.

23 Let\'s look a little bit at the prognostic

24 markers. I just talked a little bit about BNP.

25 Well, it is very interesting. If we divide BNP in

48

1 quartiles here, depending on the amount of BNP that

2 you have circulating, your survival will go down.

3 It is a prognostic marker, as well as a treatment.

4 Norepinephrine, the same way. So, these are

5 markers of prognosis.

6 It is very interesting. These are levels

7 of BNP, and if you can decrease them, decrease to a

8 less degree, or here, we have an increase. So,

9 depending on which direction your BNP goes, your

10 survival varies as well, and that is an important

11 concept.

12 Let\'s look at another different kind of

13 marker. Exercise capacity, peak oxygen consumption.

14 In the transplant world, this is very important.

15 Here you see the number 14, so a peak oxygen

16 consumption greater than 14 or less than 14 has

17 very different prognostic indicators and in many

18 centers, this serves as a marker threshold for one

19 of the criteria for entering the patient into a

20 transplant program.

21 You can see here a difference in mortality

22 from 53 percent mortality over two years in

23 patients that have an NVO2 of less than 14, to that

24 of 11 with greater than 14, so this is another

25 important number in patients with heart failure.

49

1 Then, if we look overall and look at

2 symptoms and hospitalizations, here is a New York

3 Heart Class I to IV, and this is fairly intuitive,

4 but as we get more symptomatic, we have an impact

5 on survival, and as we are getting more

6 symptomatic, we have an increase in

7 rehospitalization.

8 What about ejection fraction? I just

9 talked about ejection fraction, and you can see

10 here, similarly to NVO2, ejection fraction can

11 divide prognostically how patients will do. Here

12 we see more than 20 percent, less than 20 percent.

13 Here you see a two-year survival, 54 percent, so

14 half the people dying that have an ejection

15 fraction less than 20 percent. At one year, that

16 is a little over 20 percent.

17 The same thing, this is a large randomized

18 clinical trial, ejection fraction less than 40

19 percent. Over time, people die more frequently.

20 Now, let\'s add a little arrhythmia to

21 this. Looking at different levels, the first two

22 are greater than 30 percent ejection fraction, here

23 less than 30 percent, so that stratifies that out,

24 but then if we just add the amount of extra

25 ventricular beats to this, and if we have less than

50

1 10 per hour, more than 10 per hour, and then with a

2 poorly contractile ventricle, your survival goes

3 down as we add extra ventricular beats.

4 One attempt that has been made to sort of

5 graph this problem, because now I have shown you

6 many different prognostic markers and different

7 things we can use to classify these patients to

8 decide what to do and how to follow them.

9 One of them is a heart failure survival

10 score. There is an invasive model, there is a

11 non-invasive model. So, things like cause of heart

12 failure, resting heart rate, EF, mean blood

13 pressure, if there is a conduction delay

14 electrically in the heart, oxygen consumption, and

15 serum sodium can enter into a risk classification.

16 Here, you just basically have a graph that

17 shows according to low, medium, and high, your

18 survival will vary according to the risk.

19 In our little schema here, that leads

20 symptoms and low ejection fraction to heart

21 failure, what are really the things, though, that

22 are driving mortality? They are going to be pump

23 failure, on the one hand, and arrhythmia, on the

24 other, because sudden death, as I talked to you

25 about before, is a very prominent problem in people

51

1 that have heart failure.

2 So, it is the combination of these three

3 things that will pretty much drive patients to a

4 lethal exit.

5 Let\'s talk a little bit about treatment

6 now. What are the goals of treatment of heart

7 failure? You want to delay the progression or

8 reverse remodeling, which you can do in some

9 patients, and delay the progression and reverse

10 myocardial dysfunction.

11 You want to reduce mortality, relieve the

12 symptoms, improve functional capacity, and reduce

13 disability, also decrease the intensity of medical

14 care and hopefully reduce economic cost.

15 I have shown you we go from initial

16 injury, initial infarct, we suffer remodeling, we

17 get a remodeled heart that now has a low ejection

18 fraction, and over this course of time, we have a

19 worsening of symptoms, so how are we going to

20 impact this in terms of treatment?

21 Well, the two mainstays are neurohumoral

22 blockade, we have kind of gone over some of the

23 things that we can do, and we will look at those,

24 and the other is revascularization. So, many times

25 with the use of medication or with the use of

52

1 revascularization, we can reverse some of this

2 remodeling in some patients, and in some patients

3 we don\'t.

4 One thing that is very important in terms

5 of being able to recover patients that have

6 remodeled hearts, and that are in this road of

7 heart failure, is identification of viable

8 myocardium.

9 Myocardial viability has clearly been

10 shown to influence the prognosis of people that are

11 undergoing revascularization procedures, so if you

12 have a viable myocardium, you are going to do

13 better. You have a chance of improving more than

14 someone who doesn\'t.

15 Just to shift gears for just a second

16 here, these are electromechanical maps. These are

17 representations of the left ventricle. This is

18 from a patient in our Brazil stem cell study.

19 This is an electrical map, this is a

20 mechanical map. Let\'s just look at the electrical

21 map because I just talked to you about viability.

22 Very simply, if your cells are alive, they have an

23 electrical signal that is high. If you have a big

24 scar with no cells, you have no electricity, you

25 have a low electrical signal.

53

1 We put it on a little color scale. Red is

2 dead or red is very little voltage. Purple is

3 high. Here, you see on this electromechanical map,

4 an area of myocardial viability. Again, just as it

5 is important to understand viability when you are

6 vascularizing patients that have heart failure,

7 that have coronary disease, it is also going to be

8 important, in my view, to understand myocardial

9 viability when we are applying some of these

10 therapies, and I think there will be differences in

11 bone marrow therapies and myoblast therapy, but

12 that is something to keep in mind.

13 I just wanted to show you an example of

14 the very common things that we deal with, so this

15 is not some esoteric difficult patient to find. We

16 come across people like this all the time in the

17 hospital every day.

18 This is a patient who was 41 years old, he

19 had bypass, he stopped up all his vein grafts and

20 his memory artery, and he had ejection fraction of

21 20 percent, very similar to the one that I showed

22 you, and Class IV congestive heart failure.

23 This gentleman was really delightful. He

24 was actually a pilot for a major airline, and

25 because of his bypass, he had to be put off the

54

1 flying, and he was actually in charge of all the

2 simulators, and he was the guy that graded all the

3 pilots when they had to come in and do the

4 simulation testing.

5 Basically, here, we have a 41-year-old

6 guy, very active man who has gone bypass, he has

7 lost his graft, he obviously has very aggressive

8 disease, and why I hear the talk about why some

9 people have more aggressive coronary disease than

10 others.

11 You see this is his right coronary, it is

12 completely blocked up, X\'s mean that you can\'t see

13 anything on angiography, so this kind of fills from

14 the other side by collaterals, see these little

15 twigs down here.

16 Then, the circumflex is completely

17 occluded. This is a floating marginal branch.

18 This is supposed to be connected, but this is

19 totally occluded, as well. The only artery he has

20 got left is the one down the front of his heart,

21 but this is very much infarcted, and has a very

22 significant blockage here, as well as the takeoff

23 of this.

24 So, this patient, there is really nothing

25 to do, and we are faced with this a lot every day.

55

1 This patient, as I have shown you these curves of

2 mortality, this patient at our hospital wound up

3 going for an LVAD type procedure and died, and that

4 is what we see again and again, so this is a very

5 serious problem.

6 So, looking of an overview of treatment of

7 heart failure, let\'s see, we have medical-based

8 therapy, on one hand, we have device-based therapy,

9 on the other.

10 On the medical side, we need neurohumoral

11 blockade, we can have a hemodynamic approach and

12 also antiarrhythmic approach, so we are going to

13 use these drugs, ACE inhibitors, aldosterones,

14 diuretics, beta blockers, and then antiarrhythmics,

15 such as amiodarone, and then we are going to use

16 more potent i.v. inotropes that improve

17 hemodynamics, and asaratide \[ph\], which is

18 basically similar to BNP, it is like giving the

19 patient BNP.

20 On the other hand, we are going to have a

21 device-based approach using resynchronization

22 therapy. It really hasn\'t shown a benefit in

23 survival, but in combined endpoints. We are going

24 to put defibrillators into people, and I will show

25 you how that has improved survival.

56

1 Then, we will have ventricular assist

2 devices, and when all this fails, we have an option

3 of heart transplant, that is very little available

4 actually, and as you saw, it is a very little

5 sliver of what we are able to do.

6 But as you cumulatively add these

7 therapies, you are able to impact on survival and

8 make patients live longer. Here, you see sort of

9 adding digoxin and diuretic, adding an ACE

10 inhibitor, and then adding a beta blocker, we get

11 progressive improvement. So, this is pretty well

12 established in terms of medical therapy.

13 When we look at defibrillators, here is a

14 curve. This is from the MADA-2. This is primary

15 prevention, defibrillator in patients, previous MI,

16 LVF less than 30 percent, a very significant

17 survival difference in the patients that get a

18 defibrillator, so treating the arrhythmias is also

19 important.

20 Back to our schema of the different

21 classification of stages of heart failure. You see

22 that we can gradually, we start with ACE inhibitors

23 and gradually add different medications, but

24 everybody kind of goes up these stairs and ends up

25 here at the top, and that is why we have increasing

57

1 mortality from heart failure, because we are

2 getting people to get to this point where before

3 they really didn\'t reach that stage.

4 Then, we get to a stage of basically

5 refractory symptoms, so they have been bypassed,

6 they have had stents, everything has been done for

7 them, and they have that bad heart, it doesn\'t pump

8 well, they have a lot of symptoms, they can\'t

9 breathe very well. Many of them have angina. I

10 want to want to give you a little bit of my own

11 perspective on that.

12 If we look at current trends, this was

13 published last week in JACC, very interesting.

14 Heart failure treatment\--this is the survival

15 curves\--heart failure treatment in 1994 to 1997.

16 Here is a survival curve. We have improved the

17 treatment of heart failure.

18 1999 to 2001, gee, we are doing a lot

19 better, and this is comparable actually to

20 transplant from 1993 to 2000, and it really raises

21 the question if transplant, with the modern

22 management in medical management of heart failure,

23 how important is it and what the role of transplant

24 really is.

25 Really, there is a gap between a very

58

1 invasive transplant or LVAD and the medical

2 therapy, there really is, and we are here to talk

3 about stem cell therapy. There is a gap of

4 something that could be done that is not quite as

5 invasive and traumatic as an LVAD or transplant,

6 and that can improve the patient significantly

7 since we are doing so well with medical therapy.

8 I want to talk to you a little bit about

9 my perspective on end-stage ischemic heart disease.

10 Basically, as I have told you, we have improved the

11 medical management, so we have longer survival, we

12 have improved the vascularization treatments of

13 coronary disease, we have improved the survival

14 following a heart attack, and that is why we have

15 more patients, and now we are using widely

16 defibrillators, and that is why people are living

17 longer.

18 So, this is sort of my understanding of

19 this end-stage patient. You progress with coronary

20 disease until you get to the Stage III and Stage

21 IV, Class III/Class IV heart failure.

22 If we look at these patients, sometimes

23 there will be a little surprise, because some

24 patients really just have shortness of breath, so

25 this is a variable. This may occupy the whole

59

1 square or angina may occupy the whole square.

2 So, some patients predominantly have heart

3 failure, and these patients that predominantly have

4 heart failure probably weren\'t very good at forming

5 collaterals when they had heart attacks and

6 developed a lot of scar tissue, and have a very low

7 ejection fraction. These are the sickest patients

8 and the patients that are going to have a very high

9 mortality.

10 On the other hand, but also in the Class

11 III or Class IV, and sometimes we pool these people

12 together in trials and that is why I am making this

13 distinction, some people have angina more than they

14 have heart failure. These probably have a much

15 better collateral formation when they had these

16 events, so their ejection fraction is a little more

17 preserved.

18 I have had many patients that have lived

19 on one artery. Their whole heart is beating okay.

20 That one artery feeds everything by collaterals,

21 but they are in really bad shape. I mean it\'s an

22 illusion that they are doing okay, but they do have

23 a preserved ejection fraction, and their

24 manifestation is a lot of chest pain.

25 So, symptoms can vary from one side to the

60

1 other and some patients have a balance here, and I

2 think we need to keep this in mind when we are

3 designing these trials.

4 So, there is a predominant angina, and

5 this is the kind of patient that got, let\'s say,

6 these TMR type procedures. That is the kind of

7 population you are dealing with. The predominant

8 aspect is disabling angina, preserved EF, 100- to

9 200,000 new cases per year, and constitute about 5

10 percent of the patients undergoing angiography at

11 tertiary referral centers. This has been studied

12 in this particular case at the Cleveland Clinic.

13 One year mortality is still very high.

14 Then, that other group, predominantly heart failure

15 symptoms, very low EF, myocardial ischemia, though,

16 is still present, but with more scar. No option

17 really for any kind of revascularization. One year

18 mortality, 20 to 50 percent. I have shown you one

19 curve where it is up to 80 percent, I mean it can

20 be really bad.

21 Here, we have ICD therapy trials. If we

22 look at secondary prevention trials, very sick

23 patients in this study, treated with amiodarone,

24 you see here one year mortality 44 percent. I mean

25 heart failure can be worse than cancer.

61

1 Here is the REMATCH trial. This is an

2 LVAD. This is the impact of LVAD, and there is an

3 impact of survival, but again you are dealing, in

4 this case, with Class IV patients that are

5 unresponsive to medical therapy, so these very sick

6 patients, but again an invasive, costly, not widely

7 available kind of therapy, but it does have an

8 impact on failure.

9 I want to finish now talking a little bit

10 then, hopefully, I have given you an overview of

11 the problems with heart failure, and how are we

12 going to look at adverse events.

13 Well, what are the things that are going

14 to drive the adverse events here, are going to be

15 arrhythmia, ejection fraction, and symptoms, and I

16 think if we focus here, we can pretty much decide

17 what we need to look at in these patients over time

18 as we use new therapy towards these patients.

19 Let\'s look at low ejection fraction, how

20 are we going to monitor that? Well, we need to

21 look at cardiac function, cardiac size, and the

22 perfusion status of the ventricle. We can do that

23 very simply, if you take a simplistic approach,

24 with echocardiography.

25 I empirically have placed this here based

62

1 on my own limited experience here, but I read in

2 the document that you wanted some more practical

3 advice, so I will give you my own sort of practical

4 feel for what I would do.

5 If we did echocardiogram on these

6 patients, we could do it monthly for the first

7 three months and then at six months follow-up. We

8 can do SPECT, we know that we don\'t need it too

9 early, and that is a very simple way of doing it,

10 three to six months. Clinical visits, which will

11 be very frequent, and I will talk about that, and

12 BNP can be done for that, as well.

13 Now, we can get fancy and use alternative

14 imaging strategies, we can use MRI,

15 electromechanical mapping, PET, depending on the

16 institution, and depending on what we are really

17 looking for and want to find.

18 Cardiac arrhythmias, it is important to

19 monitor cardiac rhythm. Holter monitoring is very

20 simple, probably should be done after the

21 procedure, one, three, six months later. Q-T

22 interval when the patient comes in for his clinic

23 visit is a strong predictor of survival, just a

24 plain-old, good-old 12-lead EKG, and that should

25 always be looked at.

63

1 In the patients I guess that are getting

2 myoblast therapy, there may be a little bit more

3 concern about this, and this is really not my area

4 of expertise, but these patients, many of them

5 already entering with an AICD, that have sort of a

6 built-in little computer that is already monitoring

7 their rhythm as it is. If they don\'t, you might

8 want to consider event monitoring.

9 For symptoms, well, clinical visits

10 biweekly for 8 weeks, monthly up to 6 months. We

11 are going to look at heart class, we are going to

12 look at EKG, CBC, CRP, look for inflammation.

13 Exercise capacity, ramp treadmills, as you know, if

14 you put a patient that has end-stage heart failure

15 on a graded treadmill test, every time the

16 treadmill bumps up and goes a little faster, he

17 just may not be able to exercise at that point.

18 So, the advantage of a ramp treadmill

19 protocol is that you have a gradual continuous

20 increase, so these people that really can\'t do very

21 much at all, they will be able to tolerate the

22 exercise and probably get further than they could

23 in any other kind of exercise test.

24 There is a very simple way of evaluating

25 an exercise test, a 6-minute walk test. You just

64

1 define a distance, walk the patient walk for 6

2 minutes, see how fast he can go. You can do that

3 at a clinic visit, and it is very simple to do.

4 So, you can do something like this at one, three,

5 and six months.

6 Rehospitalization. We look at the

7 rehospitalization rates. It is important to look

8 at the use of i.v. medications that are used to

9 control symptoms, because this is, as you saw, the

10 biggest part of the pie in terms of costs, and is a

11 real problem in the end-stage patients.

12 Quality of life, it is important to assess

13 quality of life, for example, SF36, Minnesota

14 Questionnaire.

15 Just some suggestions. I want to wrap

16 this up and saying I hope I have given you a

17 general idea and scope of this problem. We deal

18 with a very, very serious problem, which is heart

19 failure, specifically, that which is ischemic heart

20 failure and specifically, end-stage ischemic heart

21 failure.

22 I hope I have given you a flavor of this

23 and set the stage for the discussions.

24 Thank you very much.

25 \[Applause.\]

65

1 DR. RAO: Thank you, Dr. Perin.

2 There is time for questions, and we can

3 open it up to the committee.

4 Q&A

5 DR. SCHNEIDER: Emerson, one of the things

6 that you did very nicely was lay out the clinical

7 spectrum for people who may not be familiar with it

8 in this context.

9 I wanted to follow up on that point

10 because work presented at international meetings

11 recently by the Frankfurt group of Andreas Sire and

12 Stephanie Dimler suggests that bone marrow derived

13 cells and circulating progenitor cells from

14 patients with established heart failure may be

15 deficient relative to the performance of bone

16 marrow derived and circulating progenitor cells

17 from patients with an acute infarct.

18 So, while it is not quite an apples and

19 oranges comparison to envision cardiac cell

20 grafting immediately post infarction or in the

21 first week post infarction in patients without

22 severe ventricular dysfunction versus patients,

23 let\'s say, two to four months out with mild or no

24 ventricular dysfunction versus the end-stage heart

25 failure patients who have been a focus in your talk

66

1 this morning, it does seem to me that that clinical

2 heterogeneity introduces a couple of problems.

3 I am curious to know how you have worked

4 those through in your own work. One of them is

5 because what we are discussing today and tomorrow,

6 is autologous cell therapy, I believe that there is

7 a serious issue of patient-to-patient cell

8 heterogeneity which has been relatively little

9 discussed in the field except in these still

10 unpublished or perhaps one paper has come out in a

11 secondary journal from Stephanie and Andreas about

12 the defects.

13 So, one question is what kinds of

14 standards should a proposed production center be

15 required to meet in terms of their ability to

16 generate cells that perform in accordance with some

17 standard when there is patient-to-patient variation

18 of this kind.

19 Secondly, if you are envisioning putting

20 cells of different kinds into a so severely an

21 ischemic background as the 41-year-old former pilot

22 that you mentioned, doesn\'t it become important to

23 clearly distinguish, as the prefatory remarks did,

24 between mechanisms of action for proposed donor

25 cells that are aimed at regeneration specifically

67

1 versus benefits that are achieved through entire

2 different mechanisms, such as angiogenesis?

3 If you put new cells into an ischemic

4 background, they will surely die, and if the goal

5 is to achieve angiogenesis in a background where

6 the native coronary circulation has failed and the

7 graft has failed, then, it seems to me we need a

8 clearer resolution of the problem of which cells do

9 which things well, and really fine-tune much better

10 than the field has to date, you know, which are the

11 cells that we want where the spectrum is normal

12 vasculature, insufficient muscle cells versus the

13 hypothetical ischemic patient that you described

14 where revascularization is the major goal.

15 DR. PERIN: Well, that\'s fantastic.

16 \[Laughter.\]

17 DR. PERIN: I think the basic answer to

18 your question is I don\'t know, but, you know, these

19 are all very good points, starting with the cell

20 type, we really don\'t know.

21 Actually, we have submitted a manuscript

22 in which we have had the pathology of one or our

23 patients in our study in Brazil who received

24 autologous bone marrow, died 11 months later, and I

25 really can\'t preempt I guess our publication, but I

68

1 think we will be seeing some evidence of myogenesis

2 and angiogenesis from autologous bone marrow cells,

3 but we really don\'t know what we are getting when

4 we are putting, let\'s say, autologous bone marrow,

5 and even in that patient that has, let\'s say he has

6 predominantly ischemia, if we want to

7 revascularize, can we get a predominantly

8 angiogenic effect, so we really don\'t know, and we

9 need to define that.

10 Mononuclear fraction of the bone marrow is

11 a very simple approach, the one that we have taken,

12 and it seems to initially, and we haven\'t really

13 done efficacy studies and we are continuing on, but

14 there is a suggestion that it does, so I think that

15 we need to take every step that we take should be

16 put one foot in front of the other, and if the

17 mononuclear cell fraction works, I think we can go

18 from there and keep investigating that.

19 Now, the average age in our trial was

20 about 58, and you mentioned the problem\--

21 DR. RAO: Can I interrupt? These are

22 really important questions, but they discuss data

23 which was not presented in the talk right now. I

24 would like to at least focus the questions

25 initially on the issues that relate to the

69

1 presentation right now.

2 We should really come back to these

3 questions tomorrow when we discuss exactly these

4 sorts of issues.

5 Do you think that that would be okay with

6 you, Dr. Schneider?

7 DR. SCHNEIDER: We will certainly return

8 to them tomorrow, but I was discussing issues that

9 were raised in this talk, which was clinical

10 heterogeneity.

11 DR. RAO: Let\'s then focus, not on the

12 cells per se, and the choice of cells, because none

13 of the presentation was related to the production

14 facility or how the cells would be, or the quality

15 would be, or how you would choose the mechanism,

16 but maybe how do you choose patients for a trial or

17 is there some reasonable way of selecting patients,

18 that there would be consensus on.

19 DR. PERIN: Okay. So, we will get back to

20 your first question and really, that is something

21 that actually, we are working on trying to

22 understand, is there a thumbprint or is there a

23 profile in the study by Dimler and their colleagues

24 looking at the characteristics of cells in certain

25 patients, and obviously, they may not be the same

70

1 in a diabetic, in a severe heart failure, we don\'t

2 know, so there is another important we don\'t know.

3 Age obviously is a very important thing,

4 so harvesting cells from a 75-year-old may be very

5 different than doing that in a 55-year-old, so

6 these are all questions that need to be answered.

7 DR. RAO: Dr. Mul.

8 DR. MULE: Given the slides you showed of

9 the steps toward progression of heart failure, and

10 given the current interventions along that pathway,

11 from your perspective, where would you see

12 cell-based therapy intervention falling into that

13 step toward complete heart failure?

14 DR. PERIN: Right now, at close to the

15 last few steps, I think ethically, we are propelled

16 to really study the problem in the patients that

17 really don\'t have a proven conventional option for

18 treatment. In brief, I would say in the patients

19 who can\'t be revascularized, because really medical

20 therapy, we are going to apply to everyone, so then

21 we are left with revascularization.

22 Well, can we revascularize? Well, we do,

23 and we do it again and again, and there is a point

24 where you are out of revascularization options, and

25 I think that is one place we are initially now,

71

1 then, you could think about applying this kind of

2 treatment.

3 DR. HARLAN: Building upon what Dr. Rieves

4 mentioned when he gave his introductory comments, I

5 want to just congratulate you on, it seems like our

6 task is to weigh the risk-benefit, and you have

7 outlined very clearly the risk, and I accept that

8 it is severe, and I also want to congratulate you

9 on mentioning the JACC paper that was just

10 published, that showed how dangerous it is to look

11 at historical controls, because we are making such

12 rapid progress.

13 My question is along those lines, not in

14 this field, I just read in the journal, the

15 Washington Post, about the great advance that has

16 been made in super-high statin therapies, and I

17 wonder if you could comment on that study, that

18 these super-physiologic statin doses seem to have a

19 major impact on mortality.

20 DR. PERIN: I really don\'t have an

21 expertise in a lot of things, and that is not one

22 of them, so it is really hard for me to comment on

23 that. I know that it looks like giving people HDL

24 in the future may be a very exciting thing, and we

25 may be able to finally find our liquid plumber kind

72

1 of solution for people.

2 Then, again, statins are just\--more and

3 more if you study statins, you have probably come

4 to the conclusion it should be in the water pretty

5 soon, I mean the patient benefit is on every single

6 aspect of cardiovascular disease.

7 DR. RAO: Dr. Kurtzberg.

8 DR. KURTZBERG: You mentioned some

9 practice-based methods to evaluate outcomes and

10 function in these patients, but I think the

11 challenge is to determine what the cells are doing,

12 you know, are they differentiating into other kinds

13 of cells, are they mediating inflammation, are they

14 mediating angiogenesis, and I don\'t see how you can

15 sort that out by clinical-based study.

16 Do you know of other technologies that are

17 on the horizon that may help with that, that are

18 non-invasive, or would you consider serial biopsies

19 in patients like this to answer those questions?

20 DR. PERIN: That is a good question. I

21 don\'t know that serial biopsies would be a very

22 efficient way of evaluating that. You would have

23 to have a very precise way of being able to

24 identify where you put the cells and be able to go

25 exactly to that same spot.

73

1 We do have that technology. Dr. Lederman

2 is going to follow me eventually here. The MRI

3 field, I think is very promising in that regard in

4 terms of labeling and following cells.

5 Now, I really don\'t know that even

6 labeling a cell, even if it died, if the label

7 stays there, you still see the label, so I think

8 that we have to even go a step further and be able

9 to prove the functionality of the cell that is

10 alive and was implanted.

11 That can be done on an experimental basis,

12 so we figure ways out to do that, but this is a

13 very intriguing problem and a very difficult

14 problem to evaluate. I think you have put your

15 finger on something that is going to be hard to

16 know.

17 DR. DINSMORE: Jonathan Dinsmore from

18 GenVec.

19 I just had a question on your angina heart

20 failure continuum. I was confused because most

21 heart failure patients present without angina, with

22 symptoms of fatigue, so what percentage of heart

23 failure patients actually experience angina?

24 DR. PERIN: If we are talking about

25 ischemic heart failure, we are not talking about

74

1 other kinds of heart failure, actually, idiopathic

2 heart failure, you kind of get the same remodeling

3 and everything except you didn\'t have that infarct

4 in the beginning, but you go through the same sort

5 of pathophysiologic processes.

6 So, we are talking about ischemic heart

7 failure. People that have ischemic heart failure

8 have coronary disease. Coronary disease is

9 narrowing of your coronary arteries.

10 Depending on what your response is, you

11 will or will not have angina, but angina is one of

12 the manifestations of coronary disease, and it is

13 really not a good thing to base a lot on, because

14 the expression of angina is very variable.

15 It depends on your pain threshold. I mean

16 if you are a diabetic, you may not have as much

17 pain. It is a subjective thing subject to

18 interpretation by the actual patient, so it is

19 something that is very difficult to evaluate, and

20 that is why I put the continuum, because it is all

21 there and you really shouldn\'t take a patient

22 population based on angina or based on shortness of

23 breath.

24 I think you have got to bring both of

25 these things together to understand they are sort

75

1 of in the spectrum of a similar underlying

2 pathophysiologic process.

3 DR. SIMONS: I would like to come back to

4 the issues of the differences among the patients

5 having these kind of therapies. We have learned

6 from a number of trials of growth factor therapies

7 that there is a very large difference in how the

8 patients respond.

9 This issue that there are different

10 subgroups that we are not defining is fairly

11 critical to the field. You mentioned one or two

12 biomarkers, but there seemed to be a general

13 association of markers as opposed to really

14 identifying which patients respond in which manner.

15 What would you suggest as a way of trying

16 to sort of stratify these patient groups? Not

17 suggest ejection fraction, that is probably in a

18 way sort of crude measure, but in terms of

19 biological responses.

20 DR. PERIN: If we look at the trials of

21 devices, I think that probably a common way to look

22 at these patients is exercise capacity.

23 I think that probably is one of the

24 unifying parameters that we cannot only use at

25 entry, but also you are able to follow as a patient

76

1 goes along, and if he has a response to therapy, he

2 will have a positive response in terms of what he

3 is able to do in terms of function.

4 That has a very practical translation into

5 quality of life and people feeling better. I would

6 say in a broad sense, that exercise capacity, peak

7 oxygen consumption might be something that I might

8 consider an important thing to follow in these

9 patients, and not just ejection fraction, which is

10 dependent on a lot of things, how much loading the

11 ventricle has that day, the amount of mitral

12 regurgitation, et cetera, so there is a lot of

13 things that will make that extremely variable.

14 DR. RAO: As an extension of that, it\'s a

15 very general question. Is there any problem with

16 many of these studies which are in high-risk

17 patients enrolling people for the placebo arm of

18 the trial? Not in cell therapy, but maybe when you

19 do devices or you do assists, has this been

20 historically a problem for the cardiovascular

21 field?

22 DR. PERIN: Well, it has been done as you

23 can see, so I have showed you a bunch of studies

24 where it has been done, and it can be done.

25 Personally, the way I like to see it is I

77

1 want to offer patients that get in the placebo arm

2 some kind of a treatment, so in our future upcoming

3 study, what I am going to do is I will tell a

4 patient you are going to get randomized to maybe

5 not getting treatment, but if you don\'t get that

6 treatment at an X period of time, six months, you

7 will cross over to get the treatment.

8 I think that is a humane way of doing it,

9 in which these patients are very ill and desperate

10 to get something to help, so again, if you can

11 cross over, sometimes these placebo patients at

12 some point after you have achieved your assessment,

13 then that makes it a more palatable or fair way to

14 do things maybe.

15 DR. RAO: Dr. Cunningham.

16 DR. CUNNINGHAM: I just wonder, in your

17 data, if you see any difference by either

18 socioeconomic status or by gender, or by any way of

19 culture, dividing populations, whether it would be

20 race or ethnicity or any other factor like that?

21 DR. PERIN: You mean in our own\--

22 DR. CUNNINGHAM: Yes, reading the JACC

23 data, was there anything by gender, for instance,

24 or by subpopulation?

25 DR. PERIN: Females, there are some

78

1 differences in the female population in which there

2 are some differences. There is the catch-up

3 phenomenon in the end, but socioeconomic

4 differences, I am not aware that it would have an

5 impact on that, as well, but maybe gender

6 differences, yes.

7 DR. RAO: One question to sort of follow

8 on Dr. Simons\' question, in at least the way I

9 understood it, it is really kind of difficult to

10 stratify patients or to extrapolate from one class

11 of patients to the other. Historically, that has

12 always been a problem.

13 Again, it\'s a general feeling when one

14 conducts studies in the cardiovascular field, is

15 there some consensus that everybody says that,

16 well, if you measure by ejection fraction, and we

17 take patients, which is what it seemed like a lot

18 of studies have done, that that is a reasonable

19 criteria that you can extrapolate from one

20 classification of that kind to the next, or one

21 cannot? Just as a general statement.

22 DR. PERIN: It has been done, and it is a

23 general way of separating\--there is definitely a

24 correlation with your ejection fraction and your

25 survival, so it is probably not the most refined

79

1 way of dividing patients, and it depends where you

2 make the cutoff, so if you make a fairly high

3 cutoff, let\'s say, patients that had ejection

4 fraction less than 40 percent, then, you are

5 including most of the population of patients that

6 have heart failure, so it\'s a general way to divide

7 things.

8 If you start decreasing that number of

9 that cutoff, then, you are really selecting out

10 more I think subpopulations we were talking about,

11 maybe some different kind of subpopulations of

12 patients with heart failure.

13 DR. RAO: Dr. Borer.

14 DR. BORER: Dr. Rao, a few minutes ago you

15 made a point, and I would like to restate it in

16 another way, because what Dr. Perin did, as I see

17 it, is very well present an overview as an outline,

18 was a scaffold upon which we can conduct subsequent

19 more specific discussions.

20 I think that right now we are getting into

21 a series of questions that are way beyond the data

22 that exist, and you couldn\'t expect Dr. Perin to

23 respond to them in a meaningful way because the

24 data don\'t exist.

25 In specific response to your question,

80

1 which was a very fundamental one, I think we are at

2 a point now with this form of therapy where if we

3 could define any group in which we saw a response

4 which seemed credible, which was statistically

5 valid, we would then have a series of hypotheses

6 that would have been generated that would allow one

7 to move further, but I think that is the level we

8 are at.

9 The idea of defining a general population

10 in which to test therapy the way we do with drugs,

11 we are not there yet, so I think the specific

12 questions have to come a little later in this

13 forum.

14 DR. RAO: I just wanted to get it clear to

15 people that that was the case, but your point is

16 very well taken.

17 Dr. Neylan.

18 DR. NEYLAN: Thank you.

19 That was a very nice clinical overview,

20 and I wanted to ask you from your perspective as a

21 clinician, there are obviously many parameters

22 whose relief or improvement would be significant in

23 terms of the lives of individual patients, and many

24 of these could be utilized as endpoints for proof

25 of concept.

81

1 But ultimately, what do you believe is the

2 most relevant clinical endpoint for defining

3 registration criteria for this form of therapy, is

4 it patient mortality or something else?

5 DR. PERIN: I don\'t know if we are going

6 to be impacting patient mortality. That is a very

7 difficult question. I would go back and what I had

8 said earlier, and use an endpoint, I would use

9 something like the LV02 as an endpoint.

10 I think that is a little bit more

11 palpable, and obviously, looking at mortality, this

12 is such an initial incipient field in which we have

13 barely treated any patient, so to think about

14 looking at mortality, which involves a much larger

15 number of patients, I think that is probably

16 getting ahead of ourselves a little bit.

17 We need to first verify if this is

18 efficacious and if there is some objective

19 improvement in these patients, and one of those

20 objective ways of doing that would be something

21 like exercise capacity, like I mentioned.

22 DR. RAO: Dr. Ruskin.

23 DR. RUSKIN: Just two quick comments on

24 Dr. Perin\'s very nice presentation.

25 One is that we have learned from drug and

82

1 device trials that both ejection fraction and heart

2 failure classification are critically important

3 predictors, but that they are not necessarily fully

4 interactive, that is, they are independent, so

5 using both, I think in any classification with

6 regard to these kinds of interventions would be

7 critical because the outcomes are very, very

8 different in Class III and IV even with the same

9 EF.

10 The other relates to a question that Dr.

11 Rao raised about recruitment and controls. I think

12 that given the excitement in this area, but the

13 unknown issues that have already been raised, doing

14 trials that have adequate controls perhaps is more

15 important here than anywhere else one can imagine

16 given the severity of the illness that we are

17 dealing with and the kinds of outcomes that Dr.

18 Perin has described.

19 As someone who recruits for device trials,

20 though, I can tell you that it is not easy, and

21 randomizing patients to acceptable controls in this

22 kind of illness is going to be a huge challenge,

23 but I think it is important for this group to

24 emphasize that there is no place where this could

25 be more important, otherwise, we will never get an

83

1 answer, and I think that mortality ultimately will

2 have to be a critical part of any trial that is

3 done.

4 DR. RAO: Go ahead, Dr. Borer.

5 DR. BORER: I agree completely with Jeremy

6 that controls are essential in this kind of

7 research and really in any clinical research, but I

8 think again to put this whole area in context, and

9 in response to Dr. Neylan\'s point and question, we

10 are at the point now of looking at physiological

11 variables and what we would call in drug

12 development \"surrogates,\" to see whether cardiac

13 performance, cardiac perfusion, this, that, and the

14 other thing, is affected in one way or another, so

15 that one could extrapolate to the point where it

16 would be legitimate to define hypotheses about

17 clinical outcome.

18 We are not there yet, and the clinical

19 outcome, just to put it in context from the drug

20 world, is perfectly legitimate in the view of most

21 people who deal with this area and these agents to

22 think of a therapy as being approvable if it makes

23 people feel better, but doesn\'t make them live

24 longer.

25 If it makes people feel better, even if it

84

1 makes them live a little bit shorter, as long as

2 you know how much shorter that is, and if it makes

3 people live longer while not making them feel too

4 much worse.

5 I don\'t think we are at a point yet again

6 to define what the outcomes variables should be. I

7 think we are at the point of defining physiological

8 and pathophysiological surrogates, and that is what

9 is being done in the studies to date, and then we

10 can decide what the outcomes are, clinically

11 important for registration.

12 DR. RAO: I guess that leads us to the

13 fact that many of these things should be discussed

14 tomorrow, just like you pointed out.

15 If there are no critical questions

16 remaining, I will thank Dr. Perin.

17 \[Applause.\]

18 DR. RAO: We are going to take a short

19 break.

20 \[Break.\]

21 DR. RAO: We are really extremely

22 fortunate in having Dr. Menasch here to present

23 his findings, and I look forward to a really

24 interesting talk.

25 Clinical Experience of Autologous

85

1 Myoblast Transplantation

2 DR. MENASCHE: Good morning. First of

3 all, I would like really to thank you for the

4 privilege of this invitation and this unique

5 opportunity of sharing some data on the clinical

6 myoblast transplantation.

7 What I would like to do in this talk is

8 first to briefly touch on the preclinical data

9 which have paved the way for these early clinical

10 trials, and then, as requested by Dr. Grant, to

11 focus on the various aspects of the clinical

12 experience which has accumulated so far before

13 drawing some perspectives which may have clinical

14 relevance in the near future.

15 Now, I think just to make things clear,

16 that the basic assumption is fairly

17 straightforward, and the objective of this therapy

18 is really to try to repopulate areas of dead

19 myocardium with new contractile cells with the hope

20 that these areas can regain some function, and

21 given the close relationship between function and

22 survival, which has been already mentioned, the

23 ultimate hope is obviously that it can have a

24 significant impact on clinical outcomes.

25 The reason why we initially started with

86

1 the skeletal myoblasts are actually listed here.

2 These cells are not really stem cells, they are

3 better termed precursor cells for muscular fibers

4 in that they are very committed to their skeletal

5 muscle phenotype as you will see.

6 The first advantage of the myoblasts is

7 that they can be very easily retrieved from the

8 patient himself, thus overcoming any problem

9 associated with rejection and immunosuppressive

10 therapies.

11 These cells feature a very great expansion

12 potential which is important given the relationship

13 which exists between the number of cells which are

14 injected and the ultimate functional outcome.

15 As I have just said, they are pretty well

16 committed to their myogenic lineage, and the risk

17 of tumor development is virtually negligible.

18 Finally, they are pretty resistant to ischemia, and

19 although unfortunately, many of them die shortly

20 after the injections, fortunately, some of them

21 will survive and may positively affect function.

22 So, this is type of animal model which has

23 been used initially in rodents. You see here the

24 heart and the needle injecting the cells. I just

25 would like to mention that it took us seven years,

87

1 seven years of preclinical work before I did

2 operate on the first patient June 15, 2000.

3 During the seven years, we moved from the

4 rodent models to the large animal models, which I

5 think is absolutely necessary before arriving to

6 clinical trials.

7 Just to summarize the bulk of this data,

8 we can say, number one, that when you inject

9 skeletal myoblasts into an infarcted area, they

10 retain the possibility of differentiating into

11 typical myotubes. Here is a typical myotube,

12 elongated structure, and this is a sheep heart and

13 this is a human heart.

14 This is an autopsy specimen. One patient

15 of our Phase I trial died 18 months after his

16 surgery from stroke, and we had permission for the

17 autopsy. You will appreciate the striking

18 similarity of these two slides. Here you find in

19 this human heart, a typical myotube embedded in

20 scar tissue.

21 At closer magnification, you can

22 appreciate the typical cross-striations, and I

23 think two observations are important to be made at

24 this point. Number one, these cells really remain

25 committed to their skeletal muscle phenotype. In

88

1 other words, there is virtually no evidence that

2 they can ever turn to cardiomyocytes. They will

3 not become cardiac cells.

4 Number two, they remain electrically

5 insulated from the surrounding myocardium, which

6 obviously raises major mechanistic questions

7 regarding the underlying mechanisms by which they

8 can improve function, but the fact is that there is

9 no real evidence that they develop connections with

10 the neighboring cardiomyocytes.

11 Nevertheless, when you subject them to

12 strong depolarizing currents, they show excitable

13 properties, and you see here, this is a fluorescent

14 myotube which has been grafted in a myocardial

15 scar. This is an in vivo study and definitely they

16 can respond to currents by generating action

17 potentials followed by contractions.

18 This translates into an improvement in

19 function, both regional function here in the sheep

20 model, and global function, the LV ejection

21 fraction. This improvement, as you can see, seems

22 to be sustained over time until one year in our rat

23 studies, and basically, these kinds of observations

24 have been made by several other investigators

25 already past 10 years.

89

1 So, there is a fairly good consistency

2 showing that these myoblasts can, to some extent,

3 improve function at least in animal models, and

4 obviously, the gap with the humans is a wide one.

5 So, if we now move to the clinical

6 experience, so far there are 44 patients who have

7 been included in early Phase I trials, and 34

8 patients currently included in our ongoing

9 randomized, multi-centered Phase II study .

10 This list is by far not exhaustive. I

11 have not tabulated anecdotal case or me-too cases.

12 I have just kept those studies which have been

13 published in peer-reviewed journals.

14 Basically, the inclusion criteria have

15 been fairly straightforward across all these

16 studies. Patients with low ejection fractions,

17 usually below 35 percent, patients with a history

18 of myocardial infarct, and obviously, patients

19 requiring concomitant coronary bypass surgery since

20 for ethical reasons, it is difficult to open the

21 chest just for injecting a product we don\'t really

22 know whether it is effective or not.

23 If we try to summarize the main results,

24 we can say, number one, that multiple epicardial

25 injections look to be safe. I have never seen any

90

1 bleeding from the needle holes, and overall, this

2 experience has been shared by the other surgeons

3 who have practiced the operation.

4 Number two, it is possible\--and we will

5 come back on that\--that the procedure increases the

6 risk of arrhythmia postoperatively, at least in the

7 early post-op period.

8 Number three, I will be extremely careful

9 and cautious about that, there are some data

10 suggesting that maybe function can improve, but it

11 is clear that until we have the results of the

12 ongoing randomized, placebo-controlled study, we

13 cannot make any meaningful conclusion.

14 This is the list of the studies and of the

15 patients. I have just added the last one a few

16 days ago. Professor Siminiak presented at the

17 American College of Cardiology another series of 10

18 patients who got the cells through a percutaneous

19 catheter using the coronary sinus route. I will

20 come back on that catheter in a few minutes, but I

21 will rather concentrate on the surgical

22 implantations listed here.

23 Dr. Smits also injected cells through a

24 catheter using the interventricular approach

25 similar to the one alluded to by Dr. Perin.

91

1 This goes back to the inclusion criteria

2 which have previously been mentioned. I think it

3 is important to look at all words, because as you

4 will see, differences in definition may really be

5 confounders in the interpretation of the results.

6 It is important to look at akinetic areas

7 that is really dead myocardium, not simply

8 ipokinetic or dyskinetic, really akinetic

9 myocardium, which are not amenable to

10 revascularization and obviously, it is also

11 important that the bypass surgery be done in other

12 areas.

13 For example, you will see that in one

14 study, the area which was transplanted with cells

15 was also revascularized, so when the authors

16 conclude that cell therapy improves function, it is

17 clearly meaningless since the same area has got

18 simultaneous revascularization.

19 For those of you who are not familiar with

20 the procedure, I just would like briefly to show

21 you this three-step operation. It starts with a

22 muscular biopsy. We take it at the thigh. It is a

23 very simple procedure under local anesthesia.

24 We remove a chunk of muscle, which is then

25 cut into small pieces, put in this sheeping medium

92

1 and sent to the cell culture lab where a multiple

2 tri-cell factory is being designed to allow for

3 large-scale cell production.

4 Then, there are regular morphological

5 controls. Obviously, the key point is to inject the

6 cells before they reach confluence. What you would

7 like to do is that confluence occurs in vivo

8 following the engraftment, not before, so it is

9 important to check the morphological state of the

10 cells on a regular basis.

11 This is how human myoblasts look like

12 during the cell culture process, and this is how

13 the cells look like when they are back in the

14 operating room.

15 Then, with the curved needle, we inject

16 the cells all across the infarcted area including

17 the borders. It\'s a time-consuming, I would say

18 10, 12, 15 minute procedure, rather tedious and

19 boring procedure, by the way, where you have to

20 mentally construct the grids and then go with the

21 needle from side to side, so we are working on the

22 multiple shot device, but it is more tricky than we

23 initially thought.

24 So, right now we have the requirement for

25 these multiple injections all across. This is

93

1 another view of the injections.

2 So, if we start by feasibility, I think it

3 is quite well established that this technique is

4 perfectly feasible. In other words, it does

5 demonstrate that provided you have the appropriate

6 techniques, you can take a small piece of muscle

7 which contains, say, 3- 4 million skeletal

8 myoblasts initially and expand it over two to three

9 weeks until approximately 1 billion cells.

10 These are the results of our cultures

11 during the Phase I trial, during which the target

12 numbers which have been prespecified have

13 consistently been obtained and even overshoot it.

14 You will note that you can get up to 90

15 percent of skeletal myoblasts in that\--and this is

16 an important point\--you really end up with a pretty

17 well defined cell therapy product. You really know

18 what you are injecting.

19 Importantly, what we have seen is that

20 heart failure does not prevent skeletal myoblasts

21 to differentiate into myotubes, and this was a

22 question because when we did preclinical rounds, I

23 got pieces of tissue from orthopedic colleagues,

24 but often these patient were young, and the

25 question was are the myoblasts from this Class

94

1 III/IV heart failure patients going to

2 differentiate normally, and the answer is yes, so

3 far we have had no failure.

4 The only thing is that it may take a

5 little bit more time for some patients until we get

6 the target number of cells, but at the end of the

7 day, it has always been possible to achieve the

8 prespecified target number of cells in myoblasts.

9 What about safety now? These are the

10 different adverse events we were concerned with by

11 the time we started the trial, and fortunately, I

12 must say that none of them has occurred except\--and

13 we are going to discuss that\--possibly the

14 arrhythmias, but it is important to emphasize that,

15 for example, there was never any particular

16 bleeding from these multiple puncture sites.

17 There was no unusual complication in the

18 postoperative course of these patients, and when

19 the cells were injected in newt immunocompromised

20 mice, there was never any evidence for tumor

21 formation.

22 Obviously, before we started the study, we

23 had to go through a lot of regulatory constraints,

24 indeed, what I did is to discuss with the French

25 FDA and ask them what was approved or not, and the

95

1 game was not so easy because as previously

2 mentioned, there was no precedent.

3 So, they told us, well, this is what you

4 are allowed to do. This is the kind of culture

5 medium, ancillary product additives which are

6 permitted for human use, so we immediately from the

7 onset designed our cell culture in accordance to

8 the prespecified instructions, and obviously, it

9 was timesaving because when we came back with the

10 process, there was nothing else than to accept it.

11 Well, what about the V-tachs? In the

12 initial series we had 4 patients with sustained

13 episodes of ventricular tachycardia.

14 All of them occurred during the early

15 post-op period, the early three first week,

16 postoperative weeks, and there was virtually no

17 recurrence later on because these patients had a

18 defibrillator put on and only one of them

19 experienced firing of the defibrillator one year

20 later, so it really appears to be a relatively

21 early post-op event.

22 Now, there are different mechanisms which

23 could account for these arrhythmias, in particular,

24 the differences in electrical membrane properties

25 between the grafted cells and the neighboring

96

1 cardiomyocytes. Obviously, other mechanisms can

2 also be considered, but we really favor the first

3 one because we did an EP study in which we looked

4 at the different membrane properties of the cells.

5 Here, you see a typical action potential

6 of a muscular fiber and here of a cardiomyocyte.

7 Now, if you graft skeletal myoblasts back into a

8 muscle, these cells retain a typical skeletal

9 muscle phenotype, and this is also true for

10 myotubes which grow in culture.

11 The question is how does it look like when

12 you graft the skeletal myoblasts into the heart.

13 Well, definitely it remains very similar to what it

14 was initially and different from the action

15 potential of the cardiomyocyte.

16 If you expressed it graphically, you would

17 see that the action potential duration is quite

18 different between the cardiomyocyte and the

19 myotube, and this heterogeneity might account for

20 some of these arrhythmias.

21 Now, having said that, the picture is

22 probably more complex and the reason, as you know,

23 and it has been mentioned by Dr. Perin in his talk,

24 is that heart failure by itself predisposes

25 patients to arrhythmias.

97

1 So, I think that as long as we don\'t have

2 the results of the randomized trial in which all

3 patients have been instrumented with a

4 defibrillator, it will be difficult to conclusively

5 establish a causal relationship between grafting of

6 cells and the occurrence of arrhythmia.

7 I can also tell you that we currently have

8 randomized 34 patients in the Phase II trial and

9 the incidence of arrhythmia has been strikingly

10 low, much lower than in the initial study we had

11 done, so things are probably less clear than they

12 were initially, and once again we have to wait for

13 the results of the randomized trial before we can

14 definitely say yes, there is no relationship

15 between myoblast transplantation and arrhythmia.

16 Anyway, these patients or most of them

17 would require at one point a defibrillator, so it

18 was not a big issue for us to implant those

19 defibrillators in all the Phase II patients.

20 Now, what about efficacy? Now, we have to

21 be extremely careful in the interpretation of the

22 results which are presented because of the

23 multiplicity of the confounding factors.

24 The culture conditions, for example, the

25 Spanish group has used a culture medium which

98

1 contains the patient\'s own serum, and the

2 conclusion is we had no arrhythmia, so if you use

3 the patient\'s own serum instead of fetal calf

4 serum, you prevent arrhythmia.

5 I think it is really a simplistic

6 conclusion based on 12 patients, but it can

7 introduce an additional bias. There is currently

8 no evidence that fetal calf serum is really

9 responsible for the arrhythmias.

10 Dosing has been extremely different and

11 variable from one study to the other, as well as

12 the kinetics of the grafted area.

13 Once again, any kinetic area is different

14 from a dyskinetic area, which features a

15 paradoxical motion, and, for example, in the U.S.

16 trial, some patients were included who had

17 hypokinesia, which we know can improve just because

18 of the revascularization even if revascularization

19 is not targeted at this particular area.

20 The same for bypasses. In the Spanish

21 study, for example, the cell grafted areas were

22 also bypassed, which makes the interpretation of

23 results impossible.

24 Type of surgery has also been different.

25 In the U.S. study, for example, some patients had

99

1 additional reconstructions of the left ventricle in

2 addition to the bypass surgery, which make things

3 still more complicated.

4 Finally, the method of outcome assessment,

5 in some studies, the assessment has been

6 centralized at one side, in others, each center has

7 made its own assessment, which obviously makes big

8 differences.

9 This is just to illustrate the variability

10 in the number of cells which have been injected. I

11 don\'t have the figures for the initial surgical

12 study from Professor Siminiak, but as you can see,

13 there is a wide variability.

14 The U.S. study of Dr. Dib was, as you

15 know, was a dose escalating study accounting for

16 this variability in the numbers. Dosing is

17 probably important. This is one study among others

18 showing that there seems to be a tight relationship

19 between the number of injected cells and the

20 functional outcomes.

21 This is the reason why, in our early Phase

22 I trial, we have targeted a high number of cells,

23 800 million. In the Phase II, we have two arms with

24 two different doses of cells, but the number

25 probably makes a big difference given the high rate

100

1 of early cell death.

2 The characteristics of the grafted

3 segments, as I previously mentioned, have also been

4 different from one study to the other, as well as

5 the method for assessing viability, usually,

6 dobutamine echocardiography, occasionally MRI or

7 PET scan.

8 Same variability in the characteristics

9 of injections, but you see that you can go up to

10 almost 60 injections without any concern related to

11 bleeding, and obviously, the number of injections

12 depends on the extent of the area of infarction.

13 It is also important to look at the cell

14 concentration. We extensively studied that before

15 I started doing patients. You have to find a

16 tradeoff because if you use a large needle, then,

17 you can have large holes and some bleeding

18 problems.

19 If you use a too small needle, you will

20 eliminate the bleeding problems, but the cells may

21 be packed and damaged through their passage, so we

22 ended with a 27-gauge needle which gave an

23 acceptable rate of cell viability.

24 The concentration of cells is important,

25 and probably still more important when you are

101

1 using a long catheter. We are using a short needle

2 with directly the serum hooked to the needle, but

3 if you are using a long catheter, concentration may

4 make a big difference.

5 Finally, revascularization is occasionally

6 being done in the same area as the area where cells

7 were put in, which completely confuses the results.

8 This is, for example, the Spanish study,

9 what you see is that, what they call the untreated

10 segments, that it is segments which had just

11 bypassed, the wall motion score went from 1.2 to

12 1.1 and 1, but really, this is almost normal

13 motion, so obviously, it makes it easier to

14 demonstrate that in the other segments which have

15 bypass surgery and cells, the improvement was

16 greater.

17 This is a summary of our data from the

18 Phase I trial. We had an improvement in the

19 functional status and an increase in ejection

20 fraction. These results are meaningless because

21 these patients had associated bypass surgery.

22 So, we rather looked at the number of

23 scarred segments, and I remind you these were

24 akinetic segments without viability on dobutaminic

25 echocardiography without any possibility for

102

1 revascularization. So, we looked at the changes in

2 the contractions of these segments which have been

3 grafted with cells.

4 So, initially, obviously, there was no

5 motion since it was one of the inclusion criteria,

6 and afterwards we had, at two different time

7 points, approximately 60 percent of segments

8 regaining some function.

9 I am not saying that these segments were

10 normally contracting, they were not. There was a

11 slight and modest improvement. This was a blinded

12 assessment, in other words, we blinded the dates of

13 the echo tapes and asked independent

14 echocardiographers to review them and to grade

15 them. There was a modest improvement, not normal

16 contraction, but it was sufficient to push us to

17 move forward to the Phase II study.

18 I just show you a couple of examples.

19 This is a flat exterior wall, no motion at all, and

20 this is the same wall with the systolic thickening

21 following myoblast transplantation. This is the

22 MRI study which does not project on the screen. I

23 have it on the computer, but not on the screen.

24 You see here the interior infarct which

25 has been grafted, and you can appreciate an

103

1 improvement in wall motion in the postoperative

2 period. This is an exterior infarct. You see the

3 thin wall here, which has been grafted, and this is

4 the post-op pattern with a thickening of the wall.

5 I add intentionally that these patients

6 also had bypasses in the left system. I don\'t like

7 the slides where you see pre-transplantation,

8 post-transplantation, just omitting that in

9 addition, there was either bypass surgery or

10 balloon angioplasty.

11 This is another example of an interior

12 infarct pre-transplantation and bypass to the

13 posterior descending coronary artery and the

14 post-op, with an improvement in the wall motion.

15 So, now, can it be due to the

16 revascularization of the PDA? It is unlikely, but

17 it cannot be eliminated.

18 So, basically, this is the design of the

19 MAGIC, the Phase II trial which has been initiated

20 now in Europe, in different countries in Europe.

21 It is targeted to include 300 patients in different

22 countries, and to emphasize what Dr. Ruskin was

23 mentioning earlier, it is a placebo-controlled

24 study. In other words, patients following

25 randomization have a muscular biopsy and they have

104

1 eventually injection of a placebo solution in

2 addition to their bypass surgery.

3 There are three arms, one control and two

4 treated groups, one having 400 million, the other

5 having 800 million cells. The production of cells,

6 and this is probably important, has been

7 centralized in two sites, one in Paris and one in

8 Boston, and it is exactly the same technology which

9 is used in the two sites.

10 The primary endpoint is the improvement in

11 the contractility of the segments which have been

12 grafted with cells in the core lab and in a blinded

13 fashion. In addition to that, we are obviously

14 looking at major adverse cardiovascular events at

15 the one-year follow-up time.

16 I would like to move on now before

17 finishing to some clinically relevant perspectives

18 which may have really clinical implications in the

19 near future.

20 First of all, so far we have been talking

21 primarily of ischemia cardiomyopathy, but as

22 mentioned by Dr. Perin, there are other causes of

23 heart failure in particular non-ischemic, globally

24 dilated cardiomyopathy.

25 So, we have been interested in assessing

105

1 myoblast transplantation in this particular

2 context, and use a particular genetic strain of

3 hamsters which develop a non-ischemic dilated

4 cardiomyopathy, and randomize the animals to

5 receive either autologous skeletal myoblasts,

6 because phenotypically, these myoblasts are free

7 from the disease, or culture medium.

8 To make a long story short, you see that

9 there is a definite improvement in function which

10 correlates with a major engraftment of cells in

11 this non-ischemic myocardium. I think it just

12 brings another piece of evidence that maybe

13 something good is occurring.

14 The second problem is cell death.

15 Regardless of the cell type, cell death is

16 extremely high, 80, 90 percent of cells are dying

17 shortly after the injections for a variety of

18 causes, in particular, apoptosis, but also

19 ischemia. It makes sense since we are injecting

20 cells in scar areas which receive very little

21 vascularization. So, even if myoblasts are fairly

22 resistant, they die nevertheless.

23 So, now there are several studies

24 suggesting that the co-induction of angiogenesis

25 may be an effective means of improving survival of

106

1 the cells, and ultimately, of improving function of

2 the animals.

3 This is a study comparing transplantation

4 of fetal cardiomyocytes, injection of fibroblast

5 growth factor, or a combination of both. As you

6 can see, function is improved when you combine the

7 two therapies.

8 Recently, we have duplicated this study

9 except that we used myoblasts and another growth

10 factor, and we found exactly similar results.

11 So, there are different ways of inducing

12 angiogenesis, and I know Dr. Epstein is going to

13 discuss that, but the point I wanted to make, this

14 is, you know, the difference in cell survival

15 between myoblasts alone and myoblasts plus an

16 angiogenic growth factor.

17 The point I would like to make is that

18 probably in the future, you will have to deal with

19 proposal of studies trying to combine cell

20 transplantation with some form of angiogenesis just

21 to optimize cell survival and potentiate the

22 benefits of the intervention.

23 A third point regards cycling. This is

24 the muscular biopsy of the patient who died. I

25 previously talked about this patient who died from

107

1 a stroke. Initially, in this biopsy, and this is

2 not unexpected, you find fast skeletal myosin and

3 slow type myosin. You don\'t find fibers, virtually

4 no fibers co-expressing fast and slow.

5 When we looked at the heart of this dead

6 patient, we found approximately 30 percent of cells

7 co-expressing fast and slow myosin, which means

8 that although once again these myotubes remain

9 myotubes and do not turn to cardiac cells, it seems

10 that some of them may incur some phenotypic changes

11 in response to their new myocardial environment and

12 start expressing slow myosin, which as you know is

13 a fatigue-resistant myosin.

14 So, this is important and should be put in

15 parallel with this study showing that if you

16 co-culture myoblasts in cardiac cells,

17 cardiomyocytes, this is the green myoblast, this is

18 an antibody against a cardiac troponin and against

19 another cardiac marker, some of the myoblasts, as

20 you can appreciate here, will express some cardiac

21 markers.

22 Now, what is shown here is that if you

23 stop the beating of the co-culture fetal cells,

24 there is no myoblasts which can acquire cardiac

25 cell characteristics. Conversely, if you subject

108

1 the preparation to a cyclic stretch, then, the

2 stretch makes some of these myoblasts able to

3 express the cardiac markers.

4 In other words, it is quite possible that

5 in vivo, the cyclic contraction of the neighboring

6 cardiomyocytes leads to the expression of some

7 cardiac markers and leaves the slow myosin in the

8 grafted cells, and the practical implication could

9 be that maybe combining cell transplantation with

10 ventricular stimulation, resynchronization could

11 actually improve the extent by which the grafted

12 cells express slow myosin and become fatigue

13 resistant.

14 So, once again, because this is a

15 clinically used modality, biventricular

16 resynchronization, in the future, we may have to

17 deal with studies trying to combine ventricular

18 resynchronization with cell transplantation.

19 Finally, a few words about the routes of

20 delivery, I have talked about epicardial

21 injections, we are also looking at the scaffolds,

22 which are just put on top of the infarcted area.

23 We are currently working on polyurethane, as well

24 as collagen patches. Obviously, it is less

25 traumatic and maybe it could reduce a little bit

109

1 the extent of cell death. This is a pattern after

2 a couple of weeks.

3 Now, the catheters, I know that this issue

4 will be discussed this afternoon. I just want to

5 say that from a surgical perspective, I am amazed

6 by the fact that many clinical studies have been

7 initiated in spite of the fact that we had few data

8 on cell retention, functionality, cell viability is

9 not the only issue.

10 It is not because you see myoblasts, that

11 they are going to turn into myotubes. You really

12 have also to assess the functionality, the ability

13 for these cells to become myotubes, long-term

14 engraftment, as well as the possible interactions

15 between catheter materials and the cells.

16 Most of the studies published so far in

17 the preclinical setting have dealt with technical

18 feasibility rather than functional efficacy, and

19 the various routes have not really been compared.

20 Having said that, we are very interested

21 in the percutaneous routes, and in our group, we

22 have interventional cardiologists working in that.

23 I must say that we have been primarily interested

24 by the transvenous cell injection through the

25 coronary sinus, and this is the summary of the

110

1 study which was presented last week at the ACC,

2 which is a functional study that is a sheep model

3 of myocardial infarction in which we injected cells

4 through this catheter.

5 You can appreciate that it allows a real

6 delivery of the myoblasts, which turn into

7 myotubes, and this correlated with a significant

8 improvement in function. So, this is a not a

9 feasibility study, this is a true efficacy study,

10 which is encouraging at least with regard to this

11 particular catheter.

12 So, these are maybe the challenges of

13 the future, in the setting of bone marrow, the

14 famous MAPS, the mesenchymous adult report in

15 cells, which feature distant advantages, possible

16 disadvantages. We are currently working on the

17 cells, cardiac progenitors, and I am sure Michael

18 Schneider will have a lot of things to say about

19 that. Also, possible embryonic stem cells.

20 What is also important now is to compare

21 cells between them, and not exclusively with

22 controls. This is true, for example, with the bone

23 marrow.

24 I just would like to show you a recent

25 study that we have done comparing skeletal

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1 myoblasts, CD133 progenitors or culture medium in a

2 randomized study, and the result is that there is

3 virtually no difference between the CD133 and the

4 myoblasts in terms of function.

5 If you look at histology, it is easy to

6 find the myotube. It is co-expressed, you know,

7 specific markers like myosin heavy chain. It has

8 been extremely difficult to identify the CD133. We

9 have to rely on PCR to find some of them, which

10 means that probably very few are still present

11 after one month.

12 So, I think it is important to compare the

13 cells, and to some extent, given the amount of data

14 which have accumulated over years, skeletal

15 myoblasts may provide a sort of benchmark for

16 testing other cell types.

17 A similar study is being done in Doris

18 Taylor\'s lab showing basically that there was no

19 difference between skeletal myoblasts and

20 mesenchymous cells.

21 Once again, we are back to the question

22 which was raised by Michael Schneider. This is the

23 setting of chronic heart failure, and in this

24 particular setting, current evidence will rather

25 favor skeletal myoblasts.

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1 This is a completely different setting

2 from acute MI in which bone marrow cells seem to

3 generate impressive results, but they are different

4 patient populations, and it is quite possible that

5 the acute stage of the MI, the bone marrow cells

6 receive appropriate signals which allow them to

7 improve function. The setting may be quite

8 different where you are dealing with heart failure

9 patients and old scars for which apparently,

10 skeletal myoblasts look more suitable for improving

11 function.

12 So, I just would like to close by two

13 general slides summarizing a little bit what we

14 have learned from our 10-year experience in the

15 field.

16 Regarding preclinical issues, it is clear

17 that screening experiments have to be done in

18 rodents, but I think it is critically important to

19 validate that in large animal models before

20 arriving to clinical trials.

21 We have a good example of that with the

22 combination of bone marrow cells and JCSF. You are

23 aware of the initial study by Orlig\'s group showing

24 a regeneration of mouse myocardium by combining

25 JCSF and bone marrow cell transplantation.

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1 This mouse study could now be duplicated

2 by two independent groups including Orlig\'s group

3 in primates, and then we have the Lancet paper last

4 week showing that there was a higher rate of

5 restenosis in patients receiving these two

6 therapies.

7 So, this jump from the mouse to the man

8 without an intervening large animal model seems to

9 be maybe a little questionable.

10 It is also important that this preclinical

11 study be designed just like clinical studies with

12 appropriate controls and blinded assessment, but

13 having said that, we must be aware that all these

14 models have serious limitations at what point we

15 are not able really to model the very complex

16 situation of heart failure patients with a

17 long-standing coronary artery disease. A good

18 example is that in our preclinical work, we have

19 never seen any arrhythmia in any of the animals.

20 Regarding clinical issues, it is important

21 to have a well characterized cell therapy product.

22 I am not sure that once the feasibility has been

23 demonstrated in small pilot trials, it is necessary

24 to multiply this 10-patient studies, because the

25 amount of information that can be collected from

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1 these small studies is indeed limited once

2 visibility has been established, and I think it is

3 rather important to move on to larger clinical

4 trials focusing in efficacy and safety, and

5 allowing to draw more meaningful conclusions,

6 safety, the arrhythmias with the myoblasts and

7 possibly instant restenosis with the bone marrow,

8 and efficacy obviously is left ventricular function

9 and major inverse cardiovascular events.

10 So, we are really now at very early stage,

11 as you know, in the field. We have some evidence

12 that the myoblasts, among others, may improve

13 function, but we still have a lot of basic

14 questions to answer, and in the meantime, I don\'t

15 think we can make any progress without the

16 implementation of well-designed clinical trials

17 more or less resembling those which have been

18 designed for drugs with appropriate controls,

19 randomization, blinded assessment, and so on,

20 because this is the only way really to know whether

21 hosts will be matched or not.

22 I would like to acknowledge obviously all

23 those who have participated in this endeavor with a

24 special thanks for those who really did the work.

25 Thank you very much.

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1 \[Applause.\]

2 DR. RAO: We will be open for questions.

3 Go ahead, Dr. Borer.

4 Q&A

5 DR. BORER: First of all, Dr. Menasch, I

6 have to tell you I think that was one of the most

7 exciting talks I have heard in a long time. That

8 was really wonderful.

9 I have some specific questions. I will

10 only give a couple of them, so that everybody else

11 can talk, and then maybe ask a few more later.

12 During your many years of preclinical

13 studies, I am sure you made efforts to determine

14 whether there were aspects of the preparation that

15 could increase the plasticity of the myoblasts, so

16 that they would manifest themselves more as

17 cardiomyocytes than as myocytes.

18 If you did, number one, did you find

19 anything that altered the character, that increased

20 plasticity, because if it did, that suggests that

21 maybe the current preparation isn\'t the end of the

22 line, maybe one could do better.

23 With that in mind, you mentioned that you

24 saw increased evidence of differentiation into

25 cardiomyocytes or more cardiomyocyte

116

1 characteristics in the beating setting.

2 So, I wonder\--I am sure you thought about

3 it\--but I wonder if you did culture any of the

4 cells, rather than on flat plates, on flexor cell

5 plates where periodic stress was applied in the

6 culture phase, so that you could perhaps generate

7 some of these cardiomyocyte characteristics before

8 injection.

9 That is one set of questions, and a second

10 question I would like to just put in here, because

11 it\'s a one-word answer, if these cells are

12 electrically isolated, as you mentioned, how is it

13 that they were caused to be in concert with the

14 rest of the heart?

15 DR. MENASCHE: Regarding your first

16 question, to be honest, we have not found any trick

17 during the cell culture process which could really

18 increase the transdifferentiation of these

19 myoblasts into cardiomyocytes, and really, I don\'t

20 think\--I am thinking of the works of Chuck Murray,

21 for example\--I think no one has really shown that

22 changes in the culture conditions could really make

23 them turn into cardiomyocytes.

24 The only evidence that can acquire some

25 cardiac-like characteristics is this expression of

117

1 slow type myosin. We are currently exploring the

2 possibility maybe of increasing the expression of

3 these slow myosin isoform by implantation

4 stimulation of the cells, but these are experiments

5 which now are going to be done, and this was the

6 reason why I was mentioning ventricular stimulation

7 as a potential additive in the future to the

8 clinical trials.

9 But to summarize, no, we have not found

10 any particular intervention, although maybe we have

11 not found the right one, which could increase the

12 proportion of cardiac-like skeletal myoblasts.

13 Now, the mechanisms, I don\'t know; from scratch, I

14 don\'t know.

15 There are different possibilities. One is

16 a limitation of remodeling. I am not sure if there

17 is a predominant mechanism because in our patients,

18 we have had some evidence of improved systolic

19 function, but we have never seen a reduction in

20 left ventricular diastolic dimensions.

21 Another possibility is that GAB junctions

22 are not the only ways for electrical impulses to

23 travel across the heart, and as you have seen on

24 the film, these cells retain excitable properties.

25 In order words, if you excite them, they will

118

1 contract.

2 So, it is not completely impossible that

3 in areas where physically, they are very close to

4 the neighboring cardiomyocytes, they may be

5 directly excited by electrotonic currents, and

6 there is a third hypothesis we are currently

7 exploring, and which is the paracrine hypothesis.

8 It is quite possible, and it has been

9 shown for bone marrow, for example, that these

10 cells secrete various growth factors or cytokines,

11 and so on, that can positively affect the function

12 of the host cardiomyocytes.

13 For example, in our studies we have found

14 that myoblasts and myotubes from patients secrete

15 very large amount of IGF-1, which has important

16 effects on tissue regeneration. So, maybe it has

17 nothing to do with their countertype properties,

18 but rather with the fact their behavior, small

19 factors releasing good factors for the heart.

20 So, we are currently playing with all

21 these hypotheses, but I don\'t have any definite

22 answer.

23 DR. RAO: So, it is pretty clear, like Dr.

24 Schneider pointed out earlier, that mechanism is an

25 issue that is still not clear.

119

1 DR. MENASCHE: No, it is not clear at all.

2 The only thing is I don\'t think that you could

3 infer from the lack of connexin 43 expression, that

4 improvement in function is not possible. I think

5 both should be dissociated.

6 DR. RAO: Dr. Kurtzberg.

7 DR. KURTZBERG: You mentioned studies with

8 bone marrow derived AC133 cells. I wonder what the

9 rationale behind the selection was and why you

10 thought there would be an advantage to using

11 selected cells over whole bone marrow.

12 DR. MENASCHE: The reason is that we first

13 did a large animal study with whole bone marrow in

14 the sheep model of myocardial infarction, once

15 again, a chronic infarct. So, we injected the

16 whole bone marrow and we didn\'t find anything, no

17 improvement in function, no limitation in

18 remodeling, no evidence for transdifferentiation of

19 cells.

20 So, we said, well, maybe the whole bone

21 marrow is not the appropriate medium for this

22 particular setting, let\'s try to purify the cells,

23 and we went to the CD133. The results were

24 slightly better in that. There was some

25 improvement in function compared with controls, but

120

1 the improvement was not greater than that we got

2 with the myoblast.

3 Currently, we are comparing now myoblasts

4 with AMAPCs, so we have tried to pick the different

5 populations, in a stepwise approach, test all of

6 them.

7 DR. RAO: Dr. Cannon.

8 DR. CANNON: Thank you for your talk, it

9 was most interesting. I am Richard Cannon from

10 NHLBI.

11 My question is in any of your preclinical

12 animal work, did you ever inject your myoblast

13 culture preparations or cell suspensions into the

14 circulation to see where they might end up and what

15 toxicity they might cause.

16 This may not be an issue with an

17 intra-operative injection into scar, but I would

18 imagine with a catheter-based approach, it is

19 conceivable that despite the operator\'s best

20 efforts, some of these cells might be injected into

21 systemic circulation.

22 Do you have any data on where the cells

23 end up, do they lodge in the brain or the kidneys,

24 do they cause any toxicity or injury to other

25 tissues?

121

1 DR. MENASCHE: Well, in the preclinical

2 studies we have done, we have not found evidence

3 for, first of all, all the injections were direct

4 intramyocardial injections, so it may be difficult

5 to find them in the brain or in the liver.

6 We have not found them disseminated

7 throughout the body, but I must say that maybe if

8 we had done more extensive studies, autopsy studies

9 of the brain or the lungs, or any other organ,

10 maybe we could have found some of them.

11 We have never injected intentionally the

12 cells intravenously just to see what was happening.

13 I don\'t believe it is a real issue because even

14 when you are injecting them intraoperatively in

15 humans, it is clear that some of them are escaping

16 through the lymphatic system or in the venous

17 system, and so far we have never seen any evidence

18 for unexpected or unusual complications.

19 But I agree with you that if you are

20 expecting some leakage of the cells in the systemic

21 circulation, this is probably a point that should

22 be addressed more extensively than we have done.

23 DR. RAO: I have a practical question.

24 Did you, when you looked at the cells, ever look at

25 BRD incorporation to see whether cells continue to

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1 divide at any time?

2 DR. MENASCHE: Yes, absolutely, including

3 in the human trial. In the human trial, we always

4 keep aliquot, initially, we kept aliquots of cells

5 and just let them grow, and this is why we have

6 been able to show that these cells were

7 differentiating into myotubes including in heart

8 failure patients.

9 DR. RAO: Did you ever take your samples,

10 and look at freeze/thaw? You know, you grow them

11 in cell culture, can you freeze these cells and do

12 they behave the same way when you send them to

13 another site like you are planning in the Phase II

14 trial, for example?

15 DR. MENASCHE: Absolutely, we have done

16 that and we have validated that after thawing, they

17 retain their ability to differentiate into

18 myotubes.

19 DR. RAO: Have they been done in any

20 transplant paradigm, or has it only been done by

21 looking at they are forming myotubes in culture?

22 DR. MENASCHE: Both. We have several

23 preclinical studies in rats and in sheep, in which

24 we have used cryopreserved and thawed cells with

25 apparently functional outcomes similar to those we

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1 had with fresh primary myoblasts.

2 This is the reason why actually we got

3 permission to freeze them should they become

4 necessary for logistical reasons in the Phase II.

5 DR. BORER: You mentioned that about 95

6 percent of the cells that you inject have CD56

7 characteristics. That suggests that there is some

8 alteration in some of the cells or perhaps a

9 different cell line is growing in parallel, in the

10 cultures that you are using, so I wonder, number

11 one, how many passages do you use before

12 administering the cells, and, number two, is the

13 reproduction error rate increased with passage in

14 any meaningful way, and does it make any

15 difference?

16 Obviously, a lot of these cells that you

17 inject are nonviable. When you inject them, they

18 don\'t survive. I don\'t know which ones are

19 surviving and which ones aren\'t. But it seems to me

20 that the number of passages employed may affect the

21 ultimate outcome of the injection, and I wonder if

22 you have some data on that from your preclinical

23 work.

24 DR. MENASCHE: We used three to four

25 passages, but it has been shown by Chuck Murray

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1 that if you multiply passaging, you may end up with

2 a population of differentiation of effective cells,

3 in which case you might end up with some unexpected

4 overgrowth without any functional benefit.

5 So, it is probably important not to

6 multiply passaging too much. With three to four

7 passages, we have been able to reach the target

8 numbers of cells, 400- or 800 million of cells. We

9 don\'t go beyond that.

10 DR. TSIATIS: In your randomized clinical

11 trial, do you actually have formal stopping rules

12 for either safety or efficacy, and, if so, what are

13 they, or what is the general philosophy for

14 monitoring?

15 DR. MENASCHE: It is primarily based on

16 the judgments of the DMSB given the type of

17 surgical population we are dealing with. It is

18 difficult to have stopping rules, just as you can

19 have with drugs, for example, for each adverse

20 event is reviewed by the DSMB, and based on that,

21 they would decide whether the study has to be

22 stopped or not.

23 DR. RAO: Dr. Neylan.

24 DR. NEYLAN: Thank you.

25 I have another preclinical question. I

125

1 was wondering if you had an opportunity to compare

2 the morphology and functionality of the myoblasts

3 when these are injected either into the akinetic

4 areas or perhaps into an area resected, and thus

5 undergoing a normal reparative milieu, and whether

6 perhaps under that milieu, there might be a

7 different behavior of these cells or expression.

8 DR. MENASCHE: Really, basically, our

9 model has been the model of, you know, coronary

10 ligation creating myocardial infarction, so you

11 really end up with an akinetic scar. I cannot

12 answer this question.

13 DR. NEYLAN: You never had the chance to

14 maybe resect that, maybe adhere to the natural

15 surgical tendency of cutting things out.

16 DR. MENASCHE: I try to refrain from that.

17 I have cardiologists as bodyguards, so it would

18 just refrain you from doing that.

19 DR. MULE: You had mentioned that the vast

20 majority of cells that are injected will die, and

21 clearly, there is room for improvement with perhaps

22 increasing angiogenesis, and so forth.

23 About the kinetics of myotube formation in

24 the ischemic areas, is it a dynamic process, in

25 other words, once you inject the cells, myotubes

126

1 will form over a given period of time, and then no

2 more tubes will form, or additional tubes are

3 generated over a prolonged period of time, and do

4 those tubes, when they are formed, remain viable

5 for the extension of the observation period?

6 DR. MENASCHE: It is difficult to tell

7 you. The kinetic studies indicate that although a

8 substantial number of cells die, the remaining ones

9 obviously proliferate in different shape over a

10 period which seems to extend, say, two to three

11 weeks.

12 At least in patients when we have seen

13 improvements, we have never seen improvements

14 before one month, and in animals, it is difficult

15 to see any improvement before two weeks.

16 Now, afterwards, the longest follow-up we

17 have is 14 months in animals, but we cannot know

18 whether the myotubes that we found at the end of

19 the experiments were present since the beginning or

20 whether they have been continuously regenerating.

21 The interesting observation, however, I

22 don\'t know whether it really answers your question,

23 is that these myotubes harbor new myoblasts, so

24 when you look at them with electron microscopy, you

25 clearly see, on their basal lamina of these

127

1 myotubes, newly formed myoblast cells, so they are

2 able to regenerate their own pool of precursor

3 cells.

4 Now, whether these cells participate in

5 the formation of new myotubes, I don\'t know.

6 DR. RAO: The last comment, Dr. Noguchi,

7 and then we move on.

8 DR. NOGUCHI: I am sorry to have prolonged

9 this, but it is just fascinating. Of course, FDA

10 always loves these controls, but to follow up on

11 Dr. Mul\'s question, is it a question of liability,

12 do the cells have to be alive, or you have a

13 myotube has some structure, and then I just recall

14 there is, in tumor biology, an old effect called

15 the reverse effect where if you have a few viable

16 cells with a lot of dead cells, you can actually

17 get tumors developing from one cell where normally,

18 you might need a million or 10 million.

19 I am just wondering if you have done any

20 mixtures of dead and live cells to really see how

21 much is viability, how much is surrounding stuff.

22 DR. MENASCHE: No, we have not done that

23 intentionally. We have just completed a study in

24 which we have looked more carefully at the patterns

25 not only of cell death, but also of cell

128

1 proliferation.

2 So, we know that we have this mix of dead

3 cells and living cells, but we have not done an

4 intentional mixing of them to see whether there was

5 any tumor formation.

6 Regarding oncogenicity, we have learned a

7 lot from our colleagues working in the field of

8 dystrophic myopathies, and it really seems that

9 these cells have a very low tumor-retaining

10 potential.

11 In the newt mice in which we have injected

12 our human myoblasts, we have never seen any tumor

13 in spite of the fact that many of these cells

14 expectedly died.

15 DR. RAO: Thank you, Doctor.

16 We will move on to our next speaker, Dr.

17 Epstein.

18 Bone Marrow Cell Therapy for Angiogenesis:

19 Present and Future

20 DR. EPSTEIN: It is really an honor to

21 have been asked to speak to this very august group.

22 I wanted to emphasize because I really do

23 think it is important in this, an ever-growing

24 field to make sure that disclosure is presented,

25 and I have a number of potential conflicts of

129

1 interest, which I hope in no way influences what I

2 will be talking about to you for the next 20 or 30

3 minutes.

4 I will talking about bone marrow cells and

5 angiogenesis. I wanted to start out and make a

6 careful distinction. Dr. Schneider brought this up

7 in his earlier questions, but basically, what we

8 are considering today is really the use of bone

9 marrow cells, stem cells, progenitor cells for

10 myogenesis, but also for angiogenesis.

11 It is critically important to understand

12 that these are very distinct targets with

13 undoubtedly different mechanisms and certainly

14 very, very different issues, and therefore will

15 have a profound impact on how the FDA I think

16 judges whether or not a particular proposal is

17 meritorious.

18 For example\--and you have heard this very

19 eloquently discussed by Dr. Menasch\--for

20 myogenesis, the transdifferentiation of adult

21 progenitor cells or skeletal myoblasts is a

22 critically important issue. Maybe you don\'t need

23 transdifferentiation into cardiac myocytes to

24 improve myocardial contractility, but nonetheless,

25 it is a very important issue to consider.

130

1 Also, if you think about how many cells

2 are present in a large myocardial scar, the issue

3 of adequate numbers of cells to replace the scar to

4 cause a significant biologic effect has to be

5 considered, and you have heard a very eloquent

6 presentation and some demonstration relating to

7 this.

8 Now, the issues relating to angiogenesis

9 are, as I indicated a moment ago, different.

10 Transdifferentiation is really not an important

11 factor, because what has been recognized most

12 recently is that cytocrine secretion, exerting a

13 paracrine effect can induce proliferation and

14 remodeling of existing vessels.

15 So, it is not necessary, although it may

16 happen, it is not necessary for the cells that you

17 are injecting to turn into blood vessels. They

18 could induce the development of already existing

19 blood vessels, and the adequate number of cells

20 relating to angiogenesis is not nearly of similar

21 concern secondary to these paracrine effects that

22 have an amplifying activity of the individual cells

23 that are injected.

24 So, these are very different issues, each

25 are very important. I think the path to myogenesis

131

1 is going to be a longer one. I think that there

2 are a lot of problems that still have to be solved,

3 and I think the issue of angiogenesis, we have gone

4 along that path for probably a longer period of

5 time, and my sense is that we are closer to pivotal

6 clinical trials even when one considers cell

7 therapy, but I will be focusing my remarks on

8 angiogenesis.

9 The first thing I wanted to point out,

10 which is obvious to anyone who is involved in the

11 field now, is how complex the molecular and

12 cellular mechanisms are that are involved in

13 collateral formation.

14 This is a slide I always like to show.

15 This is a cartoon showing different genes

16 expressed, either increased expression or decreased

17 expression, four different times, different

18 amounts, two actually wind up with a collateral.

19 So, there are multiple, multiple genes

20 that are necessary to actually form a new

21 collateral vessel. Just to illustrate the

22 importance of interactions between different

23 angiogenic cytokines, I wanted to show you the

24 results of a study that we did a couple of years

25 ago using a rabbit ear.

132

1 So, here is the ear. It is supplied by

2 three major vessels. This is a laser doppler image

3 which is color coded for velocity. Red is highest

4 velocity, green is intermediate, and blue is low

5 velocity. If you tie off two of these three

6 vessels, you have a marked decrease in flow, and

7 the nice thing about the rabbit ear is that you

8 could observe this hourly if you wanted, and you

9 could with this laser doppler do repeated analyses

10 of the changing flow with time.

11 What you can also do is focus on a

12 particular area of interest and measure tissue

13 perfusion and the change in tissue perfusion that

14 occurs with time.

15 Here is the tying off of these vessels,

16 resulting in a profound decrease in flow, which

17 gradually recovers over several weeks, and in this

18 particular model, it is quite interesting. It

19 plateaus off below normal flow, so this is a model

20 of chronic hypoperfusion, which makes it kind of

21 interesting.

22 In this model, we looked at what happens

23 with endogenous VEGF levels, and VEGF is a key

24 angiogenic molecule, so we measured VEGF by western

25 blot before the induction of ischemia, and there is

133

1 essentially no VEGF present, however, if we measure

2 VEGF levels throughout the course of this, and even

3 at the end, there is a low level of VEGF present.

4 So, this is further indication that we are dealing

5 with a chronically ischemic preparation that has a

6 background of VEGF present.

7 The next issue that we wanted to document,

8 we took the model during the period of chronic

9 ischemia, and we take that now as our starting

10 point for this experiment, where we had an

11 angiopoietin-1 gene within an adenoviral vector, so

12 that is the transgene, which we inject

13 intradermally in the region of hypoperfusion in the

14 ear.

15 We inject it and we see over the course of

16 time, a major increase in collateral flow and

17 tissue perfusion, but remember there is background

18 VEGF present. If we coinject with the adenovirus

19 expressing angiopoietin-1, an inhibitor of VEGF,

20 and this is a soluble VEGF receptor, so it sops up

21 and inactivates whatever VEGF is present, it

22 obliterates the collateral-forming effects of

23 angiopoietin, so it just is an example of how you

24 need multiple factors to develop your collaterals.

25 If any one of these is perturbed, you

134

1 could seriously influence the course of collateral

2 development.

3 Now, just as background for the cell

4 therapy, there have been a number of adequately

5 powered, randomized studies that have been

6 performed using individual cytokines for

7 angiogenesis, and basically, these are either basic

8 FGF or VEGF used in the coronary circulation or the

9 peripheral vasculature, either the protein was

10 injected or a gene encoding the protein were

11 injected.

12 As of the moment, there have been no

13 definitive and robust beneficial results. There is

14 trends, there is some encouragement, there is some

15 early positive results, but nothing to really get

16 excited about.

17 Of course, as I indicated, all of these

18 randomized studies to date have involved a single

19 agent to promote collateral development, and it was

20 these considerations about four or five years ago

21 that provided the impetus for developing and

22 testing a second generation of angiogenesis

23 strategies, which is the use of cell therapy, which

24 had the potential to deliver multiple

25 collaterogenic cytokines.

135

1 I just wanted to show this slide. I am

2 not an expert at all in stem cells, progenitor

3 cells, but I just wanted to indicate to you what

4 has been used in clinical trials or in late-stage

5 preclinical trials. So, hematopoietic stem cells

6 characterized by positive CD34-133, which do

7 progress to endothelial progenitor cells and then

8 to endothelial cells, which have been shown to lead

9 to an increase in collateral flow.

10 Now, more recently, monocyte lineage cells

11 have been demonstrated. These are characterized by

12 the lack of CD34, but having CD14 and 45 MAC-1,

13 these monocyte lineage cells have been shown, not

14 to produce endothelial cells directly, but

15 nonetheless, are capable of inducing collateral

16 formation.

17 We have used freshly aspirated bone marrow

18 cells that have been filtered and directly

19 injected. These are autologous into pig ischemic

20 hearts, as well as patients. You have heard about

21 monocyte-derived bone marrow cells. Dr. Perin used

22 these in his study.

23 We have been also working now with

24 mesenchymal stem cells or stromal cells. There are

25 multiple terms that have been used to describe

136

1 these. These are CD34-negative, 45-negative cells,

2 and these have been shown to produce collaterals.

3 Now, I won\'t get into this in any detail,

4 but you should be aware of the fact that some of

5 these cells are believed to incorporate into

6 developing collaterals, with that being a major

7 mechanism by which they enhance the development of

8 collaterals, whereas, other interventions are not

9 believed to have that as a major mechanism, but the

10 major mechanism being the secretion of all sorts of

11 cytokines and growth factors that lead through a

12 paracrine effect to the development of either new

13 collaterals or the enhancement of existing

14 collaterals.

15 What I will be talking about, because all

16 of our recent work has been done with these

17 mesenchymal stem or stromal cells, I will be

18 talking about that for the next few minutes, and

19 these we refer to MSCs.

20 So, these MSCs, just to start or justify

21 our further studies, were cultured in vitro and

22 assayed. The conditioned medium was assayed, and

23 here is our control cells which produce small

24 amounts of VEGF MCP-1 and FGF, but the MSCs produce

25 really quite large amounts of these angiogenic

137

1 cytokines.

2 So, we were very excited about that,

3 thinking that they could be little factories that

4 might enhance collateral development. So, this is

5 the mouse hind limb model. This is laser doppler

6 imaging, as I showed you with the rabbit ear. Here

7 is the mouse\'s tail and the two hind limbs, and the

8 femoral artery is ligated at day zero, and this is

9 followed now every few days, and you can see there

10 is some return of function under control

11 conditions.

12 This is just injecting media that had not

13 been exposed to cells, and here is what we see with

14 media alone and with the control cell. This is

15 mature aortic endothelial cells.

16 But then when we inject into the hind limb

17 MSCs, we see a quite marked improvement in

18 perfusion, and this can be quantitated as shown in

19 this slide. So, this was a very exciting

20 demonstration for us, which was repeated multiple

21 times in different experiments.

22 Now, I won\'t belabor the number of studies

23 that have been done here. It is in your handout

24 that was distributed, I think it is page 20 and 22,

25 but I will just go over a couple of the highlights.

138

1 There have been 7 or probably 8 published

2 studies in either chronic ischemia\--and this is

3 angiogenesis studies, no myogenesis\--in chronic

4 ischemia or in acute myocardial infarction.

5 The points to be made are, number one, all

6 of these studies have shown safety, they have shown

7 feasibility, and they have all showed positive

8 trends, they have been encouraging, but as Dr.

9 Menasch very carefully pointed out with his own

10 myogenesis studies, when you are dealing with such

11 small numbers of patients, none of these studies

12 was randomized, double-blinded. There is no way

13 you could draw any conclusions regarding efficacy.

14 So, it is encouraging and it certainly

15 would indicate that additional studies are

16 necessary, but we can\'t make any inferences whether

17 the strategies that work in an animal model very

18 reproducibly necessarily work in humans.

19 Now, I want to point out, and I think we

20 have to be aware of this upfront, and any

21 investigator who is involved in the field has to be

22 aware of it, that there are potential problems with

23 any angiogenic strategy including cell-based

24 strategy.

25 For example, genetics. Here are some

139

1 beautiful studies done by Birgit Kantor in

2 collaboration with us. This is microscopic CT

3 imaging of two different strains of mice. This is

4 the femur, tibia, and this is the femoral artery,

5 and the femoral artery had been ligated, and you

6 can see that the C57 black 6 mouse has an

7 extraordinary capacity to develop collaterals,

8 however, about C. mouse, same ligation site, has a

9 paucity of collaterals. Well, clearly, the same

10 thing must relate to humans.

11 Another thing that was raised earlier is

12 the enormous variability amongst patients to

13 respond to angiogenic interventions. These are not

14 patients, these are mice, and this is the typical

15 experiment that I showed earlier, looking at laser

16 doppler perfusion.

17 The mouse has the femoral artery ligated,

18 and there is a gradual recovery of flow in young

19 mice, however, it you look at knockout mice that

20 have high cholesterol levels, their capacity to

21 develop collaterals is significantly impaired.

22 Now, if you take an old mouse\--these are

23 mice about 18 to 20 months of age\--they are really

24 having trouble developing collaterals, and then if

25 you take an old mouse who has high cholesterol

140

1 levels, they are really in bad shape.

2 Now, no one has demonstrated this

3 relationship in humans, but I am certain that it

4 occurs. So, there is going to be different

5 capacities of different individuals to develop

6 collaterals, and undoubtedly reflecting different

7 potential to respond to angiogenic interventions.

8 Now, here is something I would like Dr.

9 Schneider to look at, because he said this has not

10 been published before, but it has been published.

11 This is in I think JACC in 2002, but when we did

12 our clinical study, injecting autologous filtered,

13 freshly aspirated bone marrow cells into ischemic

14 myocardium of patients, we took an aliquot of these

15 cells and cultured them, and looked at VEGF

16 production, as well as other cytokine production,

17 and over the course of time, one sees an increase

18 in VEGF production, so these cells do have the

19 capacity to produce different angiogenic cytokines

20 including VEGF, but that is the mean data.

21 If you look at the individual data, there

22 is marked patient-to-patient variability in the

23 capacity to express VEGF, so here is a patient who

24 really has a great capacity to produce VEGF,

25 whereas, this is a patient who hardly can produce

141

1 VEGF at all, and it is not a great stretch to think

2 that this patient may not respond as vigorously to

3 cell therapy as the patient whose cells have a

4 great capacity to produce VEGF.

5 Now, we didn\'t look at enough patients to

6 be able to make such correlations, but I am sure

7 that this is an issue that has to be addressed, as

8 Dr. Schneider really pointed out before.

9 Now, let\'s look at the cells we are

10 injecting, and this was also raised earlier, so we

11 are looking here at a HIF-1\--I will just get into

12 that in a moment\--but it is a transcription factor

13 that is a key modulator of the cells response to

14 ischemia, so let\'s take it for the moment that it

15 is a key angiogenic factor, so this is a Western

16 showing HIF levels, and the first thing I want you

17 to concentrate on is under normoxic conditions,

18 young and old, HIF is not present, it is mostly

19 absent as a matter of fact, in the absence of

20 hypoxia.

21 Now, in the young mice, if you expose

22 these cells to hypoxia over 12 hours, there is a

23 major increase in HIF protein, and then that has

24 important compensatory effects on the cells\'

25 response to hypoxia, however, cells derived from

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1 old mice have a markedly impaired ability to form

2 HIF in response to hypoxia, so there are

3 age-related changes in the capacity of cells\--these

4 are MSCs\--to perform in a way that we would expect

5 them to if they were going to have a potent effect

6 on collaterals.

7 So, HIF is a master switch gene in the

8 presence of hypoxia, a heterodimer is formed,

9 HIF-1-alpha, and HIF-1-beta, which attaches to the

10 promotor of many genes and turns these genes on,

11 and amongst the genes are multiple genes related to

12 angiogenesis, VEGF, VEGF receptor, FGF, et cetera.

13 Now, just to show the biologic effects of

14 what I just showed you before, that is, the

15 inability to increase HIF protein in response to

16 hypoxia, here are young and old mice, and we are

17 looking at VEGF levels. These are cells, MSCs

18 growing in culture, and here is the intrinsic VEGF

19 production.

20 When we expose young cells to hypoxia,

21 there is a major increase in VEGF production,

22 mediated mainly by HIF-1 reduction, but old mice

23 not only have the lower levels of HIF-1, but they

24 have a lower target production of HIF-1, that is,

25 VEGF production.

143

1 So, these are real phenomenon I think that

2 we have to be aware of and begin to start thinking

3 about when we are dealing with any angiogenic

4 intervention, but certainly with the cell

5 therapies.

6 So, these considerations provided the

7 impetus to test another generation of angiogenic

8 strategies, and this relates again to one of the

9 earlier questions of the panelists, and we are very

10 much involved in genetic manipulation of these MSCs

11 to see if we could further enhance their ability to

12 secrete angiogenic cytokines and so to improve

13 collateral flow.

14 This is a construct of Genzyme. They have

15 been very helpful in working with us in this. In

16 the absence of severe hypoxia, these two dimers of

17 HIF, HIF-1-beta and alpha, there is no heterodimer

18 formed because HIF-1-alpha is rapidly degraded.

19 So, to overexpress HIF-1-alpha, so that

20 the heterodimer can form, we transfect these cells

21 with an adenoviral vector that has the HIF-1-alpha

22 transgene and that has a deletion insertion,

23 putting on a herpes sequence VP16, which stabilizes

24 the protein under normoxic conditions, so we are

25 able to overexpress HIF-1-alpha, the heterodimer

144

1 can be formed, and the genes can be transactivated.

2 So, we then looked at the capacity of this

3 intervention to cause these MSCs to secrete

4 angiogenic cytokines, so this is VEGF. The cells

5 now are exposed to just hypoxia, and you can see

6 there is about a doubling of the amount of VEGF

7 present, but when we transfect these cells with the

8 HIF-1-alpha, there is a huge increase in VEGF

9 production, and the same thing is true for

10 fibroblast growth factor.

11 So, this was really exciting to us because

12 we saw that we could genetically manipulate these

13 cells to make them at least in vitro more like a

14 better collateral enhancer.

15 We then went to our mouse ischemic hind

16 limb model to test this concept. Here are our

17 control cells, mature aortic endothelial cells.

18 Here are our non-transfected MSCs, and here are our

19 transfected MSCs. So, not only do we see an effect

20 in vitro, but we see what would have been predicted

21 from the in vitro effects in vivo.

22 I think that this is probably something

23 that we have to think about given the effects of

24 various risk factors on the ability of cells to

25 achieve their desired effects.

145

1 I did want to point out for the panel that

2 down the line, we are not only going to be talking

3 about cells, but cell-derived products in

4 cardiovascular therapy, and I will just spend a

5 moment on this, and mention the effects of

6 conditioned medium on collateral development.

7 As I showed you before, if you grow cells

8 in culture and allow them to produce whatever

9 goodies they are producing, and then you take the

10 media and you inject that media into the ischemic

11 hind limb of mice, you get\--well, that will be the

12 next slide\--but here is what I showed you before,

13 so these in the media contains more VEGF, more

14 MCP-1, more FGF, and multiple other gene products

15 that we haven\'t tested, but when we put this

16 conditioned medium into the ischemic hind limb of

17 the mouse, here is the control again, here is our

18 control.

19 Here is the injection of the MSC

20 conditioned medium. We see that the media alone

21 has the capacity to increase collaterals. So, this

22 undoubtedly is going to be something that you are

23 not going to see an application to this, I don\'t

24 think in the next few months, but in the next six

25 months or a year, I think that the cell products is

146

1 another very interesting way to use the angiogenic

2 potential that bone marrow cells have.

3 Just to show that this is biologically

4 important, we looked at the number of collateral

5 vessels the media increased, the number of

6 collateral vessels, the strength of the leg as an

7 ambulatory score, the media increased that, and

8 also the amount of atrophy that occurs in the calf

9 as a result of ischemia, and the media injection

10 decreases that, so it was a biologically relevant

11 intervention.

12 I just wanted to mention safety concerns,

13 and I was interested in Dr. Menasch\'s comment

14 about this. There are multiple well-known ones

15 that are usually tracked, and I won\'t get into

16 this, but I would just alert you to something that

17 is more theoretical than proven, but I think you

18 have to be aware of it and at least consider it

19 when you are considering the safety of angiogenic

20 interventions.

21 That is\--and this is a general rule that I

22 have come up with\--whatever induces angiogenesis,

23 induces atherogenesis, and I refer to this as the

24 Janus phenomenon. Janus was a Greek god with two

25 heads, so that when he looks out one way, he is

147

1 also looking at the other. That goodness can also

2 be badness, there are no free lunches.

3 So, I would think that any angiogenesis

4 intervention, one of the potential side effects

5 that one should look for is the acceleration of the

6 atherogenic process.

7 This is just a slide that is still in

8 development, but basically, it shows that when you

9 induce ischemia in a hind limb, you have decreased

10 PO2. This activates cytokine release, which

11 activates bone marrow cells, splenocytes, many

12 inflammatory cells.

13 We now know that inflammatory cells are

14 critically important to the development of

15 collaterals. Macrophages have been shown to be

16 critical. We have shown that both CD4,

17 t-lymphocytes, and CD8 t-lymphocytes are critical

18 to collateral development, but these same factors,

19 these same inflammatory factors also have been well

20 described to lead to, much longer than

21 angiogenesis, an acceleration of atherosclerosis.

22 So, if it causes angiogenesis, think very

23 hard as to whether it might be worsening the

24 atherosclerotic process.

25 The conclusions: single molecule-based

148

1 strategies to improve collateral flow, although

2 effective in animals, have yet to be proven

3 efficacious in patients. Cell-based strategies

4 have great promise because of the ability of bone

5 marrow derived progenitor cells to secrete multiple

6 collaterogenic cytokines.

7 However, cell-based therapies also have

8 the potential of achieving suboptimal effects

9 because of the effects of aging and other risk

10 factors on cell function. The optimal strategy has

11 yet to be identified, but genetic manipulation of

12 cells would appear to hold great promise, and use

13 of cell products, such as conditioned medium

14 derived from cells, will also undoubtedly be

15 explored as a therapeutic strategy in the near

16 future.

17 Thank you.

18 \[Applause.\]

19 DR. RAO: Thank you, Doctor.

20 We have time for a few quick questions.

21 Q&A

22 DR. SCHNEIDER: Steve, as an exploratory

23 tool, conditioned medium for angiogenesis makes a

24 lot of sense for the reasons that you articulated,

25 but as a therapeutic product, that would be true

149

1 if, and only if, conditioned medium contained

2 products that could not be identified or could not

3 be added combinatorily from defined factors.

4 So, it seems to me it will be especially

5 useful in those conditioned medium experiments to

6 test the effect of specific blockers and find out,

7 at a reductionist level, what the components are.

8 If it were as simple as angiopoietin and VEGF, one

9 could use angiopoietin and VEGF.

10 DR. EPSTEIN: Right, it\'s a very good

11 point. My own feeling is, having been in this

12 field now for 12\--more than that\--14 years, it is

13 so complex and the number of factors that are

14 involved in collateral development are not 4 or 5,

15 but they are dozens, and maybe even more than that,

16 that I personally will not waste time trying to

17 figure out what two products are enough or what

18 three products, I don\'t believe that, but the

19 cells, they know how to develop collaterals, I mean

20 they are doing it all the time, so I will go with

21 cell therapy, and I will allow someone else to look

22 at what combination of three factors might be

23 optimal.

24 It may be that you could find such

25 factors, but don\'t forget, not only do you have to

150

1 know what factors are present, but you have to know

2 in what concentrations, and so on.

3 I think the cells, they are eliminating so

4 many of the issues that if we were going to look at

5 individual cytokines, we would have to explore for

6 years, so it is a good point, but I think that the

7 practical issues, given the huge complexity of

8 this, would be overwhelming.

9 DR. SCHNEIDER: To follow up on your

10 comment, which I would share, that it is extremely

11 likely that engineered cells will outperform naive

12 cells, I would like to ask the participants from

13 FDA what additional hurdles are seen in the

14 consideration of gene-engineered cells to be

15 applied to these therapeutic situations.

16 DR. NOGUCHI: I think we can answer in

17 general that actually, we have a fairly rich

18 experience with gene-modified cells that have been

19 given to individuals for a whole variety of

20 diseases, not too many for cardiovascular, but I

21 wouldn\'t expect that we would have very much

22 difficulty in really being able to handle that.

23 DR. RAO: Dr. Mul.

24 DR. MULE: Combining your presentation

25 with Dr. Menasch\'s, I was sitting here wondering

151

1 what is known about, if one takes whole bone marrow

2 cells and perhaps Dr. Kurtzberg can add to this,

3 with Dr. Menasch\'s studies, if there are cells

4 within the marrow that can give rise to myotubes,

5 and you combine that with a population of cells

6 that could be responsible for collaterogenesis, the

7 issue is are they the same cell or are we at a

8 period in time where we can identify two subsets

9 within the marrow that conceivably could be

10 combined to overcome some of the issues of

11 viability that Dr. Menasch has talked about.

12 If the answer is no, we are not there yet,

13 then, it begs the question if one were to use an

14 adenovirus to introduce a gene to improve

15 collaterogenesis into the cell population, that is

16 identifiable for producing myotubes, the issue is

17 does that manipulation adversely impact the ability

18 of that cell to create myotubes.

19 DR. EPSTEIN: Well, those are sensational

20 questions, and I have never thought of this last

21 one before, but it is certainly\--you know, it is so

22 easy to do harm, and it is so hard to do good, so I

23 mean your question is very apt, I mean does the

24 very expression of the cytokines that enhance

25 angiogenesis, might it interfere with myogenic

152

1 potential, I don\'t think anybody has done that

2 experiment. Hopefully, the answer will be no, but

3 it certainly is an experiment that has to be done.

4 The MSCs that we are deriving from the

5 bone marrow do not differentiate into myoblasts,

6 and it would be a very interesting experiment to

7 take Dr. Menasch\'s approach and mix these in with

8 the skeletal myoblasts to see, because I can\'t

9 understand how, if you have a scar, and you are

10 injecting cells into the scar, and you do nothing

11 about the blood flow, why those cells won\'t turn

12 into scar. The blood supply clearly was

13 demonstrated to be inadequate because you have got

14 scar.

15 Of course, Dr. Menasch is actually doing

16 some experiments now using the same molecule that

17 we are using to induce collateral formation, so it

18 certainly is a very, very important strategy to

19 test.

20 DR. KURTZBERG: In answer to the other

21 question raised, I don\'t personally think we know

22 which subsets are important yet, or whether subsets

23 are more important than whole cell preparations. I

24 think that all should be the focus of questions

25 going forward.

153

1 Evan Snyder has an interesting model of

2 spinal cord injury and repair. It\'s a rat model.

3 They ligate, take a hunk of spinal cord and then

4 inject allogeneic cells and look at repair, and

5 they see repair and re-formation and re-connection

6 of nerves, but when they went back and looked to

7 see what cells did it, it turned out they were host

8 cells that were facilitated by something that the

9 allogeneic cells brought to the table, although

10 they don\'t know what.

11 To me, that just points out how much we

12 don\'t know and how complex the process is, and how

13 much more we need to study.

14 DR. RAO: One last question. To me, and I

15 am somewhat naive in this field, there is a

16 difference between new vessel initiation and

17 collateral formation of regrowth in terms of the

18 factors which had acquired, and so on, and it seems

19 to me in some models of cardiac ischemia, what we

20 are looking at are completely ischemic regions and

21 long term, which there is no regrowth, and if you

22 had to do anything, it would be new vessel

23 formation.

24 Would it be fair to say that we can\'t

25 extrapolate from the current models that you talked

154

1 about in terms of the religation and

2 revascularization, or is it reasonable to be able

3 to extrapolate from those models to what you think

4 might happen in a cardiac model?

5 DR. EPSTEIN: Well, I think it is

6 reasonable to extrapolate because we demonstrate

7 that we are able to improve perfusion, but your

8 question is really a very interesting one, and that

9 is, there used to be a major emphasis that

10 increased perfusion is just due to angiogenesis or

11 the development of new capillaries.

12 Well, I think most people involved in the

13 field would agree at this point that capillaries

14 don\'t increase flow. They facilitate the

15 distribution of flow, and what you need is an

16 increase in conductance vessels or arteriogenesis

17 to truly produce an overall increase in flow.

18 However, we have some preliminary data to

19 suggest that both processes are real, that

20 angiogenesis is a part of arteriogenesis, and that

21 you need the development of capillaries, that the

22 development of new vessels, capillaries, can

23 remodel to form collaterals.

24 That is why I don\'t use the term anymore

25 of angiogenesis. I say \"collaterogenesis,\" because

155

1 it gets away from the mechanistic aspects, which

2 are critically important, and we still don\'t have

3 the answer what cytokines produce angiogenesis,

4 what are important in terms of arteriogenesis, and

5 are both important to actually optimize the

6 development of collaterals, so we still have a

7 couple of years I think to go to answer that

8 question.

9 DR. RAO: One last question

10 DR. HARLAN: When you showed the

11 adenoviral HIF transfected cell lines and showed

12 that those were more efficient at elaborating

13 cytokines and inducing vessel growth, it adds

14 another question, that then becomes, however,

15 potentially anyway, a less well refined product,

16 the cell-conditioned medium from those cells, and

17 in view again of what we heard when we started

18 today, as we move forward with thinking about

19 delivering products to people, you want them to be

20 defined, and I wonder if you would comment on that.

21 DR. EPSTEIN: Well, if this field is to

22 move forward, I think that criteria is going to

23 have to be eliminated because there is no way you

24 are going to be able to define\--maybe I am

25 exaggerating\--the hundreds of molecules that these

156

1 cells are producing. We don\'t even know how to

2 measure them.

3 But I am sure the FDA allows the infusion

4 of serum and plasma from one individual to another.

5 Do you know what is in that serum?

6 DR. HARLAN: I am not the FDA.

7 \[Laughter.\]

8 DR. EPSTEIN: So, there is a precedent for

9 not knowing what you are injecting. To be honest,

10 we are injecting cells, and we know a few of the

11 molecules that they are secreting, but we have no

12 idea of the concentration, and whether it is going

13 to vary from one patient to another, and if a

14 patient has diabetes or has hypercholesterol, so

15 believe me, if you going to be compulsive and say

16 we have to know the concentration, when, over the

17 course of time, those molecules are up, and what is

18 their interaction, we have to stop this field right

19 now, it can\'t move forward, and it is too bad. I

20 mean you would like to know everything, but this is

21 not a characteristic of cell therapy.

22 DR. RAO: On that note, we will break for

23 lunch. We broke a little late, so we will try and

24 come back a little bit later, but not too much, so

25 we will shoot for 1:00.

157

1 \[Whereupon, at 12:15 p.m., the proceedings

2 were recessed, to be resumed at 1:00 p.m.\]

158

1 A F T E R N O O N P R O C E E D I N G S

2 \[1:12 p.m.\]

3 DR. RAO: Good afternoon.

4 Before we begin with the talks, I would

5 like to introduce three more members of the

6 committee who have just joined us. I like to let

7 them do it.

8 DR. HIGH: My name is Katherine High. I

9 am on the faculty at the University of

10 Pennsylvania. I am a hematologist with an interest

11 in gene transfer for hematological disease.

12 DR. BLAZER: My name is Bruce Blazer. I

13 am at the University of Minnesota in the Department

14 of Bone Marrow Transplantation with an interest in

15 immunobiology.

16 DR. RAO: We also have Dr. Grant from the

17 FDA.

18 DR. GRANT: Hi. I am Steve Grant. I am a

19 cardiologist. I am also a clinical reviewer within

20 the Office of Cellular Tissue and Gene Therapies.

21 DR. RAO: We will continue with the series

22 of talks that were scheduled.

23 The next speaker is going to be Dr.

24 McFarland.

25 Cellular Therapies for Cardiac Disease

159

1 DR. McFARLAND: Thank you, Dr. Rao, and

2 welcome back from lunch.

3 \[Slide.\]

4 This slide is intended to remind me to

5 answer the implicit question which may have been

6 raised, and the question is: Isn\'t the FDA putting

7 the cart before the horse?

8 The answer is, well, yes, in a way. We

9 thought that it would be good to focus and give

10 people a peek at what is in the cart below those

11 flat-screen monitors, I suppose, before we spend

12 the afternoon dealing with the horse which is

13 pulling the cart, the draft horse of product

14 development being preclinical studies and product

15 characterization.

16 \[Slide.\]

17 As Dr. Rao said, I am Richard McFarland,

18 and I am in the Office of Cell Tissue and Gene

19 Therapy in CBER.

20 \[Slide.\]

21 What I am going to do, I have been charged

22 with providing a perspective, FDA perspective to

23 the preclinical and manufacturing issues of cell

24 therapies for cardiac diseases.

25 First, I am going to describe the general

160

1 framework in which the FDA conducts our

2 science-based assessment of safety of novel

3 cellular therapies prior to allowing clinical

4 trials to proceed.

5 Second, I am going to describe the goals

6 of preclinical testing, safety testing in general,

7 and then a little specific about how that applies

8 to cellular therapies for cardiac disease, and

9 finally introduce the speakers for the rest of the

10 afternoon.

11 \[Slide.\]

12 FDA review is product-based, and it

13 parallels prudent product development. This is in

14 contra-distinction to the NIH grant process,

15 which is more based on diseases and organ systems,

16 which is illustrated just in the administrative

17 structure of the two agencies, NIH being primarily

18 divided by institutes, and FDA being primarily

19 divided by products that we regulate.

20 That means that our FDA review is

21 dependent on the characteristics of a specific

22 product, and the preclinical studies are designed

23 to support the use of specific products, and the

24 clinical trial design that we review is designed to

25 be supported by manufacturing and preclinical data.

161

1 That product-based review is framed by

2 regulations. I think I am the one designated to

3 get ready for regulations from the FDA.

4 \[Slide.\]

5 These are selections from the

6 Investigational New Drug regulations. I want to

7 highlight a few things, that being that regulations

8 stipulate there is adequate information about

9 pharmacological and toxicological studies, that the

10 sponsor has concluded that it is reasonably safe,

11 and that the kind, duration, and scope of those

12 required tests vary with the nature of the proposed

13 clinical investigations.

14 \[Slide.\]

15 A little further down in the regs, for

16 each toxicology study that is intended primarily to

17 support the safety of the proposed clinical

18 investigation, a full tabulation of data suitable

19 for detailed review should be submitted.

20 This is kind of critical to the way that

21 we do the review in that we need to get enough data

22 to do a detailed review.

23 \[Slide.\]

24 Let\'s back up for a minute and go to

25 preclinical evaluation in general. What are the

162

1 goals of preclinical evaluation with a perspective

2 of supporting trials?

3 One is to provide a rationale for the

4 proposed therapy, discern mechanism of action,

5 identify \"at risk\" patient populations, recommend

6 safe starting doses and escalation schemes for

7 humans, do a preliminary risk/benefit assessment,

8 and to identify parameters for potential clinical

9 monitoring.

10 \[Slide.\]

11 I will talk a little more specifically

12 about use of preclinical models for cellular

13 therapies. Preclinical models are used to provide

14 the scientific rationale with the cellular product

15 intended for clinical use, to understand cell

16 function, trafficking, and differentiation as all

17 these factors impinge on safety, as well as

18 modeling of routes of administration.

19 \[Slide.\]

20 If we had an ideal animal model for

21 cardiac cell therapies, it would have a similar

22 pathophysiology to humans that would improve the

23 predictability of human risk from the models,

24 similar anatomy to humans, which would allow us to

25 use various routes of delivery including catheters

163

1 of various types, with actually the clinical

2 catheter, it would also allow us to do extensive

3 dose exploration of cells, and it would be

4 immune-tolerant to human cells, so you could

5 actually use a human cellular product.

6 \[Slide.\]

7 Well, such an animal model doesn\'t really

8 exist, so we often use syngeneic animal models of

9 cardiac diseases because they can provide us useful

10 data for assessment of safety.

11 That would be cells from analogous cell

12 source processed in a similar way from animals,

13 autologous cells in the animal species or syngeneic

14 species, gives rise to potential processing,

15 formulation, and storage differences, and limited

16 product characterization both preclinically and

17 clinically leads to some uncertainty in addition to

18 the uncertainty that is inherent in the modes

19 themselves.

20 \[Slide.\]

21 Add to that, we have added complexity due

22 to innovative delivery systems, many of which have

23 not been tested for delivery of cells. Common

24 delivery systems that we have seen, intraoperative

25 transepicardial injection usually during CABG,

164

1 catheter-mediated transendocardial injection, and

2 catheter-mediated via cardiac vein, all of which I

3 think were discussed this morning.

4 \[Slide.\]

5 I only want to present a short scaffold of

6 the animal models that have been published so far,

7 and our speakers this afternoon will much more

8 extensively discuss the data.

9 There have been data in small animal

10 models, often cryoinjury, occasionally coronary

11 artery ligation to give an ischemic area damaged

12 myocardium. One of the advantages of the small

13 animal systems is that you have them available, at

14 least in mouse and rat, to use a human cellular

15 product to give you an idea of function and safety

16 of those cells. Primarily, it has been in mouse,

17 rat, and rabbit.

18 \[Slide.\]

19 Large animal models, typically, dog,

20 sheep, and pig, and we have seen some of those data

21 this morning, as well, an ameroid constrictor used

22 to generate an ischemic area has been a popular

23 model of disease. They are amenable to catheter

24 administration and more amenable to clinical

25 monitoring modalities, however, you are stuck with

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1 using syngeneic cells in this situation.

2 \[Slide.\]

3 The regulations say that the data should

4 be adequate and extensive. What are the potential

5 sources of data to support initiation of clinical

6 trials?

7 Well, the gold standard would really be

8 preclinical studies specifically designed to

9 support a specific trial with a specific cell.

10 We also have data from other potential

11 sources: existing animal studies that were

12 designed to answer other questions, in-vitro

13 studies, clinical trials using the \"same\" product.

14 \[Slide.\]

15 However, we use data that are published

16 and unpublished. Using published data either from

17 animal studies or human studies as sole support for

18 initiation of clinical trials raises some

19 questions, some complexities, because often these

20 studies are not designed to answer a toxicologic

21 question, and therefore, adequate toxicology

22 endpoints may not have been incorporated into the

23 design.

24 If they were incorporated into the design,

25 but not in the publication, we need access to those

166

1 data. Published reports may provide sufficient

2 information for independent review.

3 \[Slide.\]

4 There are some limitations in using

5 published studies. Protocols in the studies need

6 to be sufficiently detailed. We need to be able to

7 do our independent review as per our regulations.

8 We need to see specifics of the route of

9 administration.

10 We need to see catheter specifics, such as

11 identity of the catheter, flow rate, pressures,

12 effects of catheters on cells, location of

13 injection in relation to the ischemic area, and

14 protocols, either animal studies or human

15 protocols, we need the control details of the

16 \"routine\" monitoring and analytical plans.

17 \[Slide.\]

18 The data must be presented in sufficient

19 detail.

20 In-process and lot-release data from

21 manufacturing need to be presented in sufficient

22 detail for us to know exactly what the product is,

23 and complete study reports for both animal and

24 clinical studies.

25 \[Slide.\]

167

1 Cellular products used in published

2 reports may not be comparable to the intended

3 clinical product. Often, in published reports,

4 there is insufficient data to allow us to make a

5 comparability assessment, and that is either

6 because the data don\'t exist or due to editorial

7 constraints of the publication.

8 \[Slide.\]

9 So, given the limitations of the framework

10 or the window at which FDA is required to look at

11 these, and the detail that we are required to look

12 for prudent product development raises some

13 regulatory challenges.

14 These are rather recurrent regulatory

15 challenges. This is does the submission contain

16 sufficient information to assess risk to the

17 subjects in the proposed trial. It is a question

18 that we ask at the end of our review. Were

19 adequate preclinical studies performed? If they

20 were performed, were the data submitted in

21 sufficient detail to conduct an independent review?

22 If sufficient data are present, then we

23 get to the question, are the risk to human subjects

24 reasonable and significant?

25 \[Slide.\]

168

1 That gives you an idea of the framework

2 with which we need to look through to the window of

3 science and what we are obligated to do as we make

4 an assessment.

5 There is discretion, there is ability to

6 be flexible within the regulations, and what we are

7 asking the committee to do over these two days is

8 to give us an idea of what the state of the science

9 is. It will be reflected in the questions that we

10 will be discussing tomorrow afternoon.

11 What is the state of the science? What

12 is a reasonable amount of data for us to be looking

13 at?

14 This afternoon, we are going to have two

15 speakers that are focusing primarily on cells.

16 Doris Taylor from the University of Minnesota and

17 Silviu Itescu from Columbia.

18 After the break, followed by Dr. Nick

19 Jensen from the Center for Devices and Radiologic

20 Health at the FDA, who will focus on delivery

21 devices and some of the issues that are related to

22 development and testing of delivery devices, which

23 are an integral part of our preclinical

24 development.

25 Then, Robert Lederman from the NIH will be

169

1 discussing some of his experiences from being in

2 the trenches of doing studies with devices, cardiac

3 diseases.

4 DR. RAO: Thank you.

5 We will now have Dr. Taylor.

6 Guest Presentations

7 Myoblasts: The First Generation Cells for

8 Cardiac Repair: What Have We Learned

9 DR. TAYLOR: Thank you. I have to confess

10 that if I had seen those previous slides before I

11 had signed my talk, it would be a completely

12 different talk, so bear with me.

13 I am going to be talking about some of our

14 data and some of the data from the rest of the

15 field, but I think what I really want to focus on

16 is myoblasts for cardiovascular repair and what

17 lessons we can learn from the cells that have been

18 used for the longest period of time preclinically,

19 and I think another way to think about this is gene

20 therapy revisited, are we going to do it all over

21 again.

22 I think the point that I want to make is

23 that there are a lot of lessons that we can learn

24 from the gene therapy field as we are going forward

25 with cell therapy, and I think it is important to

170

1 take those lessons away from this. I will be glad

2 to talk about that in more detail if people have

3 questions later.

4 I like to start with this because this is

5 what I tell the people in my lab, and I think it is

6 true - make everything as simple as it is, and no

7 simpler, from Einstein.

8 Philippe will recognize this. This is an

9 image I borrowed from his Lancet manuscript in

10 2001, showing the first patient into whom myoblasts

11 were actually delivered clinically. I actually use

12 it to illustrate what I think is the salient point

13 here, which is that most of us are dealing with

14 animals as well as in patients, infarcted

15 myocardium, where a process of events has occurred

16 that starts with inflammation, moves to cardiocyte

17 apoptosis, a remodeling and compensation process

18 that you heard about in extreme detail this

19 morning, scar expansion, decompensation, and

20 progression to failure.

21 The truth of the matter is we are trying

22 to intervene in this with either cells, genes, or

23 devices, but we don\'t know where in this cascade we

24 are actually intervening, nor do we know where we

25 should be intervening.

171

1 I think most of us who got into this field

2 envisioned it, first, as a field where we would

3 intervene early after an acute myocardial

4 infarction to try to prevent the slippery slope

5 here of remodeling, scar expansion, decompensation,

6 and failure, but the truth of the matter is that

7 most of the patients in whom studies have been done

8 are patients who have already progressed to some

9 degree of failure.

10 Although we initially started to begin to

11 look at prevention and repair of not only cardiac

12 injury, but also vascular injury, we are now really

13 trying to understand whether or not we can move

14 back up this scale in a reverse remodeling way, or

15 to grow new cells, and I think those are the

16 questions that are really out there in the field

17 right now.

18 The Holy Grail in this field then is that

19 transplanted cells cannot not only engraft, but

20 restore blood flow and contractility to injured

21 myocardium, and all of you know, because you have

22 seen some of the data from Philippe and are fairly

23 well versed in this field or you wouldn\'t be here,

24 that there is more than 15 years of preclinical

25 data in rabbit and dog, there is at least 1 to 5

172

1 years of preclinical data in pig, rat, mouse,

2 sheep, and now hamster, all of which showed in our

3 preclinical models that transplanting autologous

4 skeletal muscle derived cells was safe, effective,

5 and feasible, and therefore, Phase I, surgical and

6 intervascular studies were initiated worldwide.

7 I think the future that we will probably

8 ultimately all try to examine is what is ultimately

9 the best cell for cardiac repair. I have been

10 asked to focus on myoblasts, so I am going to do

11 that although a little bit later this afternoon, I

12 am going to talk necessarily a little bit about

13 comparisons among cell types because I think it

14 really begins to ask questions about mechanism

15 that we have to address as we are going forward.

16 Obviously, the best cell may be some

17 autologous bone marrow-derived product. It may be

18 a cell plus or minus a therapeutic gene to either

19 promote angiogenesis or some other signaling

20 cascade that maybe promote cell survival.

21 Ultimately, it may be embryonic stem cells although

22 I would submit at least in this country, we are a

23 number of years away from those cells, not only

24 because we don\'t understand how to regulate their

25 differentiation, but because we also don\'t

173

1 understand how to make them stop dividing

2 appropriately.

3 Then, obviously, you can\'t be from the

4 University of Minnesota without talking about

5 adult-derived stem cells.

6 So, what are the questions in the field?

7 I am just going to put my opinions out there. I

8 think just keep these in mind as a background as we

9 go forward.

10 Is there a best cell? I don\'t think there

11 is a best cell. I think it really depends on the

12 patients, the time after injury, the dose and a

13 number of other factors.

14 Is there just a better way to get it

15 there? One of the questions that we keep coming

16 back to over and over and over is whether or not

17 cell therapy is just going to ultimately be another

18 local drug delivery problem, and whether or not we

19 are really going to be able to get the cells to

20 where we need them or whether they have the

21 capacity to actually migrate there or home there,

22 and I think that is going to be an important

23 phenomenon to begin to explore.

24 The other side of that is do the cells go

25 where we want them, or do the cells go where we

174

1 want them and somewhere else where we don\'t want

2 them, and I think we will come back to that.

3 Should we just use growth factors and

4 forget cells? I am not going to focus on that.

5 Is there a future for biologic devices,

6 and is the real question dose, timing, and choosing

7 the right patients for the right cell? I would

8 submit that it probably is.

9 So, where are we in this field? Well,

10 this is a table that I copied from a review by

11 Loren Field, and the table goes on for slides, just

12 to show that in terms of preclinical myoblast

13 transplantation, there is a huge amount of data out

14 there, and what the data really begin to show is

15 that there are a lot of different cells that have

16 been used, there are a lot of different species

17 that have been used from mouse, rat, rabbit, pig,

18 dog, and sheep, that these cells have been injected

19 either into normal heart, cryo-injured heart,

20 hearts where vessels have been occluded, and that

21 surprisingly, most people didn\'t actually measure

22 improvements in function or in angiogenesis or in

23 survival.

24 I think that is important to consider as

25 we really try to pull together the summary of data

175

1 from myoblast cell therapy.

2 So, how do we really do this? Well, for

3 myoblasts, you basically take a chunk of muscle and

4 you grow cells in vitro, and you end up with cells

5 in a dish, and then you deliver these cells to the

6 injured myocardium and you measure the effect.

7 So, you inject them, you deliver them, and

8 you measure the effect. So, I am going to go

9 through the different parameters here, talking

10 primarily about myoblasts and what exists in each

11 of these areas.

12 So, the cells are typically referred to as

13 myoblasts, but the bottom line is these are

14 muscle-derived cells that contain, not only

15 myoblasts, but also often more fibroblasts than

16 myoblasts, a number of cells called SP or site

17 population cells, and then a whole lot of other

18 cells that we don\'t necessarily know how to

19 characterize yet.

20 So, this is a very heterogeneous cell

21 population, and you do take these cells and you

22 grow them in a dish for several weeks, and what

23 that means is that all of us are exposing these

24 cells to serum-containing medium, and that what

25 Arnold Kaplan learned years ago from mesenchymal

176

1 cells is that when you are using serum, what is in

2 that serum, FDA regulations notwithstanding,

3 matters, and that the lot number of serum is going

4 to give you a different outcome in terms of the

5 numbers of these cells and what their phenotype is

6 when you are done at the end of the day.

7 In terms of injury models where myoblasts

8 have been delivered, the primary injury models are

9 either acute myocardial infarction, acute being

10 anywhere from a week to one month, and primarily

11 that has been a cryoinjury model or coronary artery

12 ligation.

13 The question that really arises\--and then

14 those have varied dramatically in size, the timing

15 after creation of this infarction to the delivery

16 of cells matters, and inflammation, so are these

17 the same as a clinically relevant injury? Well,

18 the size differs, the timing differs, and the

19 inflammation differs with regard to what is

20 actually seen in patients.

21 In terms of cells, the myoblasts,

22 primarily how they have been delivered, more than

23 90 percent of the cells preclinically and

24 clinically have been delivered by surgical

25 approach.

177

1 Some preclinical data exists in terms of

2 delivering cells via catheter in a pig, but again

3 there are open questions about dose, about where

4 those cells are delivered, about when those cells

5 are delivered, and there is virtually no data out

6 there about the vehicle in which the cells are

7 given.

8 Typically, people either say they inject

9 the cells in saline or they inject them in the cell

10 growth medium minus the serum.

11 As I said up here, lot number matters, so

12 the vehicle differs dramatically.

13 The next issue that you have to deal with,

14 if you have got cells and you grow them and you

15 inject them into an animal, is how you measure the

16 outcome, and the question of safety is obviously an

17 open one, and none of us really have addressed the

18 safety question in preclinical models.

19 We didn\'t really know that there was going

20 to be a safety issue. I was talking at lunch about

21 the fact that, you know, we had a number of animals

22 drop dead over the course of our studies when we

23 were doing these experiments early on, but we

24 assumed it was because we were doing open-chest

25 surgeries on these animals to create the infarct,

178

1 to deliver the cells, to measure cardiac function,

2 not that it could have anything to do with a

3 potential electrical effect of the cells in vivo.

4 So, we had to then go back and evaluate

5 whether or not safety was even at all compromised

6 or relevant in these animal models, so none of us

7 are really measuring safety. There have been two

8 studies reported, one in a pig model of holter

9 monitoring animals, and we just presented some data

10 at ACC in rabbit, monitoring electrical effects of

11 myoblasts, and I will show some of those data in a

12 minute.

13 The other issue in terms of measuring

14 outcome is function. Typically, in rodents,

15 isolated heart preps have been used to measure

16 function although in some cases, sonomicrometry is

17 used. I have actually put the methods here in

18 order of I believe their ability to actually give

19 you useful information.

20 I think the isolated heart prep is the

21 least useful because it is subject to a lot of

22 variability, it is subject to baseline drift, it is

23 subject to flow and rate factors, and it is subject

24 to ischemia in vitro.

25 Echocardiography is obviously used

179

1 clinically, as well as experimentally, but it

2 varies dramatically with the operator and the

3 orientation of the probes, so you can make echo

4 tell you just about anything you want.

5 If you really want to measure work done in

6 a region of the heart, I submit that you have to go

7 back and do sonomicrometry in that area and use

8 crystals to actually measure the ability of that

9 region of the myocardium to move in an electrically

10 and mechanically meaningful way.

11 Then, more recently, cine MRI has really

12 come to the fore in terms of our ability to make

13 measurements in not only people, but animals, as

14 well.

15 But measuring function is pretty useless

16 unless it correlates with histology, and we begin

17 to ask questions about angiogenesis and myogenesis,

18 and we don\'t always correlate histology with

19 outcome, and, in fact, one of the issues that comes

20 up over and over is clinically, as well as

21 experimentally, there is a disconnect between the

22 number of cells we can find in the heart and the

23 functional improvement we see, which begins to ask

24 questions about mechanism.

25 So, in terms of myoblasts, what are these

180

1 cells? As I said, there are myoblasts or

2 fibroblasts and there are SP cells. I propose that

3 the mechanism of repair of these cells depends on

4 the number of cell types that you have present and

5 the percentage of each.

6 I believe myoblasts know to become muscle,

7 and they are capable of myogenesis. I believe the

8 fibroblasts not only secrete an angiogenic factor

9 FGF, but also act as a growth factor and a mitogen

10 for myoblasts.

11 That has been known for years. Judy

12 Swain\'s data, they actually keep myoblasts alive

13 and keep myoblasts proliferating over a fairly

14 extended period of time, FGF does.

15 SP cells, I believe are more likely to be

16 angiogenic and also possibly to fuse with other

17 cells in the myocardium. That is based on data

18 primarily from our group showing that the more

19 immature a cell is, the more likely it is to fuse.

20 So, in terms of our animal models, I think

21 the question we have to ask is what do the patients

22 look like first, and in the myoblast trials, those

23 patients have been post infarction, usually greater

24 than one month, up to many years. The average in

25 one of the studies was 6.7 years.

181

1 Most of those patients are in need of

2 revascularization, they have an ejection fraction

3 of less than 35 percent, and they have heart

4 failure.

5 If you look at the European experience,

6 Philippe has already talked about some of this, and

7 I am not going to go over these in detail. This is

8 a slide that was given to me by Peter Smits from

9 Rotterdam.

10 You look at Spain and Poland, and

11 obviously the French study, and then the U.S.

12 studies, both Arizona heart and Bioheart Mount

13 Sinai study, and then the Bioheart study in Europe,

14 and I should say for the sake of disclosure that I

15 have had a relationship with Bioheart, so take

16 these data with a grain of salt, that all of these

17 patients are heart failure patients.

18 The average in this study is 6.7 years

19 post-infarction, so these are patients who are

20 already pretty sick, and who have significant

21 electrical abnormalities already.

22 How do the preclinical patients compare?

23 Well, for myoblast studies, these animals all have

24 acute cardiac injury, either, as I said, cryoinjury

25 or coronary artery ligation. Very few, if any,

182

1 studies deal with occlusion reperfusion, which is

2 what happens clinically.

3 You can open up virtually any artery in

4 the heart now, but nobody is doing preclinical

5 studies where we do ischemia reperfusion. We are

6 just now moving in that direction.

7 I think the reason initially for at least

8 us, and I believe for other people, was that we

9 wanted to kill everything that was there, so that

10 anything we found was due to something we put in,

11 and so we started with cryoinjury where we applied

12 a minus 70 degrees C probe to the surface of the

13 heart to wipe out that region of the heart, but it

14 raises questions about the inflammatory process,

15 which we are coming to understand is critical in

16 terms of potential homing of cells and the

17 potential mechanism.

18 Most of the preclinical studies, the cells

19 are delivered two to three weeks post-injury, and

20 there is a one- to three-month follow-up.

21 Clinically, this isn\'t exactly relevant.

22 I just told you that every clinical study

23 is at least one month post-injury and sometimes six

24 to seven years post-injury, so we are not looking

25 at the same milieu into which we put these cells.

183

1 In term of heart failure models, there are

2 a few models out there. Dan Burkoff\'s group has

3 recently published a study in dog where they used

4 microspheres to actually create a heart failure

5 model, and have gotten data with myoblasts that

6 actually look very similar to some of the data

7 gathered in earlier models.

8 None of the studies to date have really

9 used any animals with LVAD support, and yet there

10 are clinical trials beginning to move forward in

11 that, and actually, one has already been completed

12 in that context. So, we would expect that with a

13 completely unloaded heart, we might have very

14 different phenomena.

15 In terms of cardiomyopathy, there is a

16 hamster model and mouse genetic models that have

17 begun to be used for myoblast transplantation, and

18 Magdia Koob\'s group has actually published

19 reasonable data in terms of a rat model of

20 adriamycin toxicity. So, we are beginning to get a

21 plethora of models in which we can look at myoblast

22 transplantation.

23 I put up here actually the little bit of

24 physiology that I could pull together about some of

25 the different animal models just to make a few

184

1 points.

2 One is that when you start looking at

3 these different injury models in mouse, rat,

4 rabbit, dog, pig, sheep, and humans, that there

5 really are significant differences in rodents and

6 the larger animals.

7 Mouse and rat, you know, the hearts are

8 pretty darn small. Their heart rate consequently

9 is very high. These animals have very few

10 collaterals, and they express completely different

11 contractile proteins than are expressed in the

12 majority of the heart.

13 Moreover, the action potential in the

14 electrical capacity of the mice and rat is very

15 different. There is no plateau phase in the action

16 potential in mice, and the action potential

17 duration is on the order of 10 milliseconds. In

18 humans, it is on the order of 250 milliseconds.

19 Rabbit is the first animal model where you

20 begin to get numbers and conditions that resemble

21 humans, and that is why we chose rabbit early on,

22 and I would submit that in terms of feasibility

23 studies, rabbit is a good entry level animal under

24 most conditions except where you are trying to do

25 stem cell work.

185

1 When you are trying to do stem cell work,

2 we don\'t have the markers for stem cells in most of

3 these other species that we do in rat, mouse, and

4 humans, so that is when mice, rats, and humans, or

5 maybe pigs, some of the human cytokines and

6 antibodies cross to pig, but not all of them, so

7 you can begin to do some of those studies in pig.

8 Nonetheless, in terms of feasibility

9 studies, I think rabbit is a good model, and you

10 move up from there.

11 So, let\'s look at each of these. In terms

12 of myoblast in the mouse, you can begin to track

13 the cells because you can use genetic models of

14 where the cells actually express different markers

15 that are unavailable in the animals in which you

16 inject the cells.

17 You can begin to isolate and characterize

18 stem cells including the stem cells in muscle, but

19 you can\'t characterize from larger species. You

20 can use immunocompromised mice for human cells, but

21 you are missing an important component, which is

22 the inflammatory component, and we are beginning to

23 understand again relates to homing and perhaps even

24 recruitment of cells.

25 What is the advantage of a rat versus a

186

1 mouse? Well, the main advantage is it is larger

2 than a mouse, so you can do a few more things, but

3 you can still track the cells and isolate stem

4 cells, and you can still have an immunocompromised

5 model.

6 The rabbit, the bottom line about rabbits

7 is most people make antibodies in rabbits, not

8 against rabbits, so it is really hard to find the

9 tools that you need to do some of the evaluations

10 downstream, but it is still a relatively

11 inexpensive model with cardiac characteristics very

12 similar to humans.

13 The pig, obviously, the size is good, and

14 the geometry is good for catheter-based studies.

15 One of the points I want to make about delivery of

16 these cells, and I think Philippe showed it when he

17 was talking about his clinical trials, and we have

18 done the same thing in terms of our preclinical

19 studies, surgical studies, is that when you inject

20 these cells surgically, most of us have delivered

21 the cells parallel to the surface of the heart.

22 We have done that for years because we

23 really thought it was going to increase the number

24 of cells that we could get into the myocardium.

25 Yet, all of the catheter-based studies deliver the

187

1 cells perpendicular to the surface of the heart,

2 and it is not completely unexpected that geometry

3 may make a difference in terms of how these cells

4 actually function in the myocardium.

5 So, I think it is important to evaluate

6 the geometry of the cells in some of these larger

7 animal models.

8 So, how do you choose an animal model for

9 the myoblast studies? I think feasibility and

10 costs are obviously important, whether or not you

11 are going to do high throughput studies and need to

12 track your cells. Rodent and hamster I think are

13 best for those. Rabbit is best in terms of

14 beginning to be physiologically relevant to humans

15 in terms of heart rate and scalability. I will

16 show some data in a minute in terms of scalability.

17 The large animal models are obviously much

18 more physiologically relevant. You can get a sense

19 of dose. You can use conventional delivery methods

20 that you would use in humans. You can do the right

21 functional assessments, and the heart size and

22 geometry is very similar to a human.

23 So, what exists for myoblasts? Well, as I

24 said, the route of administration has been

25 primarily surgical or percutaneous. Intravenous

188

1 and intracoronary studies are just beginning

2 although we published some intracoronary data, that

3 was the first thing we published in \'96.

4 Myoblasts are unlike stem cells or unlike

5 bone marrow mononuclear cells. They are very much

6 like stromal cells. They are big. When we put

7 myoblasts in the coronary circulation, what we

8 found is that we got profound ST elevations, and we

9 saw transient ischemia every time we injected these

10 cells.

11 So, we actually think that with large

12 cells, that the way they are actually having an

13 effect in the myocardium is creating essentially a

14 microinfarct clogging the vessels and then getting

15 out of the vessels as a result of that.

16 Mononuclear cells are much smaller, and I

17 think don\'t have the same effect.

18 In terms of dose, in a mouse, typically,

19 you give about 1,000 cells. Some people go as high

20 as a million, but typically, 1,000 is enough to

21 begin to see an effect.

22 In our hands, in rabbit, the lowest dose

23 at which we see an effect is 3 x 10

7 cells. We

24 tried 10 7, 3 x 107, 108, and

3 x 108, and this is the

25 range in which we see the most effect.

189

1 In pig, it is about 3 x 10

8, and what we

2 found, you know, pig is about 10 times bigger than

3 a rabbit, you need about 10 times as many cells.

4 Rabbit is about I think about 4,000 times the size

5 of a mouse, and we found that we need many more

6 than 4,000 more cells in a rabbit than we do in a

7 mouse. So, I don\'t think you can really

8 extrapolate from mouse, but I think you can begin

9 to extrapolate at the size of rabbit and go up.

10 In terms of cell location and where

11 myoblasts have been injected, you pick a surgical

12 fellow who is doing the experiments, and you will

13 get a different location of injection virtually

14 every time, I guarantee it, and you are not going

15 to convince them otherwise that their way isn\'t the

16 right way to do it. It is completely ignored in

17 most of the preclinical studies.

18 There might be mention of one injection or

19 two injections or three injections, but in terms of

20 the exact location, I couldn\'t tell you, I couldn\'t

21 find in the literature where the majority of

22 injections occur. I know in my own lab, it is not

23 consistent from study to study.

24 In terms of timing, myoblasts have been

25 injected two to four weeks post-injury. The

190

1 vehicle has been PBS cell growth medium minus

2 serum, or it is completely ignored, there is no

3 mention of it.

4 So, this is the slide again I borrowed

5 from Philippe\'s work to illustrate how he did some

6 of the early injections with a bent needle again

7 parallel to the surface of the heart, and also that

8 the injections are done, not just in the center of

9 the infarct, but in the periinfarct region, as

10 well.

11 Similarly, with a percutaneous approach,

12 and this is another slide from Peter Smits, in the

13 first patient who received cells, and in the

14 majority of cases now with percutaneous myoblast

15 delivery, cells are delivered in the periinfarct

16 region, in the normal region of myocardium, and

17 very few of the injections percentagewise actually

18 end up in the infarcted cell, and that may have an

19 effect on safety, and I will show some preclinical

20 data that support that.

21 So, the majority of injections are

22 surgical, and you can inject the cells and find

23 them in the center of the infarct. What you get

24 surgically when you inject these cells is one or

25 two things.

191

1 On a great day when you are really lucky,

2 you get what looks like a chunk of steak in the

3 center of the heart. On a typical day, you see

4 something that looks more like this, where you have

5 patchy regions of cells distributed through the

6 infarct rather than these large fibers that you see

7 here, and these patchy cells distributed throughout

8 the infarct are not necessarily talking to each

9 other, but they are all oriented with the

10 extracellular matrix.

11 You can see there are some small vessels

12 here, here. We often see large vessels in the

13 infarct, as well.

14 These are preclinical data from my group,

15 but they don\'t look too dissimilar from what you

16 see from Pagani\'s paper from myoblasts in an

17 LVAD-supported human heart or, in fact, the data

18 that Philippe showed you earlier of the myoblasts

19 surrounded by scar in a patient 17 1/2 months after

20 injection.

21 I am not going to talk about stromal cells

22 because that is not my job today, but what I am

23 going to begin to talk about is delivery.

24 Assessing delivery requires that we be able to

25 track the cells. In vivo, we have chosen SPECT or

192

1 MRI most recently, although I think PET is going to

2 be a good method, as well.

3 That has to correlate in vitro with

4 histology and appropriate markers. If I don\'t make

5 any other point today, take home the fact that

6 using desmin, using phospholamban, using GATA-4,

7 using all of these markers that people claim are

8 cardiac markers, are not cardiac-specific markers.

9 You find these markers in other muscle cells, you

10 find these markers in undifferentiated progenitor

11 cells.

12 If you look in C2C12 skeletal muscle

13 cells, you can see phospholamban, you can see

14 connexin 43, you can see in some cases, in

15 progenitor cells, you see GATA-4. You have got to

16 use markers that are specific for cardiocytes if

17 you are going to call these cells cardiac cells,

18 and the only markers that I know of right now, that

19 I believe are specific for cardiocytes, are channel

20 markers that are actually not expressed in skeletal

21 muscle.

22 As skeletal muscle matures, it expresses

23 many cardiac-specific proteins, and as skeletal

24 muscle matures, it expresses cardiac markers, as

25 cardiac muscle matures, it expresses skeletal

193

1 markers. So, we don\'t know where in that process

2 we are, so we can\'t really use those markers.

3 This is an image showing that we can begin

4 to visualize these cells in the heart. These are

5 indium-111 labeled myoblasts present in a short

6 axis view by SPECT imaging of a rabbit heart

7 showing that we can actually co-deliver

8 tetrofosmin, see perfusion, see the dropoff in

9 perfusion here with the infarct, and then see the

10 indium-labeled cells in the center of the image.

11 So, we are beginning to believe that we

12 can actually track cells over time now. This is

13 also a cine MRI of a rabbit heart, and these are

14 data that were all gathered at Duke. This is a

15 rabbit heart, so at the level of rabbit, although

16 we can now do the same thing in a mouse, we can

17 iron label our cells in a way that we believe

18 doesn\'t affect proliferation or viability of the

19 cells, and begin to see them in the center of the

20 infarct region, the infarct region here being

21 contrast-enhanced in white.

22 So, we can start now to label these cells.

23 We have followed these cells out to four months in

24 this way and can still find them. When we kill the

25 cells and then inject them, the iron label goes

194

1 away over about two to three days, so we are fairly

2 convinced that the iron is present in viable cells.

3 So, the other issues in terms of measuring

4 outcome, I think I have already made this point,

5 safety is an open question, and I think what I take

6 away from the field so far is if you don\'t look,

7 you won\'t find it, and that we didn\'t look, and now

8 I think it is important that we begin to do holter

9 monitoring and other electrically relevant studies,

10 and those are going to require large animals, pig,

11 rabbit. You can\'t do those in mouse and rat, not

12 at 300 to 600 beats per minute. You are really not

13 going to be able to see a VF or a VT.

14 In terms of function, I think if the goal

15 here with myoblasts is really to find an

16 ischemia-resistant cell that is electrically

17 compatible with a healthy heart, we have got to

18 also look at electrical activity of these cells

19 over time.

20 This is again a slide that Peter Smits

21 provided showing clinical data and the number of

22 VPCs per visit in some of the early patients who

23 had cells delivered, and I modified the slide a bit

24 to show times at which patients have actually died

25 after cell delivery.

195

1 What you begin to see is that there is a

2 window of time from about a week to a month where

3 there seems to be an increased incidence of

4 electrical abnormalities. When we have done animal

5 studies now, we see that same sort of window from

6 about 3 days to about 3 1/2 weeks, and then it

7 drops off and we don\'t see the incidence after that

8 period of time.

9 So, the safety may depend on the cell

10 dose. We have found that if we just look at PVCs

11 in our animal models, that as we increase dose, we

12 increase the number of PVCs, and it may also depend

13 on location.

14 We have found that if we inject cells in

15 the center of the infarct and we measure PVCs, and

16 this is actually 10

8 cells, 107 is not functionally

17 relevant, 10 8 is, 109

is, we found that if we inject

18 cells in the center of the infarct, we see PVCs and

19 no monomorphic VT.

20 If we begin to inject cells in the border

21 zone, we not only increase the number of PVCs we

22 see, but we start seeing runs of ventricular

23 tachycardia. If we inject cells in both the center

24 and the periphery, we see essentially the same

25 thing, and more up-to-data were just presented at

196

1 the ACC from my lab.

2 What is interesting is we began to take

3 these cells back out of heart, what we found is

4 that their action potential duration changed, that

5 initially, the cells had an action potential

6 duration of on the order of 20 seconds, and over

7 time it increased to something on the order of 120

8 milliseconds, but it is still not compatible with

9 the surrounding heart.

10 We have also done some modeling data.

11 What we believe is that if you have these cells

12 coupled to each other, that is a good thing in the

13 center of the infarct, but that you don\'t want them

14 coupled to the remainder of the heart until they

15 are electrically compatible with the remainder of

16 the heart, and yet clinically, very little

17 attention has been paid to location.

18 Again, it is an issue that we didn\'t know

19 we were going to have to address, and now we have

20 got to go back and address. In fact, some of the

21 locations of injections could explain why there

22 have been ventricular tachycardia in some of these

23 patients.

24 The only possibility is, you know, over

25 that window of time, we don\'t know if these cells

197

1 are integrating, dying, or changing their

2 phenotype, we have no idea, and I think we really

3 have to begin to elucidate that.

4 So, in a standardized model where we know

5 how myoblasts function, we have now got to look at

6 location, dose, and route of administration.

7 I think I have already said this, so I am

8 not going to really belittle, spend time on

9 function especially other than to say we have

10 begun to collect a lot of data now with a lot of

11 different cell types and a lot of different growth

12 factors, and what we have begun to realize is that

13 virtually anything we put into the heart, cells,

14 myoblasts, fibroblasts, bone marrow stromal cells,

15 bone marrow mononuclear cells, growth factors

16 including VEGF and other growth factors, improve

17 the mechanical properties of the scar, and change

18 diastolic performance.

19 They do that first, before they have any

20 effect on systolic performance, usually by several

21 weeks. What we figure is that having something

22 alive in the scar is better than having just this

23 dense collagen matrix, and it really doesn\'t seem

24 to matter what you have alive in the scar, if is

25 vessels, if it is muscle, if it is whatever, you

198

1 improve compliance.

2 But we don\'t see the corresponding

3 improvement in systolic performance, at least not

4 with fibroblasts in our hand, but we do with

5 myoblasts, we don\'t with VEGF, but we have now with

6 bone marrow stromal cells and bone marrow

7 mononuclear cells.

8 These are some data that just came out in

9 Circulation showing that, that if we use crystals

10 to measure regional stroke work in our sham-treated

11 animals, regional function gets worse, but in our

12 myoblast-treated animals, function goes from pretty

13 bad to better, and in our bone marrow stromal cell

14 animals, the same thing is true.

15 I think that really raises a question

16 about mechanism, but the positive outcome in our

17 hands at least is dose dependent, 10

7 no effect, 108

18 positive effect, sham continues to get worse.

19 What is interesting is this is not just

20 improvement versus cell number. This is log of

21 injected cells, but this is the percentage of

22 animals that actually improve.

23 So, what we found is that the percentage

24 of animals increases with cell dose, as well.

25 So, will myoblast transfer work in

199

1 patients? Philippe already has told us that it

2 will, and we have begun to believe that, in fact,

3 that there are different mechanisms of action for

4 these cells.

5 I think I will just very quickly go

6 through the last couple slides. We believe that

7 myoblasts improve both regional and global function

8 in the heart based on our preclinical data.

9 If we use cine MRI and actually measure

10 thickness in the wall of the myocardium over time

11 and global wall thickening, so areas where cells

12 were not injected, we use contrast to define where

13 the infarct is, and this is area that has no

14 contrast in it, so the remainder of the heart

15 actually gets better, wall thickening improves in

16 the cell treated, but not in the control vehicle

17 injected animals.

18 Regional wall thickening where we actually

19 inject the cells gets better to a greater degree,

20 so we only measured this where there was a

21 transmural infarct. We didn\'t measure it in

22 regions at the periphery of the infarct where there

23 can be tethering going on. So, we use contrast and

24 only measured it in the region.

25 Diastolic volume decreased, heart weight

200

1 decreased, so global indices of failure also

2 improved.

3 Every cell we and virtually anyone has

4 injected seems to work, which either means the

5 myocardium is easier to repair than we thought or

6 we don\'t understand what is happening and we aren\'t

7 looking at the data correctly.

8 I would like to believe it is this, and I

9 am actually going to posit this in a little bit,

10 but I have a bad feeling. I would also submit that

11 they work despite the fact that we don\'t know how

12 to get the cells there in large numbers, and we

13 can\'t always find them histologically, and that we

14 don\'t really know what to look for.

15 These cells may be promoting angiogenesis,

16 myogenesis, they may just be unloading the heart,

17 changing wall stress. They may be secreting

18 paracrine factors that recruit other endogenous

19 stem cells to the area of injury, either cells from

20 the heart, if that\'s your fancy, of cells from the

21 bone marrow, or maybe a combination thereof, or

22 maybe they work because we are lacking long-term

23 follow-up in both animals and patients, and we

24 haven\'t asked the right questions.

25 So, I will just stop by saying we have

201

1 started in small animals, we have moved to large

2 animals, and we have moved to patients, and these

3 are the cells that we knew the most about, and we

4 have had to say you know what, we missed a lot, and

5 we have had to go back and take the safety and

6 functional effects that we have seen and reevaluate

7 them in all of these animal models again, and I

8 think that should be a lesson for going forward.

9 Is it time for randomized trials, and, if

10 so, who? The think the questions we have to ask is,

11 is it safe, who are the right patients, what are

12 the appropriate endpoints.

13 I show this every time. If we do this

14 wrong, we are really going to doom what I think is

15 an exciting field. I don\'t want to be standing up

16 here talking to you about the gene therapy lessons

17 that we could have learned and didn\'t.

18 I think what we can learn from gene

19 therapy is in 6 open-label trials, they were all

20 positive, and 4 out of 5 randomized, double-blind,

21 control trials, they weren\'t. Is that because

22 patients got better care, or because there was a

23 better placebo effect? Well, we won\'t really know

24 until we do the randomized trials, but we need to

25 under-promise and over-deliver, not conversely.

202

1 These are the people who did all the work,

2 and I thank you.

3 \[Applause.\]

4 DR. RAO: Thank you, Doris.

5 We are open for questions.

6 Q&A

7 DR. ROSE: Dr. Taylor, I enjoyed your

8 talk, but I must say, as a clinical cardiologist, I

9 just have difficulty in envisioning how these

10 myotubules, that presumably result from the

11 injection of the myoblasts into scar, increase

12 systolic wall thickening, which is what I would

13 accept as evidence of improved regional function.

14 Unfortunately, many of your images weren\'t

15 showable, but was the increase in thickening that

16 you showed on that on that one graphic slide, was

17 that from your larger animal models as opposed to

18 your smaller animal models?

19 DR. TAYLOR: No, that was from rabbit.

20 DR. ROSE: You mentioned early in your

21 talk about a disconnect between the numbers of

22 cells and the functional improvement. Is that true

23 for the myoblast injection? In other words, there

24 was probably a range of responses, I would imagine,

25 some had perhaps better systolic thickening

203

1 improvement than others, and by histologic

2 examination, were there more myotubules or were the

3 myotubules oriented in a different direction than

4 the animals that perhaps had less improvement in

5 systolic thickening?

6 DR. TAYLOR: Have we quantified that

7 unequivocally? No, because it is very difficult to

8 quantify the number of cells in an infarct and know

9 that they are actually myoblasts in the cells you

10 injected because we are giving autologous cells.

11 One of the rate limiting steps in this

12 field is having good markers for the cells, and

13 that is one of the reasons we went to iron, so that

14 we could actually stain our sections later for

15 Prussian blue and look for iron and say, okay,

16 these are the cells we injected. We shouldn\'t see

17 that in normal cells.

18 So, we are starting to now look and try to

19 answer that question. What we do know is that with

20 a number of studies that have been done by a number

21 of groups, the number of cells that you retain in

22 the scar after you inject them is about 15 to 25

23 percent of the cells you initially put in there

24 probably. On a good day, maybe 30, 40 percent of

25 the cells you put in there, but that is rare.

204

1 What we also know is that numbers of cells

2 can die over time, but we also know that these

3 cells can proliferate over time, and when we look

4 for proliferation, we in fact can find that in

5 these scarred regions.

6 I don\'t actually think that we are seeing

7 evidence of long myofibers necessarily. We see

8 cells that line up with each other and connect with

9 each other, and we believe that these cells are

10 being mechanically induced to contract.

11 We know when you stretch muscle, it

12 contracts. Do I think they are electrically

13 coupled with the rest of the heart? No. Do I

14 think they are mechanically coupled? Yes. What we

15 see is we measure left ventricular pressure going

16 up, we see pressure plateau, and right at the time

17 that pressure within a few milliseconds after

18 pressure plateaus, we see our crystals move in that

19 region of the heart, and then pressure decreases,

20 and then we see that again.

21 We can track that beat after beat after

22 beat in the scarred region. We can go from

23 negative work loops where the rest of the heart is

24 being pushed by the remainder of the heart to

25 positive work loops when we put the

205

1 cells\--actually, I have got it backwards\--negative

2 in this direction versus positive work loops, that

3 actually correspond to systole.

4 So, I believe that there is wall

5 thickening and actual contraction going on in that

6 region, and I think our sonomicrometry data are the

7 most convincing data.

8 Is it possible to get the image to show,

9 so I could try to show one of the functional wall

10 thickening images, so you can actually see that?

11 If you just give me the slide thing back, I will

12 try.

13 I am going to try this first.

14 So, this is, in theory, it is beating

15 here. No, apparently I can\'t, maybe you can, but

16 we are able to actually show thickening and I will

17 pull my computer out to show anybody later who

18 wants to see it.

19 You can actually see in the sham-treated

20 animal, here is the scarred region and only in the

21 very center of the scar is it not thickening

22 anymore. The scar was from about here to here, by

23 contrast.

24 The scar of the sham-injected animals over

25 here is not thickening at all. Here, we only don\'t

206

1 see thickening right in the very center of the

2 scar, and we used contrast enhancement, so we took

3 10 slices through the rabbit heart, in a long axis

4 view, we used contrast, and only where we saw

5 contrast gadolinium did we call that infarct, and

6 then measured thickening in that region, and we did

7 that in a blinded way.

8 We are pretty convinced that we see wall

9 thickening in that region.

10 DR. BLAZAR: You made a good point that

11 the larger animals allow you to assess function

12 much more directly and extrapolate to humans. With

13 the smaller animal models, you have a higher

14 throughput. So, the question is, what is the data

15 that says that the smaller animals extrapolate, the

16 large animals extrapolate to the human as you go

17 through all of these different examples, because

18 one would hear your presentation and think that it

19 really just should be restricted to large animals

20 minus a few, more esoteric.

21 DR. TAYLOR: What I can say is we have

22 made measurements in rabbit and pig for many, many

23 years, and I swore we would never use mice or rats,

24 and then we decided that we wanted to start making

25 comparisons with bone marrow derived cells, and we

207

1 didn\'t have the markers to isolate those cells in

2 rabbit or pig, so we went back and did the same

3 experiments in mice, and we got the same results.

4 So, now I have to bite my tongue and show

5 mice data even though I swore we never would. I am

6 convinced at least with MRI, that we can make

7 meaningful measurements that show us the same sort

8 of thing.

9 DR. BLAZAR: Although you also made a

10 point as to the dose, location, et cetera, it would

11 seem that that is going to be extremely difficult.

12 DR. TAYLOR: It is very difficult. That

13 being said, what we do is we inject the cells in

14 the center of the scar in a mouse. Do I believe we

15 get the same percentage that we get in larger

16 animals? No. We really don\'t know what number we

17 actually get in.

18 We have started doing some biodistribution

19 studies to try to answer that, and we are mostly

20 doing those in rabbit and pig, because I have no

21 confidence for the numbers we get in mice. In

22 terms of doing stem cell studies, though, I think

23 it is critical that you use something where you can

24 clearly define the cells.

25 DR. ALLAN: A little bit of a follow-up,

208

1 which is use of nonhuman primates. Somebody

2 mentioned that there were some primate studies.

3 This morning, I think somebody just referred to

4 them.

5 DR. TAYLOR: Right, with bone marrow

6 cells.

7 DR. ALLAN: Is it with bone marrow cells?

8 Because you can use those markers on many of the

9 nonhuman primates, you can look at stem cells.

10 DR. TAYLOR: Right.

11 DR. ALLAN: Maybe to use that model as a

12 step between \"large\" animals and humans, because

13 anything that you have derived that looks promising

14 could through that nonhuman primate.

15 DR. TAYLOR: I think that is good point,

16 and we have actually started collaborating with

17 some people in California who do nonhuman primate

18 studies. I can tell you that they are God-awful

19 expensive, they are hard to do, and I personally

20 find them hard to do.

21 Sometimes we just have to get over it, but

22 I think if the pig data are good enough, I would

23 rather stick with pigs, and the fact that we can

24 use some of the same stem cell markers has made me

25 focus more in that area.

209

1 DR. BORER: That was a very interesting

2 presentation. I have a question that sort of falls

3 into are we looking at the data the right way or

4 oversimplifying box. You can\'t argue with success,

5 and I believe that some contractility is probably

6 better than done and perfect is the enemy of good,

7 and all that stuff, but, you know, you have put in

8 skeletal muscle cells, and they contract, and yet

9 to get useful work from the heart, forced

10 generation is just part of the equation, you have

11 to have forced transmission, as well, and I haven\'t

12 heard anything yet about remodeling at the

13 extracellular matrix and regeneration of dystrophin

14 ECM hookups, and whatever, that might demonstrate

15 that we are actually developing a forced

16 generation/forced transmission system.

17 I don\'t know how important that is

18 ultimately, but it makes me wonder, the fact that

19 we haven\'t heard about that, and I am not sure that

20 I could expect it would happen, that we are

21 actually looking at the data the right way, so

22 could you talk a little bit about that?

23 DR. TAYLOR: Sure. The one piece of data

24 that we do have, that I think addresses that, is

25 the compliance data where we looked at changes in

210

1 strain with the different cell types, and that

2 begins to address matrix and what is going on in

3 that matrix - does it look at signaling, does it

4 look at MMPs, does it look at any of that? No.

5 I think the bottom line is when we started

6 this 15 years ago, our goal was to show it worked,

7 and then once we showed it worked, we thought we

8 would go back and figure out how it worked.

9 As soon as we showed it worked, God and

10 everybody wanted to do it clinically, and so we are

11 having this discussion rather than understanding

12 how it worked, which we have had to develop in

13 parallel. I think we are still catching up in

14 terms of trying to develop, trying to understand

15 the mechanisms by which it works.

16 Five years ago, at American Heart, Michael

17 organized a session, and there were probably five

18 or six people talking about this. If you look at

19 American Heart now, there are two days of people

20 talking about this. People didn\'t believe it five

21 years ago.

22 Now we have a critical mass in the field

23 and we can start asking those questions, and I

24 think the data will emerge over the next couple of

25 years.

211

1 DR. RAO: Dr. Ruskin.

2 DR. RUSKIN: Doris, you described a very

3 significant prolongation of action potential

4 duration in the myoblasts from about 20 to 120

5 milliseconds. That suggests a change in iron

6 channel expression. Do you have any information as

7 to how that came about?

8 DR. TAYLOR: Gus Grant told me it did?

9 No.

10 DR. RUSKIN: I will buy that.

11 DR. TAYLOR: Short answer is no except

12 that we know that we started seeing a plateau phase

13 which wasn\'t there before, and we didn\'t change the

14 rate of rise of the action potential. Do I know

15 any more than that? No.

16 Have we looked for channel markers? You

17 know, here I am saying, well, the only thing I will

18 believe that if you tell me it\'s a cardiocyte, is a

19 channel marker, have we looked for channel markers?

20 No, because we did all of that in rabbits, and the

21 darn markers don\'t exist.

22 Are we now trying to figure that out in

23 some of these other animal models? Yes, and I

24 think that is where some of the mice genetic models

25 of changes in electrical activity in the heart may

212

1 actually be really useful in terms of trying to

2 dissect what myoblasts can do.

3 DR. HARLAN: You had a great quote from

4 Einstein. I will have to give you a quote I have

5 from Osler, where he talks about stern iconoclastic

6 spirit that we need, and that you reflected.

7 My question is I think I misunderstood,

8 you implied that there was not the cellular

9 specificity that people assume, that you have seen

10 some contractility with myoblasts, but also with

11 bone marrow. I wonder how extensively you have

12 studied that with other cell types.

13 DR. TAYLOR: We have looked with

14 myoblasts, we have looked with bone marrow stroma,

15 and we have looked with bone marrow mononuclear

16 cells, and we see an improvement with all of those.

17 We haven\'t gone back and dissected the bone marrow

18 mononuclear cell populations, we are starting to do

19 that now.

20 Other people have seen the same thing with

21 MAPC cells. So, have we dissected that in detail?

22 No, but by the criteria that we have used, which is

23 sonomicrometry and MRI, we see the effect, and yet

24 we don\'t, when we look at histology, we don\'t see

25 the same degree of muscle formation with all of

213

1 those different cell types, which begins to argue

2 that mechanism is more complicated than we thought.

3 DR. HARLAN: Let me ask it this way. Are

4 there cells that you have looked at, that you

5 inject, where they don\'t work?

6 DR. TAYLOR: Fibroblasts.

7 DR. HARLAN: Fibroblasts don\'t work.

8 DR. TAYLOR: Fibroblasts actually improve

9 compliance, but make systolic function worse in our

10 hands.

11 DR. RAO: Dr. High.

12 DR. HIGH: I want to ask one question to

13 try to reconcile some of your data preclinical

14 studies with some earlier clinical work that we

15 heard about.

16 Was that in rabbits or pigs that you said

17 that you needed at least 10

8 cells to see an

18 effect, was that rabbits?

19 DR. TAYLOR: That was rabbits\--no, 3 x 10

7

20 in rabbits, 108 was in pigs, I am sorry.

21 DR. HIGH: Okay. Then, the 10

9 cells that

22 are being injected in the clinical study would be

23 roughly appropriately correlating in terms of\--

24 DR. TAYLOR: Right, we actually see a

25 better effect with 3 x 10

8 cells in pig. We

214

1 haven\'t gone up to 10

9 cells yet although we have

2 plans to do that. Philippe can tell you, you start

3 dealing with massive numbers of cells, and when you

4 are doing this in an autologous way, and you are

5 dealing with cells that you have to keep at low

6 confluence, it gets out of hand pretty quickly.

7 DR. HIGH: You said that in, is it the pig

8 studies, about 15 to 20 percent of the injected

9 cells are retained?

10 DR. TAYLOR: That is actually not data

11 from my lab, that is data from other groups, and

12 that has been in some pig studies, and I believe in

13 some\--I know of pig, and I can\'t remember what

14 else.

15 In our hands, in rabbit, we see a little

16 bit higher than that, on the order of 20 to 25

17 percent, but that has only been in a few studies

18 with indium-labeled cells, so I don\'t trust those

19 numbers yet.

20 DR. HIGH: Is that known to be a function

21 of time after injury?

22 DR. TAYLOR: Actually, what we found is

23 that the longer we wait after injury, the easier it

24 is to get cells to hang around in the heart, that

25 if we inject cells at 2 weeks, we get fewer cells

215

1 retained than if we inject cells at a month.

2 What we also know is if we inject cells in

3 normal heart, they all go in the cardiac vein and

4 get carried elsewhere, that the junctions in the

5 myocardium are so tight that it is really hard to

6 get those cells into the normal heart.

7 Again, it gets back to injection. We

8 have come up with a way where we inject the cells,

9 we see a bleb, we wait for the bleb to go away, we

10 inject more cells, but that is just empiric,

11 because it works for us. Do we know how it is

12 being done by other groups? No clue.

13 DR. SCHNEIDER: Doris, you summarized

14 nicely both the cellular complexities and the

15 technical complexities that are involved here, and

16 I wanted to comment about one in each of those

17 categories.

18 You talked about cryoinjury versus

19 coronary artery ligation, and I wanted to agree

20 with your comment that relatively little of the

21 work in the field is being done with ischemia

22 reperfusion injury, which more closely resembles

23 the clinical situation particularly in an era of

24 stenting and reperfusion therapies.

25 A further complication there, though, is

216

1 that much as was learned over a period of years in

2 investigations of stunning, it may be necessary to

3 distinguish between open-chested ischemia

4 reperfusion injury and the chronically instrument

5 close-chested animal that undergoes ischemia

6 reperfusion injury, which is something that a few

7 labs have been able to develop as a means to

8 minimize potential artifactual effects of the

9 surgical procedures.

10 DR. TAYLOR: Right.

11 DR. SCHNEIDER: With respect to cellular

12 heterogeneity, you talked about the possibility

13 that SP cells in the skeletal muscle population

14 might be important. Michael Rednicke has identified

15 in skeletal muscle, and investigators at Indiana

16 have, as well, a scar-positive, LIN-negative,

17 CD34-negative, CD45-negative population that lacks

18 any ability to undergo hematopoietic

19 differentiation and very closely resembles the

20 scar-positive cells we found in adult heart.

21 DR. TAYLOR: I have seen data from the

22 University of Minnesota like that, as well, and I

23 also should say that Johnny Heward at Pittsburgh

24 has found that as you increase the passage number

25 of cells, and these are actually old data, from \'98

217

1 I believe, that as you increase the passage number

2 of cells in vitro that you see differences, I think

3 passages 3 and passage 5, or something like that,

4 give you much better functional results in

5 engraftment than passages 1, 2, and 4, and the

6 desmin staining of those changes.

7 So, I think we probably are selecting for

8 different cells.

9 DR. SCHNEIDER: Along those lines, do you

10 know if the scar-1-positive fraction goes up or

11 goes down in your skeletal muscle cells over time?

12 DR. TAYLOR: Scar-1, we haven\'t look at

13 scar-1 in our population of cells. We have looked

14 primarily for SP cells. What I will say is that we

15 grow our cells a little differently than most

16 people. As you know, we make an explant and

17 actually allow our cells to grow out from the

18 explant, and we are getting a much higher

19 percentage of more immature cells than other people

20 as a result, and I think that is because we are not

21 throwing them all away when we do the filtration

22 and enzyme digestion, and those kind of things, and

23 that may impact some of our functional data.

24 DR. RAO: We will take on last question.

25 Dr. Simons.

218

1 DR. SIMONS: Doris, you mentioned that up

2 to 90 percent of cells that they injected in the

3 heart die soon thereafter. So, the cells that are

4 still there, do they need to be concentrated at a

5 certain per sort of square area of the cell, or can

6 you spread them as much as you like, so is it

7 really a mechanical effect or is it something that

8 the cells make, because you are making a point that

9 it matters where you actually inject them?

10 DR. TAYLOR: What we have found is that we

11 can do three parallel injections, and we get the

12 same effect as if we do one injection, as if we do

13 a star-shaped injection, so we spread them out in

14 different\--I mean we have given them under

15 different geometries, and we see the same effect,

16 the same dose of cells.

17 What we don\'t know is how many die under

18 each of those conditions.

19 DR. SIMONS: So, if you can spread them

20 around, that would imply that that this is probably

21 not a purely mechanical effect?

22 DR. TAYLOR: I think there is absolutely

23 some truth to that. I don\'t think it\'s purely

24 mechanical. I also don\'t think\--that is what I

25 said when we use these muscle cells, there are

219

1 multiple populations of cells in there, and I think

2 different ones have different effects.

3 Absolutely, I think mechanism is an open

4 question, we really don\'t know.

5 DR. RAO: In the interests of time, I

6 guess we move on. Thank you, Doris.

7 I apologize for not recognizing members of

8 the public, but this is the part of the meeting

9 where we have to give priority to the committee

10 members in terms of questions. People in the

11 audience can address the committee in the open

12 session.

13 Dr. Itescu.

14 Preclinical Models - Hematopoietic and

15 Mesenchymal Cell Therapies for Cardiac Diseases

16 DR. ITESCU: Thank you very much for

17 inviting me here today. As if the talks haven\'t

18 been complex enough, I have got the difficult task

19 of speaking for 30 minutes and covering a variety

20 of animal models, as well as a variety of cell

21 types, so I hope it will be cohesive enough.

22 The issues to consider in this field, I

23 have tried to address some of them here in this

24 slide in terms of small animal models or large

25 animal models, and it is the precise

220

1 characterization of cell type and population to be

2 used to define the cell source and process for

3 isolation to determine if there is a need for ex

4 vivo culture and expansion to identify the

5 mechanisms of action for inducing cardiac repair,

6 to identify appropriate animal models, in other

7 words, small versus large, species-specific versus

8 those that use human products and those that

9 involve acute versus chronic ischemia. These are

10 all very, very important questions.

11 Finally, experiments that address

12 dose-ranging studies for functional correlation and

13 toxicity, and the last question which has not been

14 touched on yet today, which I will at the end of my

15 talk, is that between autologous versus allogeneic

16 products.

17 The adult mammalian bone marrow contains

18 two stem cell populations at least. The one that

19 we are most familiar with are hematopoietic stem

20 cells defined as being CD34-positive, and more

21 importantly, CD45-positive.

22 These cells form blood-forming elements,

23 such as monocyte and macrophage lineage cells,

24 these account for about 10 to 20 percent of the

25 CD45-positive fraction, and more recently, cells

221

1 that are endothelial progenitor cells or

2 angioblasts that express these markers plus several

3 others such as AC133 and c-kit.

4 The second population, the

5 non-hematopoietic stem cell fraction is typically

6 CD45-negative and CD34-negative, and at least three

7 cell types have been defined within this fraction -

8 the mesenchymal stem cells, which really is poorly

9 termed as stem cells since these cells are defined

10 based on their in-vitro culture characteristics.

11 The way they are isolated is very crudely, very

12 grossly based on density properties and plastic

13 adherence. In fact, the population cells that is

14 pulled out initially is very heterogeneous.

15 A second stem cell type is the MAPC that

16 you have heard about. These cells are potentially

17 more homogeneous in nature, however, the fact that

18 they are dependent on negative immunoselection

19 means that again we don\'t really know what the cell

20 type that is ultimately derived is, and the cell

21 culture conditions are very laborious and require

22 low density for outgrowth.

23 Finally, there is a third population of

24 mesenchymal precursor cell or progenitor cell which

25 can be defined on the basis of several surface

222

1 markers and can be selected by immunoselection,

2 positive immunoselection freshly from bone marrow,

3 and we are using these cells currently in my

4 laboratory, and I will touch on them a little bit

5 towards the end of the talk.

6 In respect to the hematopoietic stem cell

7 fraction, the CD34/CD45 fraction, there is high

8 frequency within the bone marrow compartment, as I

9 have mentioned, 10 to 20 percent of macrophages, 1

10 to 2 percent of the CD34 progenitor cells that can

11 be freshly isolated without any requirement for

12 in-vitro culture and expansion, and are well

13 characterized for many years with established

14 isolation protocols.

15 In this slide, I tried to summarize the

16 interaction and cooperation between the

17 CD45-positive and 45-negative subsets in terms of

18 vascular network formation, so that in the bone

19 marrow, the primordial circle stem cell may give

20 rise to the CD45-positive/34-positive endothelial

21 progenitor cells, which egress to the embryonic

22 organs where you get the initial vasculogenic

23 small, thin-walled capillary development.

24 At the same time, the CD45-negative

25 parasite fraction derived from a mesenchymal

223

1 progenitor cell produces a variety of factors

2 including VEGF, FGF, angiopoietin, and SDF-1, which

3 can interact with these vasculogenic capillaries to

4 give rise to the more mature vascular network

5 through a process of angiogenesis.

6 In addition, the parasites may also

7 migrate and give rise to the smooth muscle outer

8 wall, so that you really have this cooperation

9 between both cells and factors to give rise to the

10 permanent new vessel formation.

11 With this as a background, I will like to

12 address several studies that have looked at how

13 angiogenesis per se can improve cardiac function.

14 This study from Kobashi \[ph\] and Collins in 2000, I

15 think was the first to demonstrate or one of the

16 first to demonstrate that whole bone marrow in a

17 small rodent model could induce transient

18 angiogenesis and improve cardiac function.

19 You can see here the implantation of whole

20 bone marrow into the ischemic myocardium of a rat

21 gives an increase in local production of VEGF

22 protein within 24 hours, but by 7 days this protein

23 production is gone, and in parallel, the

24 angiogenesis that is occurring in the heart is

25 again transient, maximal at 1 week

224

1 post-implantation, gone by 4 weeks.

2 So, I think this particular model gives us

3 some pause and suggests that the whole bone marrow

4 approach may not be a way to give rise to permanent

5 vasculature. Others have used this sort of

6 approach now in larger models, in pigs. Work from

7 Kemiharder \[ph\] and colleagues demonstrated that in

8 a LAD-ligated porcine model, again whole bone

9 marrow implantation gives rise to angiogenesis.

10 This is within 6 weeks post-implantation, you can

11 see improvement in collateral flow associated with

12 some degree of improvement in function.

13 In parallel studies they published about

14 two years ago now, the same group demonstrated that

15 in a more chronic ischemia model in pigs using the

16 ameroid constrictors, they were able to again

17 induce angiogenesis using mononuclear cells from

18 bone marrow, as well as mononuclear cells from

19 peripheral blood to a lesser extent. You can see

20 improvement in regional blood flow and improvement

21 in global parameters of cardiac function including

22 ejection fraction DPDT, and a reduction in the

23 ischemic area.

24 But again, all of these studies were done

25 in the setting of an acute ischemic or perhaps more

225

1 subacute ischemic model in this scenario, and the

2 animals were followed up for no more than 6 weeks,

3 so if we take that into consideration that in the

4 smaller rodent, the neovascularization and the

5 cardiac function improvement was transient, but I

6 think the 6-week follow-up in these larger models

7 is really not adequate especially in a pig where

8 the physiology much more closely resembles that in

9 man.

10 The next approach is to look at perhaps

11 subsets of some of these cells, and I would like to

12 show you some work on endothelial progenitor cells

13 defined by surface markers.

14 Isner and colleagues originally described

15 these cells demonstrating that endothelial

16 progenitor cells were present in the bone marrow,

17 were released to the circulation under certain

18 signaling and present from growth factors under

19 ischemic conditions, and that these cells can

20 incorporate into regions of ischemia.

21 So, we asked the question if one could

22 identify these cells in the adult marrow of humans,

23 could they potentially improve function through a

24 process of neovascularization.

25 The model that we use in my lab is one of

226

1 using the incompetent nude rat, which is an athymic

2 rat model that lacks T cells and B cells, continues

3 to have some degree of natural killer cell

4 function, in other words, it is a linking model,

5 but it is able to tolerate certain types of human

6 cell injections, particularly human cells that have

7 not been in vitro cultured and expanded.

8 Our objective was to cause a permanent

9 occlusion of the anterior descending left coronary

10 artery, in contrast again to other models that

11 perhaps are using the reperfusion type of model.

12 Our objective was to induce an infarct and see

13 whether we could then protect the subsequent

14 territories of myocardium still at risk in the

15 periphery.

16 We mobilized in the progenitor cells from

17 healthy human donors using GCSF, think that, in

18 fact, in the future this would have been a way to

19 move towards clinical trials, in other words, being

20 able to harvest large numbers of progenitor cells.

21 In our studies, we then immunoselected

22 these on the basis of surface markers, CD34 and

23 CD117, injected these into the tail vein of acute

24 ischemic rats to see whether they homed to the

25 myocardium.

227

1 I would just like to pause for a minute to

2 say that given the recent paper in the Lancet, I

3 think a week or two weeks ago, I think that

4 certainly raises a question about how such cells

5 are going to be accessed in large numbers if they

6 are to be used at all at the time of an acute

7 myocardial infarct because I think under a local

8 anesthetic, a bone marrow aspirate gives rise to

9 extremely few numbers of these types of cells, and

10 you really do require either large harvesting or

11 large numbers to be mobilized, and I think at this

12 point in time, GCSF is not the agent to be used

13 post-infarct.

14 In any case, the point of our studies were

15 to see whether intravenous administration at the

16 time of acute infarct would lead to selective

17 homing to the myocardium, and we have met these

18 trafficking pathways based on the type of chemokine

19 expression that occurs post-infarct, and we found

20 that B cells selectively migrate to the

21 peri-infarct region, where they within two weeks

22 induce both incorporation into vessels, and this is

23 staining with antihuman CD31, and you can see cells

24 that are previously labeled with dye/Dil,

25 incorporate into vessels of the peri-infarct

228

1 region, but in addition, a very dense induction of

2 angiogenesis at the more distal areas as defined by

3 rat-specific anti-CD31 antibodies.

4 So, again, akin to the type of data shown

5 by Dr. Epstein, it appears that these cells are

6 able to not only incorporate and induce

7 vasculogenesis, but presumably secrete a variety of

8 antigenic factors.

9 From a pathophysiologic perspective, we

10 see that when we induce a vascular network at the

11 peri-infarct region, you can see these large

12 capillaries. There is nice viability of the

13 cardiomyocytes at the peri-infarct region as

14 opposed to these apoptotic cardiomyocytes. You see

15 reduction in scar formation and clearly a viability

16 and survival of the myocytes.

17 But in addition to that, we were quite

18 surprised to see that as early as five days

19 post-neovascularization, there seems to be

20 induction of cell cycling by endogenous

21 myocyte-like cells, and you can see these by

22 confocal microscopy small cells of the peri-infarct

23 region that express alpha-sarcomeric actin in blue,

24 but more interestingly, in yellow, the staining

25 with the rat-specific anti-KR67 antibody, which

229

1 recognizes cycling cells only of rat origin. You

2 can see lots of these type of small cells in the

3 peri-infarct region only in the situation where we

4 also have neovascularization being induced by the

5 human cells.

6 Within two weeks, in these same areas,

7 what you see is that the cells that express

8 sarcomeric actin now express troponin, and you can

9 see an example of that large mature differentiated

10 cardiomyocyte where the nucleus continues to be in

11 cell cycle, and we see about 8-fold increase in

12 numbers of these type of cells at the peri-infarct

13 region that has received the human progenitor

14 cells.

15 We think that the cells or the new

16 capillaries are secreting factors that are

17 presumably inducing cycling of these

18 cardiomyocytes, and we are looking at a variety of

19 anti-apoptotic genes that are increased in

20 expression in these cells including redox-related

21 anti-apoptotic genes.

22 The end result is a very significant

23 salvage of the anterior wall of myocardium. You

24 can see that here, and you can see in this scenario

25 obviously a very dramatic left ventricular scar

230

1 formation and aneurysmal formation here.

2 We can show a dose-dependent reduction in

3 scar formation at the left ventricle as we increase

4 the number of progenitor cells that we inject.

5 Now, others have published recently that

6 perhaps the CD34 cells may have the ability to

7 become multipotential, perhaps transdifferentiate,

8 however, the data are fairly scant, and I would say

9 that given that only 1 in 7 healthy animals

10 demonstrated HLA human troponin co-staining, I

11 think at this point, it is open to debate whether,

12 in fact, these cells do transdifferentiate or

13 whether these cells have the capability to simply

14 fuse with the donor cells.

15 Nevertheless, whatever the precise

16 mechanism, it appears that hematopoietic stem cell

17 injection, either intravenously or

18 intramyocardially, does result in significant

19 global improvement in cardiac function, as we

20 showed here, at least 30 percent improvement in

21 ejection fraction, which is permanent. This was 15

22 weeks of follow-up post-LAD ligation, and we can

23 look at fractional shortening or DPDT and

24 demonstrate similar sort of things.

25 In this scenario, you can see that there

231

1 are other cell types that we use as controls,

2 CD34-negative cells, saphenous vein endothelial

3 cells, and what this sort of study shows is that

4 you must always use different cell types when you

5 are trying to evaluate a particular therapy. It

6 cannot just be compared to saline or existing

7 control. You have got to demonstrate specificity

8 in the product that is being tested.

9 Moreover, the question that we asked was

10 what about if we combine this type of an approach

11 with existing therapies, because really that is the

12 question you want to address, and the example of

13 restenosis with stenting is just one type of

14 combination therapy between a new product and

15 existing therapies.

16 What you see here is if we combined CD34

17 cells with both ACE inhibitors and beta blockers in

18 the same rodent model, we had very significant

19 synergy in outcome, and histologically, the reason

20 for that was really not because the two products

21 worked in a similar way, but because they had very

22 separate mechanisms.

23 The ACE inhibitors and beta blockers had

24 no impact on neovascularization, but, in fact,

25 prevented fibrosis in the posterior wall as they

232

1 are known to do, so we have two very different

2 mechanisms of action working in synergy, but it

3 could have just as easily worked the other way

4 around, and that is why we did the experiment.

5 It could have been that by reducing wall

6 strain or reducing fibrous replacement, we may have

7 reduced the drive for endothelial cells to induce

8 neovascularization and improve cardiac function.

9 So, what can be concluded from preclinical

10 data using non-cultured cells? The objectives

11 that one should look for when you do these kind of

12 studies include identifying the predominant

13 mechanism by which a given cell type induces

14 cardiac recovery.

15 A comparison of efficacy of one given cell

16 type with others, identification of the tissue

17 distribution of that cell type following the

18 preferred mode of delivery, unique short- or

19 long-term risks associated with the preferred cell

20 type, unique risks associated with the method of

21 cell acquisition or isolation. I have given you

22 the example of GCSF administration, and perhaps

23 alterations in efficacy or safety following

24 coadministration with standard therapies.

25 Now, I would like to shift attention a

233

1 little bit to the second population I want to talk

2 about, the non-hematopoietic stem cells, the

3 CD34-negative, CD45-negative fraction.

4 These cells are rare. They account for

5 less than 0.01 percent of bone marrow cells, and

6 they do require, for this reason, in-vitro culture

7 and expansion.

8 I apologize about the complexity of this

9 slide. I will just take you through it a little

10 bit. I mentioned these three types of mesenchymal

11 lineage cells that people are working with. The

12 so-called mesenchymal stem cells are more likely

13 committed progenitors, but the point is that they

14 are tolemerase-negative.

15 The multipotent adult progenitor cells are

16 MAPCs, tolemerase-positive, and the stromal or

17 mesenchymal precursor cells are also

18 tolemerase-positive. These cells may be related to

19 each other, but all three types of cells give rise

20 to mesoderm lineage cells. The MAPCs have also

21 been shown to give rise to endodermal and

22 ectodermal cells. It appears that the mesenchymal

23 precursor cells can also give rise to endoderm and

24 ectoderm.

25 With respect to mesoderm, which is really

234

1 what we are interested in primarily here, cardiac

2 muscle and smooth muscle lineage cells can both be

3 differentiated from the mesoderm, but also these

4 cells can give rise to other cell types with

5 mesodermal lineage.

6 The so-called mesenchymal stem cells are

7 cells that are derived from whole bone marrow and

8 then isolated by simple density centrifugation to a

9 particular layer. These cells are then taken from

10 the interface, put down on plastic, adhered for 24

11 to 48 hours, and then the cells that continue to

12 adhere are then cultured again fairly crudely with

13 generally fetal bovine serum for weeks at a time.

14 So, if you understand how this process is

15 initiated, you understand that really, the

16 isolation of these cells is so crude that you are

17 starting out with a very heterogeneous population

18 simply based on density characteristics, and that

19 the true multipotential cell within this fraction

20 probably is not more than 1 in 1,000 to 1 in 10,000

21 of the cells you are starting out with.

22 In any case, after, say, 10 to 14 days of

23 culture and passage, you see the sort of

24 monomorphous type of fibroblastoid phenotype, and

25 you see the cells that have survived this period of

235

1 culture are fairly homogeneous in terms of the type

2 of markers that are being used.

3 There are specific antibodies that can

4 define surface characteristics of these cells, SH2,

5 SH3, endoglin, and I am not sure what SH3 actually

6 defines. But in any case, these cells, after two

7 weeks in culture, become fairly homogeneous.

8 I borrowed this slide from Dr. Epstein,

9 his recent paper in Circulation Research, which

10 shows that these cells, after several passages in

11 culture, demonstrate production following induction

12 of ischemia of a variety of factors that are

13 associated with both angiogenesis and

14 arteriogenesis, and in a nice model or rat hind leg

15 ischemia, you can see\--I think you showed this

16 slide already\--demonstration of improvement in

17 perfusion following MSC infusion in these cells in

18 ligation of the hind leg artery.

19 But in addition to secretion of

20 pro-arteriogenic factors, the interest in these

21 cells lies in the fact that they seem to have

22 multipotential capability in that under appropriate

23 culture differentiation conditions, they can be

24 differentiated to adipocytes, chondrocytes, and

25 osteocytes.

236

1 Work now since at least 1999 has

2 demonstrated that if you use appropriate inductive

3 signals in these cells, in this case, 5-azacytidine

4 to demethylate the cells, but there have been

5 reports of other agents that can do similar sort of

6 things, you can push the cells toward a

7 cardiomyocyte-like lineage with appropriate marker

8 expression and electromicroscopic criteria

9 consistent with BT myotubes.

10 Probably the best study to date to

11 demonstrate that these cells can actually do

12 something in vivo is work from Victor Dzaus\' lab

13 published last year in Nature Medicine. Here, we

14 are talking about again rat mesenchymal lineage

15 cells cultured in the way I have just defined with

16 density separation and long-term culture with

17 bovine serum, fetal calf serum, and what you see is

18 after injection of these cells into the

19 peri-infarct region of rats following LAD ligation,

20 these cells appear at least phenotypically to

21 acquire markers of cardiomyocytes, so that they

22 express myosin heavy chain, cardiac troponin,

23 sarcomeric actin, and connexin 43. This is within

24 two weeks of implantation.

25 However, the majority of these cells just

237

1 don\'t survive when you put them in. They die after

2 early transplantation. Causes appear to be

3 ischemia again, competition for oxygen nutrients,

4 inflammatory and oxidative stress in the

5 post-infarct myocardium, loss of survival signals

6 from cell to cell or cell matrix interactions, and

7 probably lack of tolemerase activity and

8 self-renewal capability by these cells, and I said

9 to you earlier that these cells just don\'t express

10 tolemerase, certainly when they are in culture.

11 The work by Mongi, et al., in fact, showed

12 that if you genetically modify these cells with an

13 AKT transgene, you could significantly prevent

14 these cells from dying following implantation,

15 significantly reduce the apoptotic index, increase

16 survivability, and overall improve function almost

17 to the level of the non-infarcted animals, quite

18 impressive, but it required essentially making them

19 resistant to death or apoptosis by AKT

20 overexpression.

21 I will just move along to the second

22 population of cells, the MAPCs. These cells, as I

23 mentioned, are defined based on negative expression

24 of all known markers, CD45 and many other known

25 markers of lineage-specific differentiation, and

238

1 following long-term culture, these cells have been

2 shown to be capable of differentiating to a variety

3 of tissue types requiring cocktails of various

4 cytokines and differentiation factors.

5 In particular, following activation with

6 VEGF and implantation into appropriate medium, in

7 this case, tumor model in the scid mouse, these

8 cells appear to be able to incorporate into first

9 neovascularization, and you can see they can induce

10 networks of capillaries, neocapillaries really, in

11 wound-healing tissues, so they may contribute to

12 vascularity.

13 With that as a backdrop, Gallegos and

14 colleagues at the University of Minnesota decided

15 to look at whether again fresh non-differentiated

16 MAPCs from dogs could be implanted into an acutely

17 LAD-ligated model and could perhaps improve

18 function in some form.

19 The model was to induce again a complete

20 LAD ligation in the dog, autologous cells were

21 taken from the marrow, they were expanded for about

22 four weeks, and then four weeks later, injections

23 were put directly into the myocardium.

24 What they found was that a month

25 post-injection of the cells, there appeared to be

239

1 increase in myocardial perfusion reserve defined as

2 perfusion under stress relative to perfusion at

3 rest compared to the controls, and this is in four

4 animals.

5 But significantly, although there was some

6 improvement in regional myocardial contractility

7 within the infarct area and the peri-infarct zone,

8 defined as circumferential shortening and radial

9 thickening, there was actually no improvement in

10 global function of cardiac measurements as defined

11 through systolic improvement by fractional

12 shortening or ejection fraction.

13 Now, just to the last area that I would

14 like to touch on, and that is the possible use of

15 cultured allogeneic stem cells for cardiac disease.

16 The issues to consider here are whether or not

17 cells constituitively or under inductive conditions

18 express surface molecules, surface HLA molecules,

19 and co-stimulatory molecules, secondly, whether

20 stimulation of recipient T cell responses occur in

21 vitro, and, thirdly, whether these cells might

22 induce inflammatory responses after in vivo

23 injection.

24 This work may or may not be familiar to

25 many people here, but many labs now have completely

240

1 demonstrated that mesenchymal lineage cells from

2 humans, from primates, and from smaller animals

3 routinely actually do not express Class II HLA

4 molecules until they are induced by

5 gamma-interferon. They certainly do all express

6 Class I, but in addition to that, do not express a

7 variety of co-stimulatory molecules, such as CD40,

8 CD80, and CD86.

9 All of these molecules are absolutely

10 critical in inducing T cell immune responses when

11 you transplant cell or an organ between

12 individuals.

13 What I want to show you here is that

14 mesenchymal stem cells clearly do not induce a T

15 cell response following standard mixed leukocyte

16 reactions in vitro. This, you can see in

17 comparison to allogeneic mononuclear cells, which

18 induce a vigorous T cell response.

19 In addition, even activation with

20 interferon gamma to upregulate Class II HLA has no

21 effect on mesenchymal stem cell induction of T cell

22 responses, where you can see the potency of

23 gamma-interferon at inducing allogeneic responses

24 if you do it to dendritic cells or to allogeneic

25 mononuclear cells.

241

1 So, there is something very special about

2 these cells, that they do not seem to express

3 co-stimulatory molecules, they do not seem to

4 induce an allogeneic T cell response at least in

5 vitro, and moreover, they seem to have the ability

6 to suppress ongoing immune responses.

7 What you can see here is that this is now

8 a third party mixed leukocyte reaction where you

9 have T cells from one donor proliferating. This is

10 to allogeneic mononuclear cells, to allogeneic

11 dendritic cells, or to PHA, and you can see that if

12 you add third party mesenchymal stem cells, they

13 will suppress this proliferative response, and this

14 suppressive effect is in a dose-dependent manner.

15 Additional studies have suggested that, in

16 fact, part of this suppressive effect requires

17 cell-cell contact inhibition, and part of it

18 requires secretion of a variety of

19 anti-inflammatory cytokines, such as TGF-beta, but

20 the precise mechanism at this point has not been

21 defined.

22 The only human trial that I am aware of

23 using allogeneic mesenchymal stem cells to date is

24 one where allogeneic stem cell transplants were

25 performed with third party allogeneic mesenchymal

242

1 stem cells, and I understand the results of those

2 were better engraftment and reduction in

3 graft-versus-host disease, suggesting again that

4 the third party mesenchymal stem cell, not only

5 does not induce an immune response, and is

6 presumably not itself rejected, but is also

7 beneficial in outcome.

8 But just to go back to the cardiac studies

9 now, using that again as the backdrop, Brett Martin

10 and colleagues from Ceros Therapeutics\--and these

11 are two slices that he has given me\--presented at

12 the American Heart Association last year, their

13 findings using allogeneic mesenchymal stem cells

14 from pigs in acute myocardial injury, and this is

15 now in a reperfusion model to persecute myocardial

16 infarction, what they was generate a panel of

17 allogeneic porcine mesenchymal stem cells that are

18 matched at blood group antigen, they used very

19 large numbers of these cells distributed over the

20 infarct zone. We are talking about a reperfusion

21 model without any immunosuppression.

22 What you see here is that within two weeks

23 of implantation, the cells were still present, so

24 they hadn\'t been rejected. You can see the

25 presence of cells two weeks later. However, these

243

1 cells did not differentiate to cardiomyocytes, so

2 they did not induce a rejection episode, but they

3 also did not appear to really do much in terms of

4 integration or differentiation.

5 With respect to function, what you see is

6 actually improvement again in diastolic parameters,

7 as we have heard earlier, with increasing diastolic

8 wall thickness at various time points including

9 well beyond six weeks of study, and a reduction in

10 N-diastolic pressures, however, again no

11 improvement in systolic function, which is again

12 consistent with perhaps alterations in matrix,

13 alterations in ground substance, but no real effect

14 on contractility or myocytes.

15 This table, what you see is really a

16 summary of the type of the cells that are being

17 used of mesenchymal lineage. The MAPCs, so far we

18 have seen only modest efficacy in cardiac models in

19 dogs in the absence of predifferentiation in vitro.

20 Mesenchymal stem cells have shown good

21 function in rats, but modest only in pigs, and

22 mesenchymal precursor cells are currently being

23 investigated. We think they may be far more potent

24 than either of these two because you are able to

25 isolate them by surface characteristics when they

244

1 are fresh.

2 The final slide here is that I think many

3 remaining hurdles is the message. The appropriate

4 clinical indication needs to be defined. The more

5 proof of principle animal studies, we have got to

6 determine optimal doses for efficacy, careful

7 registry of adverse events in our animals, let

8 alone humans.

9 We have got to optimize the ex vivo

10 culture process, which I haven\'t even talked about.

11 The autologous versus allogeneic question is

12 critical because it is going to impact both on the

13 process and on the cost of this whole therapy,

14 determine the best route of administration and

15 really how do you improve engraftments, we have

16 heard all about that before.

17 Thank you very much.

18 \[Applause.\]

19 DR. RAO: Thank you, Dr. Itescu, for a

20 really nice summary and trying to help keep us on

21 time, as well.

22 In the interests of trying to get us back

23 on track, I am going to ask people to limit their

24 questions to things which are directly relevant to

25 the talk and if there are specific burning

245

1 questions on this issue.

2 Q&A

3 DR. CUNNINGHAM: I just have one question.

4 I was curious, in all your rat studies you

5 published in Nature, did the rats die, and what did

6 they die of, you know, the ones that didn\'t get to

7 the endpoint of the experiment, were there any

8 adverse events that you noticed?

9 DR. ITESCU: You mean unrelated to the

10 initial surgery? We have something like 30, 40

11 percent death rate from the initial surgery, but

12 pre-cellular implantation, but after cellular

13 implantation is what you are asking me, we carried

14 the animals out. There was no real adverse events

15 that I am aware of in these studies.

246

1 DR. KURTZBERG: You made a passing comment

2 as you were speaking that you thought GCSF wouldn\'t

3 be safe after an MI. Can you expand on that?

4 DR. ITESCU: I think in the recently

5 published study, just to summarize my take on that

6 study, a randomized study where one group received

7 GCSF subcutaneously to mobilize the endogenous

8 population of marrow, another group received GCSF

9 subcutaneously to mobilize and then had the

10 mobilized cells harvested, not immunoselected, and

11 I think the cells were then delivered by I believe

12 intracoronary routes, whole unfractionated cells.

13 The conclusion of the study was that the

14 patients that received the cell therapy had a

15 significant improvement in cardiac function,

16 whereas, the group that received GCSF alone did

17 not.

18 However, in both populations, the study

247

1 was cut short because of this complication, but I

2 think if you pooled both populations, 7 out of 10

3 patients developed significant restenosis at the

4 site of the stent implantation.

5 Now, these are bare stents. This is the

6 pre-repamycin days. I think the anticipated

7 restenosis rate would have been maybe 25 percent at

8 most in that population. I think even with

9 repamycin, you would expect to reduce the rate\--if

10 70 percent is right\--diabetic patients actually

11 have a restenosis rate with repamycin stents still

12 of about 20 to 25 percent, 50 percent without

13 repamycin, so these patients are more severe than

14 diabetics are.

15 I wouldn\'t anticipate that you would be

16 able to lower that to below 35, 40 percent, and

17 that is obviously totally unacceptable.

18 Now, the question is why is it that GCSF

19 was associated with this high restenosis rate, and

20 I think we can all make speculations, but I think

21 at least one possibility is that the cells that are

22 mobilized, CXER-4 positive cells from the marrow,

23 many cell types express CXER-4 including smooth

24 muscle progenitor cells, and I think that is

25 probably the simplest explanation, that there are

248

1 smooth muscle progenitors that are mobilized, that

2 migrate to the site of the stent, and I am not sure

3 how one can get around that actually, but that is

4 just a guess really.

5 DR. TAYLOR: That was great. I have one

6 quick question. You didn\'t mention this except

7 that in vitro, a lot of these cells can become a

8 number of mesodermal cell types, and I think there

9 are some data in rats that showed early on that if

10 you inject these cells in the center of an infarct,

11 they, in fact, at times become adipocytes or

12 chondrocytes or osteoblasts, or something like

13 that.

14 I wondered if you would comment on what

15 cells you think are likely to do that and whether

16 all of them are.

17 DR. ITESCU: Obviously, that is the worst

18 case theoretical question in this whole field.

19 What we don\'t want is to develop bone in the middle

20 of their hearts.

21 I am not aware really of a lot of studies

22 that have demonstrated that sort of abnormal

23 differentiation. Certainly, adipocytic

24 differentiation has occurred, I am not sure that I

25 have seen bone differentiation.

249

1 It really depends on how well defined the

2 cell population is, I think, and to what extent the

3 cells have been predifferentiated or to what extent

4 they may be still very multipotential. It is not

5 clear to me whether you need to start with a cell

6 that is very multipotential or that is fully

7 differentiated, and perhaps the culturing process

8 where you are pushing the cells, and again using

9 fetal calf serum I think is an unfortunate\--it is

10 the only thing that as been done to date\--but it is

11 I think probably the worst way to be culturing

12 cells, because you don\'t know what is in your

13 culture medium, you are pushing these cells to many

14 different lineages.

15 When we look at these cells

16 following\--very few studies have been published

17 that have looked at this\--but we have looked

18 ourselves in this way, and you can see that after

19 three or four weeks of culture, you can see cells

20 that express markers of mature smooth muscle cells,

21 of mature, some bone differentiation, some

22 differentiation to cartilage.

23 Whether or not that is relevant when you

24 put the cells back in, whether there is going to be

25 differential potential for outgrowth of one cell

250

1 type over another, I think is really a totally open

2 question, but I would push towards putting cells

3 that are less differentiated in rather than more

4 differentiated, because I think the less

5 differentiated cells have got the ability to still

6 proliferate, to be pushed towards the appropriate

7 lineage under appropriate inductive signals that

8 may still be present in the heart.

9 There may be more differentiation towards

10 maybe a cardiomyocyte lineage, but at least towards

11 a smooth muscle lineage, so you might get some

12 degree of arteriogenesis, as well as I think it is

13 the more undifferentiated cells that are the ones

14 that produce the very rich supply of arteriogenic

15 factors.

16 Just getting back to what I was saying,

17 the very undifferentiated cells express markers

18 really of parasites, so if you are thinking of

19 parasite implantation, it is one way of maintaining

20 viability of endothelial cells and integrity of the

21 endothelium, and maybe it is a way of building the

22 vascular network.

23 DR. RAO: This is the last question.

24 DR. BLAZAR: The issue of

25 transdifferentiation in vivo is striking that you

251

1 can get cells there and they just sit there, and I

2 think this has been seen with a number of

3 non-hematopoietic cell sources, MSCs being one.

4 I guess the question is whether there are

5 inhibitory signals that are present that prevent

6 differentiation in vivo under certain conditions,

7 and has anyone ever taken these cells back out of

8 the heart and show that they can, in fact, be

9 induced to differentiate in vitro? Do we know

10 anything about the inhibitory factors present at

11 the site that are precluding differentiation?

12 DR. ITESCU: Those are obviously great

13 questions, very important questions, and again I

14 think it speaks to having a good understanding of

15 the surface markers of these cells because if you

16 know what they express, you can do the sort of

17 experiments that you are suggesting. You know what

18 you are putting in, and then you can actually take

19 them out again based on maybe immunoselection.

20 We are actually trying to do those type of

21 experiments to ask exactly those questions, but I

22 think in many ways you are limited then to human

23 cells, because many of the well-defined surface

24 markers, there just aren\'t enough reagents.

25 Perhaps mouse cells is the only other. The mouse

252

1 system is the one that is well enough developed and

2 the human system, and other than that, we are

3 really missing reagents where you can do those type

4 of experiments. DR. RAO: Thank you,

5 Doctor.

6 DR. HARLAN: Can I still ask a quick

7 question? You were careful to say so in your talk

8 when you were talking about allogeneic cells, and

9 you said at least in vitro, these allogeneic cells

10 don\'t activate T cells, but I just wish to

11 emphasize the point that in vivo, it is so much

12 more complicated than that, and the presence or

13 absence of B7, I thought Bruce maybe was going to

14 speak to this, you didn\'t make the point, but I

15 think it is important to emphasize that whether or

16 not a cell in vitro stimulates a T cell response is

17 really a very poor predictor of whether that will

18 be rejected in vivo.

19 DR. ITESCU: Well, I am not sure I agree

20 with that at all actually.

21 DR. HARLAN: Then, we will have more

22 discussion. I saw the pig data, that is

23 interesting.

24 DR. ITESCU: I am actually a transplant

25 immunologist. This is what I do, I take care of

253

1 cardiac transplant patients, and I am going to tell

2 you that 12 months ago, when I first heard about

3 these sort of data, I was extremely skeptical,

4 along similar lines to what you are saying, you

5 know, is it an in vitro artifact.

6 However, you go back and you see how many

7 labs are reproducing these data, which is really

8 what I am surprised about, and I can tell you that

9 the mixed leukocyte reaction is about the best

10 single assay that we have in transplant medicine to

11 predict allogeneic rejection and allogeneic

12 sensitization.

13 In the old days, it used to be used

14 routinely for kidney transplant selection,

15 donor-recipient selection, and it is still the best

16 assay. I am not sure whether it reflects whether

17 these cells are going to be accepted long term in

18 vivo, but it is certainly a very good marker for

19 biology. I don\'t know what it means, but it is

20 routinely being reproduced using these type of

21 cells.

22 DR. HARLAN: To say it is the best, and I

23 won\'t disagree with it, is fine, but it is still a

24 very poor predictor of in vivo function, and it

25 goes back 30 years to the two-signal model,

254

1 thinking if you could get rid of the antigen

2 presenting cells within a graft, that you would

3 take, and it seems to work in rodents, but it just

4 doesn\'t work in higher animal models.

5 Bruce, do you want to step in?

6 DR. BLAZAR: I think there is the point of

7 somewhere in between because clearly, there are

8 cell-to-cell contact phenomenon that happen in

9 vitro with regulatory T cells. You can show a

10 TT-dependent inhibition of responses throughout

11 TGF-beta, and depending on the models in vivo,

12 those either are true or they fall apart.

13 We know in matched sibling donor

14 transplants, mixed leukocyte reaction culture does

15 not predict graft versus host disease. So, I think

16 that if you do see suppression, it is encouraging

17 to try to go forward for in vivo, and that is

18 probably as good as you are going to do, but it

19 doesn\'t necessarily mean that those mechanisms take

20 place in vivo particularly as those cells

21 themselves change in their own ability to elaborate

22 cytokines or express other molecules.

23 DR. ITESCU: I agree entirely and I am

24 saying the exact same thing, that I think it is a

25 phenomenon that is reproducible in vitro, it\'s an

255

1 unusual and unique scenario. Whether or not it has

2 implications in vivo remains, but I think at least

3 it ought to be tested, and I think it may have

4 implications on dendritic tolerance-inducing

5 mechanism, which truly is a different way of

6 thinking about this all together.

7 It may have nothing to do with the ability

8 of these cells themselves to escape surveillance in

9 the periphery, but perhaps they end up in the

10 thymus, and they may actually be able to reeducate

11 the immune response, but I think all of that

12 remains open to test.

13 DR. RAO: Last comment.

14 DR. NEYLAN: A very quick

15 transplant-related question, again, maybe bring

16 some of these last discussions to a very practical

17 safety concern.

18 That is, given the ability of these

19 allogeneic cells to abrogate or reduce the immune

20 response of the host, is it possible that the

21 migration and homing of these cells may differ to

22 autologous cells in a way, maybe akin to the

23 micro-chimers and observations of solid organ

24 transplantation, that potentially pose a safety

25 risk to the use of allogeneic cells, homing to or

256

1 disseminating and finding a welcome home in other

2 tissues.

3 DR. ITESCU: I think that is a fair point,

4 in other words, if they are immunoregulatory and

5 you are injecting them and they find their way into

6 the thymus, for example, will they induce a state

7 of tolerance to an exogenous antigen that the

8 patient sees at exactly the same time, which is the

9 concern whenever we use an immunoregulatory new

10 drug.

11 Now, there are a couple of studies

12 actually that have just recently been published

13 that suggest that, in fact, while they induce

14 tolerance to themselves and maybe tolerance to an

15 alloimmune reaction, they don\'t seem to have

16 induced tolerance to an exogenous pathogen, for

17 example.

18 Now, again, that is two studies, more work

19 needs to be done, but I think the question is fair,

20 it\'s an absolutely valid point, and obviously, you

21 have to worry about that when you do your studies

22 and follow up the patients very closely.

23 DR. RAO: We will visit it tomorrow. We

24 should move on.

25 Dr. Taylor.

257

1 From Mouse to Man: Is it a Logical Step

2 for Cardiac Repair?

3 DR. TAYLOR: It is a logical step because

4 I think it raises actually the issues that we are

5 just talking about, whether or not data we get from

6 rodents actually can be translated to humans or to

7 larger animals.

8 I also want to say actually, in terms of

9 an apology, I realized when I sat down that one of

10 the reasons my last talk was so disjointed was that

11 the version that was up here was not the version

12 that I had on my computer, so it was a kind of

13 foreign talk to me, so I apologize and hope we will

14 do better this time.

15 From mouse to man, is it a logical step?

16 I am going to start with another comment from

17 Ghandi, which I have had on my office door for the

18 last 10 years about this field, which is, \"First,

19 they ignore you, then, they laugh at you, then,

20 they fight you, then you win.\"

21 I think it raises the point that we are

22 somewhere in the continuum in this field, and it is

23 time for us to start asking the hard questions, so

24 that we can have the fight and then win.

25 As we are talking about moving from mouse

258

1 to man, I think we have to talk about cell type,

2 and I think I would be remiss if I didn\'t say that

3 I think cell type depends on what we are trying to

4 do here, and that if we are trying to look at

5 chronic ischemia or hibernation, we are probably

6 looking at cells that are more likely to induce

7 angiogenesis, such as the cells you just heard

8 about - bone marrow mononuclear cells, angioblasts,

9 some subpopulation of stromal cells, growth

10 factors, or maybe even myoblasts plus growth

11 factors, but if you want functional repair and

12 contractile cells, you are either want cells that

13 are contractile, such as skeletal myoblasts or

14 cardiocytes, maybe cardiac stem cells, or you want

15 bone marrow cells that can become contractile

16 cells.

17 But the most important issue probably in

18 this whole field, which is why I think we are going

19 to have to talk about moving from mouse to man is

20 probably this arrow, and the fact that we need both

21 angiogenesis and myogenesis if we are going to have

22 an appropriate outcome, and that, in fact, it may

23 be not just one cell type, but multiple cell types

24 that we end up needing for cardiac repair.

25 Unfortunately, as we are looking at these

259

1 cells, we don\'t have the opportunity to do those

2 anywhere except in rodent at the present or in

3 humans, so we are going to have to move from mouse

4 to man at least with many of these cells unless

5 industry provides us with the tools that we need to

6 do the studies in between, because right now we

7 really don\'t have the capability of moving to a

8 larger animal model.

9 I guess I want to start by asking the

10 question what the appropriate preclinical animal

11 models are and how quickly can we move forward by

12 saying, you know, I presented this slide a minute

13 ago, we had 15 years of preclinical data in rabbit

14 and dog and pig and rat, mouse, and sheep, and we

15 thought myoblast transplantation was safe,

16 effective, and feasible.

17 But we missed a lot of things, and we

18 missed\--I apologize, I thought there was another

19 part down here, we are off to a great AV start, but

20 that\'s okay\--so what did we miss? We missed the

21 fact that these cells might be electrically

22 incompatible with the remainder of the myocardium.

23 We missed questions about location of

24 injection, we missed questions about some of the

25 dosing phenomena, we missed a number of things in

260

1 our early preclinical models despite the fact that

2 we used both large and small animal models.

3 So, the question then really is what do

4 you really need to do and when do you move to

5 clinical studies, and I am going to give my jaded

6 perspective for a minute and say that I think

7 sometimes you move to clinical studies because your

8 institution wants you to and kind of forces you to

9 either because there is a financial incentive or

10 that there is no such things as bad PR, but I would

11 like to say that the appropriate time to move to

12 clinical trials is when the data warrant it and

13 that again we have to underpromise and overdeliver.

14 So, if we believe that every cell injected

15 seems to work, and that thus the heart is easier to

16 repair than we thought, let\'s take, for example,

17 the possibility that that is really the case, and

18 if that is the case, when do we move to the clinic.

19 I guess I could start by saying we have

20 already moved to the clinic, but that being said,

21 if we look at the clinical data, does it support

22 the fact that the myocardium is easier to repair

23 than we thought.

24 Well, yes, everything works, but none of

25 the clinical cell studies are placebo controlled,

261

1 and 8 patients or even 53 patients can show you

2 anything, especially when follow-up is short and we

3 aren\'t considering age, gender, or heart failure

4 status.

5 I want to get back to these, and I will in

6 this talk, that we haven\'t talked at all about

7 factors like age and gender and how they may be

8 really relevant, and there is a reason that drug

9 trials involve thousands of patients with at leave

10 five-year follow-up.

11 Also, most of the clinical cell studies

12 out there were designed as Phase I safety studies

13 as they should have been, yet, many of these claim

14 efficacy despite the fact that they were either

15 revascularization studies or had other

16 co-treatments involved, and I think it really

17 raises questions about what we need to do.

18 So, if we believe that the myocardium is

19 easier to repair than we thought, what does that

20 tell us about moving to clinical studies? I am

21 sorry, those two slides are actually backwards. If

22 every clinical cell works, what does that mean?

23 I think it means that we have no clue how

24 they work, whether they create angiogenesis or

25 myogenesis, unloading of the heart, recruitment of

262

1 stem cells or whatever.

2 What questions does that raise? It raises

3 questions about patients, which raises the same

4 questions about injury models, preclinical injury

5 models. It raises questions about dose and timing

6 of cells, which then have to interact with the

7 injury models. It raises questions about route of

8 administration or location of the cells, and how we

9 measure the outcome, and those all affect which

10 animal model you can choose.

11 Well, the genie is out of the bottle, as

12 the people at Mayo have said. Clinical trials have

13 started, so what do we do? I think we educate

14 people about what the appropriate situation is.

15 There are a number of my clinical

16 colleagues that I have talked with, and it scares

17 me a little bit, who don\'t even realize that if you

18 are going to use bone marrow derived cells, that

19 the FDA needs to be involved if you are going to

20 put them in the heart, and I think that is an issue

21 that we really have to address.

22 I think we need to require enough

23 preclinical data, and then I think we need to quit

24 rewarding people for doing it wrong. What do I

25 mean by that? Well, what I mean by that is we have

263

1 to quit doing science by the Washington Post, as

2 you said earlier, and we have to quit focusing on

3 the fact that this is a multibillion dollar market

4 every year, and focus on the patients instead.

5 So, what do myoblasts tell us about moving

6 forward? Well, as I said, what we knew and what we

7 missed is that there are electrical events, the

8 route of administration wasn\'t clear, location and

9 timing wasn\'t clear, culture medium, we thought we

10 knew, but it didn\'t turn out to always be the case.

11 Autologous serum has been reported to be

12 safer than non-autologous serum. Different

13 vehicles have been used to deliver the cells and

14 been associated with different outcomes, and we

15 don\'t know anything about biodistribution, and we

16 really didn\'t look.

17 So, what issues are there? The issues are

18 safety and efficacy obviously, and as we move up

19 this continuum, we can address these issues

20 differently. Safety obviously involves cells,

21 delivery, dose, and we have to do that in relevant

22 models.

23 Efficacy involves the right model, acute

24 MI potentially, looking at various cell types in a

25 side-by-side way. We have to begin to

264

1 differentiate between diastolic heart failure. I

2 think the preclinical data that exists so far

3 suggests that everything works to begin to improve

4 remodeling and diastolic effects including cells

5 that don\'t work in systolic heart failure.

6 Systolic heart failure in our lab at

7 least, we know fibroblasts don\'t work, stromal

8 works less well than some other cell types, and I

9 think the issues are open questions that have to be

10 addressed.

11 If you then believe that the issues effect

12 are impact cells, delivery, and effect, how does

13 that translate to the animal models? Well, in

14 small animals, I think we can ask questions about

15 the cells. We can ask questions about deriving the

16 cells, we can ask questions about markers for the

17 cells, we can ask in vitro questions.

18 I think as we begin to move towards

19 delivery and effect, we have to move up the animal

20 continuum. Delivery, we really can\'t do in small

21 animal models, relevant delivery, we can\'t do.

22 Effect, again, I don\'t think we can do in small

23 animal models, we have to do it in larger animal

24 models.

25 In addition, when we start looking at the

265

1 mechanism of effect, to some degree, if we are

2 looking at angiogenesis, we can measure capillary

3 density in small animals, but it is very difficult

4 to get adequate measures of relevant vessels or

5 perfused vessels in small animals, and I think we

6 have to move up the continuum.

7 In terms of myogenesis, we can get some

8 data about ex vivo in isolated heart preparations

9 and whether or not there are gross improvements in

10 contractility. We can begin to measure wall

11 thickness, ejection fraction, and you can certainly

12 do exercise studies in small animals and move up

13 the continuum, but in terms of electrical

14 compatibility and mechanical compatibility, you are

15 never going to get it done in a rodent model, you

16 have to do it in large animals.

17 So, I think it is only relevant to work

18 with small animals when you have no choice about

19 the cells, but as you start to measure the

20 important parameters of physiology, you have to

21 move up the model.

22 So, what are the possible effects of these

23 cells? I think they can cause unloading of the

24 heart or reverse remodeling simply by altering the

25 mechanical properties of the scar. They could

266

1 possibly engraft and become muscle and contribute

2 to contraction.

3 They can obviously potentially form

4 vessels or secrete factors that recruit cells that

5 improve blood flow. You know, if you think about a

6 lot of the data we have seen so far, it is possible

7 that the cells we are putting in are doing nothing

8 but recruiting bone marrow derived cells and

9 actually ramping up endogenous repair.

10 It is possible that the cells have a

11 paracrine effect and either change the cytokine

12 supply to the scar or the remainder of the heart,

13 or recruit other stem or progenitor cells.

14 Another possibility that is not really

15 talked about, and again you can do these studies in

16 small animal models better than large animal

17 models, is fusion with existing cardiocytes. It is

18 possible that the cells we inject actually fuse

19 with cardiocytes that are hibernating and save

20 those cells, and thereby contribute to contraction.

21 If we are going to talk about the

22 different possibilities, cell delivery and effect,

23 and which animal models to use, I want to briefly

24 say that we have to define our populations of

25 cells, and that is easier in some animal models

267

1 than others.

2 Again, when you start looking at

3 myoblasts, fibroblasts, and other cells, being able

4 to do that in rodents is much easier than being

5 able to do that in larger animals. I think I have

6 beat that horse, so I won\'t keep saying it, but

7 cells, I think are studies we can do in rodents.

8 You have seen this slide before where we

9 begin to talk about how the cells might work and

10 whether or not we have an effect on reverse

11 remodeling or growing new cells, and whether or not

12 we think we can do those studies in large animals

13 or small animals. I would submit that we are going

14 to have to do those in large animals because we

15 can\'t really measure remodeling and reverse

16 remodeling in all of the new cells in the small

17 animal models.

18 Now, we took the approach that if we are

19 going to start moving back up this cascade of

20 events, that might, in fact, take a combination of

21 angiogenesis and myogenesis, and it might take

22 cells plus genes or multiple combinations of cells.

23 Initially, we looked at cells plus genes

24 by virally infecting myoblasts with VEGF

25 adenovirus, and we measured the effect of those

268

1 cells on capillary density in peripheral skeletal

2 muscle and a hind limb ischemia model, and these

3 are old data, so I am not going to spend much time

4 on them other than to say that we found that

5 myoblasts had a relatively significant effect in

6 terms of increasing capillary density greater than

7 VEGF virus alone or MT virus, but when we

8 overexpress VEGF in the myoblasts, we got back up

9 to about 75 percent of the control without the side

10 effects that we saw with VEGF virus alone,

11 angiomas.

12 So, we decided to move into the

13 myocardium, and these are data done with cells that

14 overexpress another angiogenic factor where we

15 looked at MRI, and I apologize, this is percent

16 change and ejection fraction, and our historical

17 controls, and these really are historical, they are

18 not done at the same time, the active study was

19 myoblasts versus angiogenic myoblasts versus

20 historical shams.

21 We found that we increased capillary

22 density significantly in these animals. These were

23 studies done in mice. We couldn\'t do these studies

24 actually in rabbit because rabbit cells don\'t have

25 the cell surface receptors to actually take up or

269

1 be transfected with some of the viruses that we

2 were trying to use, so that becomes a problem.

3 But we believe that angiogenesis was more

4 important than to promote cell survival or

5 proliferation, and we also looked at Victor Dzaus\'

6 data, I wanted to show briefly. You saw it when

7 Silviu presented it, that if you overexpress a

8 survival factor AKT in bone marrow stromal cells

9 and transplanted them into the heart, survival

10 increased.

11 So, we know angiogenesis increases

12 function, increasing survival increases function,

13 so if angiogenesis helps and increasing survival

14 helps, why use a gene, why not use a mixture of

15 cells.

16 We based that on data that we have gotten

17 now in our hands where we were able to show that if

18 we gave cells from young apoE animals, gave bone

19 marrow cells from young apoE animals to apoE

20 animals that were fed on a high fat diet, normally

21 develop pretty bad atherosclerosis, that we could

22 actually prevent this atherosclerosis.

23 We took, we actually have now begun to

24 take these cells and deliver them in combination

25 with myoblasts to look at effects on function. I

270

1 don\'t have a slide, but I can tell you that we are

2 beginning to see better effects on function with a

3 combination of cells and with individual cells

4 alone. We couldn\'t do these studies except in

5 mice, so there are times when mouse cells are

6 relevant.

7 We did another study where we began to

8 look at bone marrow mononuclear cells. These are

9 data that were just presented at the ACC last week,

10 where we actually infused mononuclear cells into

11 the circulation of animals where we created a

12 vascular injury.

13 We are able to show in our sham-treated

14 animals or animals treated with other cells that

15 you had neointimal proliferation, that we were able

16 to prevent with bone marrow mononuclear cells.

17 So, again, we have now started the

18 approach of delivering these cells in combination

19 with myoblasts to see if we can have a more

20 dramatic effect on not only myocardial repair, but

21 on vascular repair, as well.

22 So, what are the other factors that are

23 likely involved that are going to affect the model

24 we use? The timing after injury, whether or not we

25 can really grow old animals that replicate the six

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1 to seven years that are needed, the type of injury,

2 it is not probably going to be feasible to do

3 dilated cardiomyopathy studies in mice and inject

4 significant numbers of animals, plus when we start

5 trying to treat ischemic and chronic and acute

6 animals, mice, the issue we have to consider is we

7 have got a 1 mm infarct, we got a 1 mm infarct, and

8 the cells are microns in diameter.

9 Those cells are the same size essentially

10 in rat, rabbit, pig, human, they are not much

11 different in size, so the whole geometry of putting

12 those cells in and getting an improvement is going

13 to be much different than you are going to see in

14 larger animal models.

15 There are two issues that really don\'t

16 affect what animal you choose, but they are not

17 being discussed at all, and those are gender and

18 age.

19 Most of the preclinical data that we have

20 published are in female rabbits, so we went back

21 and started doing studies in male rabbits, and what

22 we found is that male myoblasts die under

23 conditions where female cells survive, and that is

24 true both in vitro and in vivo, and that really

25 surprised us, and we had to go back and begin to

272

1 reevaluate what we think is going on here, and we

2 are just beginning to follow up on that, but I

3 think it is an interesting point that we are going

4 to have to consider going forward.

5 In terms of age, all of the studies we

6 have done have been in old animals, but that is

7 rarely the case. It is rarely the case that old

8 mice are used in these studies, that old pigs are

9 used in these studies. In fact, typically, people

10 use young pigs because they want to keep them

11 small, and don\'t want them to grow significantly

12 over the duration of the studies.

13 I think the numbers and kinds of cells

14 that you can obtain are going to be very different.

15 Other than cells, we have to consider the culture

16 conditions, and we can\'t ignore the fact that

17 autologous serum has been touted as one reason that

18 there is a Spanish study where there aren\'t any

19 abnormal electrical events even though there have

20 been in all the other human studies.

21 So, the injury models currently that we

22 are using don\'t match the patients, and I think we

23 are going to have to really think about that going

24 forward. We don\'t have heart failure models, we

25 just don\'t. Nobody is using heart failure models,

273

1 but every patient is a heart failure patient.

2 As I said before, I apologize, in terms of

3 whether or not we can use small animals, I think

4 for an isolated heart prep, mouse and rat are fine,

5 but as you are going to start doing physiologically

6 relevant studies, you have to move up the animal

7 continuum, but there are limitations there, as

8 well, so we have actually chosen, and I think more

9 and more people are choosing, to use sonomicrometry

10 or cine MRI. Fortunately, you can use that for all

11 of the animal models that have been proposed so

12 far.

13 I am not going to show those data.

14 I actually want to end with two slides

15 that show something that I have tried to gather

16 from the clinical data that exists, but I think

17 they make a point.

18 We are talking about different animal

19 models, but we are also talking about different

20 cell types, and people are constantly saying how do

21 myoblasts and bone marrow derived stem or

22 progenitor cells compare.

23 The point I want to make is they don\'t.

24 If you look at the studies that exist so far\--and

25 these are clinical studies, not preclinical

274

1 studies\--if you look at myoblasts, the dose varies

2 widely.

3 If you look at bone marrow derived stem or

4 progenitor cells, the dose is significantly less

5 and the physiologically relevant cells are a very

6 small subset of those, as you just heard.

7 Moreover, the patients differ greatly.

8 With myoblasts, the patients are from greater than

9 one month to end-stage heart failure, but with the

10 progenitor cells, the patients are 3 to 9 days

11 post-MI or have refractory angina.

12 The delivery methods differ significantly.

13 They are intracardiac for myoblasts, surgically or

14 percutaneously. They are intracoronary for the

15 bone marrow derived cells. Yet, people are trying

16 to compare the outcomes from these, and I think

17 that is true, not just clinically, but

18 preclinically, as well, as people are trying to

19 make the argument for their cell type.

20 So, until we are doing side-by-side

21 studies with the same cell type and the same animal

22 model, I really think we can\'t draw conclusions

23 about what is going on.

24 So, the questions that I think are really

25 out there are: Is there a best cell? I don\'t

275

1 think we know that there is yet.

2 Should we use just growth factors? I

3 think the GCSF data would suggest not yet, we don\'t

4 understand it well enough.

5 Is there a future for biologic devices?

6 Probably. Are we going to be putting cells on

7 stents? I don\'t know, but we probably are going to

8 be putting them on patches and other devices in the

9 very near future.

10 There is a real question, dose, timing,

11 and patients, probably, but our models need to

12 mimic that.

13 I will stop there.

14 \[Applause.\]

15 Q&A

16 DR. RAO: Thank you, Doris. I wanted to

17 start off by asking you one question first. That

18 was that implicit in all of these talks throughout

19 has been the fact that it seems to be important

20 when choosing a model that you have to have the

21 right markers. That really is because you are

22 doing syngeneic transplants. You want to put the

23 same animal species cells back into the animal to

24 do the follow-up.

25 Nobody, at least in this field, seems to

276

1 think that you can immunesuppressed mouse and use

2 human cells directly, or do some equivalent

3 otherwise, and that that is reasonable.

4 Now, is that true? That is important

5 because this is going to be really important in the

6 future. I just wanted to get your feel for that.

7 DR. TAYLOR: I think that is fairly

8 accurate, and I think the problem has been that we

9 haven\'t had the opportunity to really measure the

10 effects of these cells in mice very well until the

11 last couple of years.

12 Until the last probably year and a half to

13 two years, you couldn\'t do MRI in a mouse reliably.

14 You certainly couldn\'t get a good enough image to

15 measure regional versus global function.

16 Sonomicrometry was hard to do in a mouse.

17 The pressure volume catheters weren\'t quite up to

18 snuff, and so the ability to make those

19 measurements weren\'t true. Moreover, most of us

20 believed early on at least that autologous cells

21 were more likely to be clinically accepted, and

22 patients certainly liked the concept of getting

23 their own cells better than the concept of getting

24 somebody else\'s.

25 So, I think we chose those cells because

277

1 they made sense to us clinically, and we were

2 technically limited by our ability to make the

3 measurements with other cells.

4 In terms of using human cells in

5 immunocompromised rodents, I think we can do that

6 now, but whether or not\--I think it is an open

7 question about whether or not we are going to get

8 the best functional outcomes.

9 DR. ITESCU: Can I just maybe add a little

10 bit to that? You know, we use the

11 immunocompromised rodents pretty well, but I think

12 what we are learning as we move forward is that

13 even the so-called immunocompromised rodents are

14 not fully immunocompromised, and you have really

15 got to start understanding which kind of lineages

16 in their immune system remain active, and what

17 impact does that have on the cells you are putting

18 in.

19 We are now at the point where we are

20 adding cocktails to try to remove even the residual

21 immune function in these kind of animals.

22 On the other hand, I think that if you are

23 trying to use a cocktail of immunosuppressive

24 agents in a normal animal, I think then you are

25 going to run into the problems of what effects all

278

1 these drugs have on the cells that we are putting

2 in, are they inhibiting differentiation, are they

3 inhibiting function. There is a whole range of

4 issues that I don\'t think we want to get into.

5 DR. RAO: Absolutely, that is important.

6 DR. HARLAN: I will make a comment on

7 that, and then I had a question. I agree, in the

8 islet transplant field that I know best, for

9 instance, in order to correct a mouse with human

10 islets, you need about 1,500 islets, whereas, you

11 need about 400 rodent islets, and presumably it is

12 because there are species differences in the

13 factors that support the growth of those cells.

14 Then, I agree and appreciate your talk,

15 but I would extend it in two ways. I think a theme

16 of your talk was that the large animal models in

17 general tend to be better than rodents in

18 predicting things, and I think that is true, but I

19 wish to point out that all models are models.

20 In our transplant studies, we did things

21 in primates, testing various immunotherapies in

22 different systems, and it worked beautifully and

23 failed miserably in the clinic, so even large

24 animal models, even using nonhuman primates, are

25 models, and they have variables that are hard to

279

1 predict.

2 DR. TAYLOR: I would add one comment to

3 that, which is all of us are looking at progenitor

4 cells and the effects of these progenitor cells on

5 cardiovascular function, and we know that the

6 animals we are treating are relatively acutely ill

7 even if we have given them heart failure, but

8 patients are very ill.

9 They have had things impacting their

10 progenitor cells for years, that we know nothing

11 about, and it is not just aging, it\'s drugs, it\'s

12 other things, so the cells we get out and the cells

13 we put back in are going to be very different than

14 cells we get out and put back in, in animals that

15 we have only made sick for a month or a year.

16 DR. HARLAN: And because we don\'t know all

17 the factors that made people sick, generating that

18 perfect animal model may be an impossible ideal,

19 that\'s the only point, I think we are agreeing.

20 The second thing is, though, that you

21 didn\'t mention is in any model, and when you go to

22 the clinic, I think it is important and we started

23 the day that way, identifying the patients for whom

24 existing therapies just have failed, so that is one

25 way where you can go. When you have got nothing

280

1 else to offer, and you have the patients in a bad

2 position, then, I think it passes the threshold.

3 DR. TAYLOR: But those aren\'t the models

4 that people are using.

5 DR. HARLAN: Well, we will talk about that

6 tomorrow, I agree.

7 Then, the third point, I say this in jest,

8 and I don\'t buy it, but I like your Ghandi quote,

9 and I will cite another philosopher, W.C. Fields,

10 who said, \"If at first you don\'t succeed, try

11 again; if it fails again, you might as well give

12 up, there is no sense being a damn fool about it.\"

13 I don\'t agree with that, it\'s just the

14 other take.

15 DR. RAO: Dr. Epstein.

16 DR. EPSTEIN: I just wanted to make a

17 point, which I think is important because I sense

18 that one could very easily come to the conclusion

19 without thinking it through further that a large

20 animal model is the really only valid preclinical

21 model.

22 I think Doris sort of made this point, but

23 it might have been lost. It depends on what you

24 are looking at. For example, if are interested in

25 myogenesis, I would agree, ultimately, you have to

281

1 go to the large animal model because the small

2 rodents, mice, become difficult to draw conclusions

3 about, but if you are looking at angiogenesis, the

4 mouse model is much better, I think, than a large

5 animal model.

6 We have excellent ways of measuring

7 perfusion now, superb ways of measuring perfusion,

8 and what comes up in our laboratory a lot is if we

9 give an intervention that has an effect, and then

10 we want to see whether we could further enhance

11 that, like stromal cells and then genetic

12 engineering of stromal cells, in a pig model of

13 myocardial ischemia, it starts out with 85 percent

14 of normal.

15 If you get it up to 95 percent of normal

16 with your first intervention, you have no room to

17 look at the next step, whereas, with mice, that

18 could be modulated much more easily, and we are

19 able to demonstrate a primary effect and then an

20 additional effect on it.

21 So, I think, you know, I wouldn\'t like the

22 FDA to go away with the conclusion that you have to

23 do an efficacy model in a pig or a dog to go to the

24 clinic. Again, it depends what you are looking at.

25 I will make a point about the

282

1 immunosuppressed animals. Angiogenesis, an

2 intrinsic component of the angiogenic process is

3 inflammation, so if you put in cells in a model

4 that is immunocompromised, it is a laboratory model

5 where you have taken away one of the normal

6 modulating influences, so you have to be very

7 careful using an immunocompromised model at least

8 to look at a process in which inflammation is a

9 very critical component.

10 DR. RAO: A point well taken.

11 Bruce.

12 DR. BLAZAR: Doris, I wanted to ask you,

13 since you have reviewed all of the preclinical

14 data, and you had the tenet that the data should

15 drive the studies, what of the preclinical data is

16 sufficiently compelling that this would have driven

17 the studies to go forward, are the models done so

18 far incomplete to be able to decide on appropriate

19 studies?

20 I know we are going to talk about that

21 tomorrow, but since you have reviewed in two talks

22 this issue\--

23 DR. TAYLOR: I think there is certainly a

24 lot more preclinical data from myoblasts than there

25 are for some of these bone marrow derived

283

1 progenitor cells. I think that is in part because

2 we have known about myoblasts since 1961 and we

3 have know about these bone marrow derived cells for

4 the last five or six years, so it is not

5 surprising.

6 I think myoblasts have been used in

7 peripheral models, they have been used in cardiac

8 models of injury, they have been used in large

9 animals, they have been used in small animals, and

10 there is a confluence of data, all of which say if

11 you give these cells, the animals get better.

12 I think most of the cardiac models were

13 relatively acutely after injury, within a month

14 after injury, but nonetheless, they said if you use

15 these cells, the animals get better.

16 I think what is starting to happen in the

17 bone marrow mononuclear cell field and in the bone

18 marrow stromal cell field, and in even the MAPC

19 field, is that we are seeing isolated studies with

20 cells that are called a given thing, but aren\'t

21 necessarily defined the same way.

22 The way one group defines an EPC, and the

23 way another group defines an EPC may be very

24 different, so there is not necessarily a confluence

25 of data yet, and you can\'t necessarily even compare

284

1 some of the preclinical studies because the cells

2 are very different, or we don\'t know what the

3 criteria are for those cells.

4 So, I think that what has to happen is we

5 have to know what the definition of the cells are,

6 and that is only now becoming true for endothelial

7 progenitor cells.

8 DR. BLAZAR: So, a corollary of this

9 question is, is it pretty well established by those

10 of you in the field, what the bar is that you have

11 to get over.

12 It seems the way you have described this,

13 it\'s a systolic function bar that is\--no.

14 DR. TAYLOR: I think it depends on what

15 you believe the mechanism of action is for the

16 cells, and I think again we don\'t know that, but it

17 looks like what is coming about generally is that

18 for bone marrow derived cells, the goal may be

19 angiogenesis, and for muscle-derived cells, the

20 goal may be myogenesis, and those have different

21 criteria and different preclinical studies that I

22 think you ought to do.

23 DR. BLAZAR: But that presumes for

24 clinical applications that you know exactly the

25 pathophysiology of the lesion you are trying to

285

1 treat.

2 DR. TAYLOR: I think it begins to argue

3 for timing after injury.

4 DR. BLAZAR: Okay.

5 DR. RAO: If anybody has a really brief

6 comment, otherwise, we will take Dr. Mul and then

7 Dr. Schneider.

8 Do you have a quick comment?

9 DR. MENASCHE: Go ahead. I just had a

10 comment, but I can wait.

11 DR. MULE: I would just like to follow up

12 on Bruce\'s questions. This has been an incredibly

13 frustrating afternoon for me from the standpoint

14 that I think the presentations have nicely pointed

15 out the strengths and weaknesses of small animal

16 models versus large animal models.

17 Overlaid on that is the fact that none of

18 these models really are good models for the actual

19 disease state in humans. What I was hoping to hear

20 this afternoon was taking the strengths and

21 weaknesses of each of the models and maybe laying

22 out, hopefully tomorrow, what an ideal, if we could

23 go that way, what the ideal recommendations would

24 be for the field to help the clinicians, such as

25 Philippe, in conducting the next generation trials.

286

1 That is the real concern to me, is that at

2 the end of the day, the horse is out of the gate,

3 and Philippe and others are going to be conducting

4 these trials rather rapidly, and I think we need to

5 help them to establish some guidelines as to

6 whether or not we should abandon animal models,

7 move on to the clinic, and design the clinical

8 trials in such a way that we get the best

9 scientific data available and the best clinical

10 situations that can be defined, and define the

11 endpoints of the clinical trial with the

12 appropriate placebo.

13 So, I just wanted to lay out that my

14 deepest concern is that years from now, we will be

15 using the same models that you have very nicely

16 summarized, small animals, large animals, fully

17 aware that these limitations continue to exist, and

18 whether or not those data that are generated over

19 the next several years will help Philippe and

20 others to characterize how we should go forward in

21 conducting these clinical trials. It is just some

22 comments I had.

23 DR. RAO: I think you are echoing what the

24 FDA is feeling, I guess, right now.

25 Dr. Schneider.

287

1 DR. SCHNEIDER: Doris, you know that I

2 share your general cautionary note and share many

3 of the specifics, but let me disagree with two

4 specific points that you made about the lack of

5 utility or impediments to the use of the mouse.

6 One of them that you singled out is the

7 impediment to using the mouse as a model for

8 studying the electrical connectivity of donor cells

9 to the host environment, which has become an issue

10 of prominence because of the issue of ventricular

11 tachycardia.

12 I think Loren Field has shown very

13 convincingly you can use 2-photon microscopy to

14 study the propagation of action potentials on the

15 epicardial surface, you can study the propagation

16 of calcium transients in the mouse heart with

17 grafted cells, so that is a non-issue as of this

18 year.

19 DR. TAYLOR: I completely disagree, but we

20 can have that discussion\--

21 DR. SCHNEIDER: Perhaps so, but

22 Circulation doesn\'t, and there is adequate

23 peer-reviewed data out there that shows that it is

24 technically feasible with some esoteric

25 instrumentation.

288

1 The same thing I think is true in terms of

2 the issue of mechanics. As you say yourself, cine

3 MRI levels the playing field across all of these

4 species, and at a few centers, cine MRI is even

5 being combined with spam, so that people can do

6 finite element analysis in the small mammals.

7 So, to me, and I share Dr. Mul\'s

8 frustration, I tend to focus myself on a different

9 issue, and that is not whether the small mammal is

10 adequately achievable for the endpoints that we

11 want to study, but is the small mammal adequately

12 predictive of the pathophysiology we want to study.

13 There, the issues include the fact that

14 neither the small nor the large mammal is done in

15 an aged animal against a background of diffuse

16 atherosclerotic disease.

17 DR. TAYLOR: I want to just comment on

18 that looking at electrical connectivity is not the

19 same thing as looking at ventricular tachycardia or

20 arrhythmias, and that it is going to be difficult

21 to do those studies when the heart is beating at

22 600 beats per minute, and that is I think one of

23 the main issues, as well as the fact that most

24 electrophysiologists tell you that the larger

25 geometry is much more conducive, of the human

289

1 heart, is much more conducive to electrical

2 abnormalities than the smaller geometry of a rodent

3 heart, so you are not going to see the same

4 properties even if you have an injury there.

5 DR. SCHNEIDER: Again, it becomes an issue

6 of whether it is a predictive biology, not an issue

7 of whether it is technically achievable.

8 DR. TAYLOR: Sure.

9 DR. MENASCHE: To some extent, Dr. Mul

10 has anticipated my comment. What I wanted to

11 emphasize from a clinical standpoint is that

12 regardless of the animal model we are going to

13 use\--and I have advocated large animal models, as

14 well\--there is not a single animal preparation

15 which can realistically model the very complex

16 situation of the patients we are dealing with.

17 There is not a single model which can

18 reproduce the situation of a 70-year-old person

19 with Class III heart failure, two previous bypass,

20 seven angiopathies, collaterals, and so on, so at

21 what point we really have to be prepared to move

22 forward and to go across the gap.

23 Now, considering skeletal myoblasts, we

24 have clear evidence regardless of the limitations

25 of the models that the technique can be implemented

290

1 easily in patients.

2 Number two, we have reasonable evidence

3 that it is safe provided some precautions are

4 taken. We don\'t fully understand the mechanisms,

5 but many interventions are currently using patients

6 without an extensive understanding of the

7 mechanisms. I think it took years before people

8 understood how aspirin was working.

9 So, as we are continuing to try to

10 understand the mechanisms, I think that given the

11 huge population we have to deal with, and the

12 number of patients without any option, it is

13 critically important and timely appropriate now to

14 move to the efficacy studies, and we are certainly

15 looking for advice and help for designing the study

16 in such a way that they can draw meaningful

17 conclusions and answers to the two fundamental

18 questions, does it improve function in the areas

19 where cells are put, does this improvement have an

20 impact on the clinical outcomes.

21 DR. TAYLOR: Philippe, the one comment I

22 would make to that is I think you are absolutely

23 right, we don\'t have good animal models, I don\'t

24 anyone in the room will argue that.

25 I think the thing I would add to what you

291

1 said is you mentioned no-option patients, but those

2 aren\'t the patients that are involved in some of

3 the studies that are going forward, and I think

4 those are the patients who should be involved in

5 the studies going forward, and I hope that is one

6 of the things that will emerge from this, that it

7 may be responsible to go forward in those groups of

8 patients, it may not be responsible to go forward

9 in some other groups without more preclinical data.

10 DR. RAO: I would like to just remind

11 everyone that part of the discussion is not

12 specific to one particular cell type, but is in

13 general, and maybe the conclusion may be that it is

14 just simply we have to have differing criteria

15 depending on the cell type or the model, or

16 whatever, when we discuss it, at least its options,

17 and that may be as far as one can go.

18 So, keep that thought in mind, that

19 nothing that you hear necessarily means this is

20 absolute or anything in that fashion.

21 DR. ITESCU: Along those lines, I think

22 the point you are making is exactly right. Some

23 cell products are based on characterization of the

24 surface phenotype, based on many years of

25 immunoselection with markers only present in humans

292

1 and not present in any other species.

2 Some cell types have been characterized by

3 very different biologists, who have used functional

4 outcomes and functional criteria. So, you are

5 talking about apples and oranges really, so you

6 can\'t have the same clinical models or preclinical

7 models for those, so you have got to define

8 appropriate models for each of those cell types,

9 because they have been characterized from totally

10 different perspectives.

11 DR. RAO: I thought we might use that to

12 start the conversation tomorrow, yes.

13 Everybody looks like they need a break, so

14 we will take a short 10-minute break.

15 \[Break.\]

16 DR. RAO: Welcome back, everybody.

17 We are going to change topics a little bit

18 and talk about devices now. It is finally going to

19 be the turn of Dr. Jensen to talk on cardiac

20 catheters.

21 Cardiac Catheters for Delivery of Cell Suspensions

22 DR. JENSEN: I am going to go ahead and

23 get started because we are running a little bit

24 slow here, and I am also going to try and see if I

25 can catch up a little bit in terms of time.

293

1 My name is Nick Jensen. I work in the

2 Division of Cardiovascular Devices in FDA\'s Center

3 for Devices and Radiological Health, and I have

4 been asked to briefly introduce the cardiac

5 catheters that have been used for delivery of cell

6 therapies intended to treat cardiac disease.

7 My presentation today will be limited to

8 potential questions that relate to the interaction

9 between cell therapy suspensions and the cardiac

10 catheters used to deliver these therapies.

11 Further, the questions that I list today

12 are among the standard questions that we currently

13 suggest to all sponsors either of cell therapy INDs

14 or for these investigational catheters.

15 As examples, we will discuss two types of

16 cardiac catheters that have been used to deliver

17 cell therapies to the heart.

18 First, is infusion of cells into a

19 coronary artery during a balloon occlusion of the

20 artery. Second, is needle-tipped injection

21 catheters designed to permit percutaneous

22 transendocardial injection into the myocardium.

23 Again, these simply represent the methods

24 and catheter types that have been most commonly

25 used to date, and they are again meant to provide

294

1 useful examples for discussion. I think it is hard

2 to predict what types of devices, what delivery

3 methods may be used in the future.

4 The first method, infusion of cells into a

5 coronary artery offers potential advantages that

6 include simplicity and ease of use. As has been

7 mentioned earlier today, this method may not be

8 suitable for all types of cell suspensions.

9 Potential limitations include the potential

10 requirement that infused cells be able to migrate

11 from the vasculature into the myocardium, and a

12 potentially increased risk of embolization or

13 microembolization if some types of cells are

14 infused using this method.

15 Published case series using this method

16 have demonstrated preliminary clinical feasibility

17 both when used within hours to days following acute

18 myocardial infarction, and this is often following

19 emergency stent placement at the site of thrombotic

20 occlusion.

21 It has also been used in patients who

22 suffer from chronic myocardial infarction and

23 ischemia.

24 In the studies reported to date, balloon

25 catheters have typically been used to temporarily

295

1 occlude the coronary artery proximal to the

2 treatment region. The desired cell suspension is

3 then infused into the artery distal to the inflated

4 balloon either using a lumen within the balloon

5 catheter, typically, a guidewire lumen, or using a

6 separate infusion catheter placed lateral to the

7 balloon catheter, such that it lies between the

8 inflated balloon and the artery wall.

9 Use of balloon occlusion permits infusion

10 of cells at pressures that exceed coronary artery

11 pressure, and it has been hypothesized that

12 increased infusion pressures may provide benefits

13 that include increased dispersion of cells within

14 the vasculature, increased adhesion of cells to the

15 vascular endothelium, and increased migration of

16 cells across the vascular endothelium and into the

17 myocardium.

18 This familiar illustration from a

19 publication by Strauer, et al., illustrates the use

20 of a coronary artery balloon catheter to infuse

21 cells into a region of acute myocardial infarction.

22 In this illustration, the balloon catheter

23 has been inserted into a large artery, directed

24 retrograde through the aorta, then, into a coronary

25 artery, and then directed distal within that artery

296

1 to the site of acute thrombosis.

2 Following inflation of the balloon to

3 obstruct the artery, a syringe is used to infuse

4 cell suspension through the guidewire lumen of this

5 catheter and into the coronary artery distal to the

6 balloon.

7 Finally, although it is not obvious in

8 this illustration, in this clinical study, the

9 balloon was inflated at the site of acute

10 thrombosis, and more specifically, at a site where

11 a coronary artery stent was placed as an emergency

12 treatment for the myocardial infarction, and that

13 is potentially important because balloon deployment

14 within a coronary artery stent can largely protect

15 the artery from one potential concern that we will

16 discuss briefly today, and that is the potential

17 for damage to the artery caused by balloon

18 inflation and subsequent stretching of the artery.

19 Studies of this cell delivery method

20 reported to date have commonly used balloon

21 angioplasty catheters to occlude the artery. The

22 catheters are originally designed to stretch the

23 lumen of an occluded fibrotic atherosclerotic

24 coronary artery to a specific diameter that has

25 been selected in advance by the treating physician.

297

1 They are also used to expand coronary

2 artery stents and again to prespecify diameter.

3 Although these catheters were not originally

4 designed for occlusion of an artery, they can be

5 used for that purpose, followed by cell suspension

6 and, additionally, as noted, if these catheters

7 have a central guidewire lumen, that lumen can then

8 potentially be used to infuse the cell suspension.

9 Potential considerations when you are

10 using an angioplasty catheter for this purpose

11 include the following. First, contact between the

12 catheter lumen materials and the cell suspension

13 can potentially adversely affect either the

14 viability or the functionality of the infused

15 cells.

16 Additionally, those cells may contact

17 various lubricants that are commonly applied to the

18 guidewire lumens during manufacturing for the

19 purpose of facilitating guidewire passage.

20 As a note, we are not aware yet of

21 published reports that have examined whether

22 catheter lumen materials may adversely affect

23 viability or functionality of cells, however, this

24 published animal study cited in the slide evaluated

25 cardiac delivery of a transgene by an adenovirus

298

1 vector.

2 The investigators found that some lumen

3 materials tested for use in a prototype needle

4 injection catheter, the second type of catheter we

5 will discuss today, adversely affected both the

6 viral activity and viral transduction.

7 They also found that a change in lumen

8 materials was sufficient to completely eliminate

9 these adverse side effects.

10 Additional considerations. Number two. A

11 second consideration when balloon angioplasty

12 catheters are used for this purpose, infusion of

13 cells, is that the balloon was originally designed

14 to stretch a coronary artery, in other words, to

15 controllably damage the artery, and for this use,

16 it must instead be used to occlude that artery,

17 hopefully, without damaging it. This is

18 potentially important.

19 The degree of artery wall stretch that is

20 typically created during balloon angioplasty will

21 also subsequently induce arterial stenosis due to

22 multiple mechanisms. We think it is therefore

23 essential that safe methods for balloon inflation

24 be developed and demonstrated if you want to use an

25 angioplasty catheter for this purpose.

299

1 Importantly, development of safe methods

2 for this new use could be complicated because

3 balloon angioplasty catheters have widely varying

4 pressure diameter relationships, in other words,

5 compliance can vary greatly between different

6 catheter models, so therefore the methods that are

7 developed for one catheter may not be applicable to

8 another one.

9 A third potential concern related to this

10 is the potential that concentrated cell suspensions

11 may clog the catheter lumen.

12 A fourth concern is that the lumens and

13 connectors of angioplasty catheters are primarily

14 designed for passage of a guidewire. They may not

15 have been tested for the ability to sustain high

16 pressures that can occur during infusion of

17 concentrated cell suspensions.

18 The second type of catheter we will

19 discuss today are needle-tipped injection

20 catheters. This method of cell delivery also

21 offers potential advantages. Notably, first, the

22 ability to directly inject cells into desired

23 myocardial locations. Second, the potential for

24 use with all types of cells.

25 The investigational cell delivery systems

300

1 developed for this therapy consists either of a

2 catheter or of a system comprised of a catheter

3 plus delivery sheaths, that include a retractable

4 distal injection needle. None are approved

5 currently for sale in the U.S., however when we

6 consider these new devices, it may be useful to

7 note that some design requirements, and thus,

8 potentially catheter characteristics, may be

9 similar to requirements for other currently

10 marketed cardiac catheters, potentially including

11 both cardiac electrophysiology ablation catheters

12 and endocardial biopsy catheters.

13 More specifically, all three types of

14 catheters or catheters plus sheaths would generally

15 require a steerable or deflectable tip in order to

16 facilitate direction of the catheter tip to various

17 endocardial locations, and all three types of

18 catheters must be sufficiently stiff to permit the

19 user to maintain stable contact between the

20 catheter tip and the moving endocardial surface of

21 the ventricle.

22 These illustrations are from a publication

23 by Dr. Perin\'s group, and they illustrate one,

24 investigational needle-tipped injecting catheter.

25 The photo on the left illustrates a complete

301

1 catheter including controls on the catheter handles

2 that deflect the tip and that extend and retract

3 the needle.

4 The needle would normally be retracted

5 into the catheter except when briefly extended for

6 each injection that is made during a therapy

7 session, and the syringe, potentially loaded with

8 therapy suspension, is attached to an infusion

9 port, also on the catheter handle.

10 The drawing on the right illustrates

11 delivery of the catheter retrograde through the

12 aortic arch, then, through the mitral valve, and

13 into the left ventricular cavity. In this drawing,

14 as you see, the catheter tip is deflected and the

15 catheter is being used to make multiple injections

16 from the endocardial surface of the ventricle.

17 There are also potential concerns that

18 attach to the use of needle injection catheters for

19 delivery of cell therapies for cardiac disease.

20 First, we believe this type of catheter

21 may potentially be particularly prone to clogging

22 by cell suspensions. Factors that might contribute

23 to this include the following:

24 The potential desirability of using very

25 small injection volumes plus highly concentrated

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1 cell suspensions, the potential desirability of a

2 small diameter injection needle that will also,

3 thus, have a small diameter lumen, and the fact

4 that injections may be made at more than 20

5 locations during a treatment session, thus,

6 increasing the potential for clogging due to the

7 repeat injections.

8 A second concern. As noted previously, it

9 may be important to ask whether cell viability or

10 functionality could be adversely affected by

11 contact with catheter lumen materials. In this

12 type of catheter particularly, we think it may also

13 be important to ask whether shear forces produced

14 during infusion of cells through a long,

15 potentially a very small diameter injection lumen

16 might also adversely affect the cells.

17 Third, it may be important to consider the

18 safety, and this was brought up in one earlier

19 session, of whether the safety of the cell

20 suspension is accidentally delivered in the

21 systemic circulation.

22 Of note, with this type of catheter, it

23 may be very difficult to maintain continuous

24 contact between the catheter tip and the moving

25 endocardial surface of the beating heart, and when

303

1 contact is broken, therefore, you could have

2 injection into the ventricular cavity and into the

3 circulation.

4 There is a note, for folks in the

5 audience, I don\'t know if you can see the

6 reference, but if I understand correctly, we will

7 have cells on the web site, is that correct? Okay.

8 Otherwise, I was going to read the reference to

9 you.

10 One note, if people want to look into

11 this, this has been studied for cardiac ablation

12 catheterization electrophysiology. I have noted

13 one good study up here where they used intracardiac

14 ultrasound imaging catheters to evaluate the

15 difficulty of maintaining continuous contact when

16 they thought contact was perfect.

17 A fourth consideration. Should we assume

18 that it is important to control or limit the

19 maximum needle extension? Factors to consider

20 might include the following: Is it critical to

21 avoid injection or laceration of the organs that

22 surround the heart?

23 It may also be important to consider

24 whether there may be safety concerns if the cell

25 suspension is inadvertently injected into the

304

1 pericardial or thoracic spaces, or if it is drained

2 from these spaces by the lymphatics and then

3 delivered into the systemic circulation. Relating

4 to needle injection, curves or bends in many

5 catheter designs, including the 180-degree bend

6 around the aortic arch that will normally be

7 present, could affect the needle extension length

8 of the catheter.

9 Finally, particularly in hearts that have

10 minimal epicardial fat surrounding the left

11 ventricle, it may be difficult to avoid occasional

12 injection completely through the wall of the

13 ventricle and into the pericardial space.

14 Factors that might contribute to this

15 could include the following:

16 First, is locally thin regions in the

17 ventricle, possibly related to myocardial

18 infarction, possibly related to the normal

19 indentations that separate the muscular trabeculae

20 of the ventricle on the endocardial surface of the

21 heart.

22 Second, is compression or stretching of

23 the ventricular wall where the catheter tip is

24 again pressed into contact with the wall, and,

25 finally, the possibility that a forceful injection

305

1 could simply potentially separate both myocardial

2 cells and epicardial cells, allowing cell

3 suspension to flush completely through the

4 ventricular wall.

5 A fifth and final question regarding

6 needle injection catheters is the following: Are

7 injection depth and the spread of injection of the

8 injected cells potentially important therapy

9 parameters?

10 For example, will injection of cells near

11 the more ischemic endocardial surface of the heart

12 provide therapy that is identical or equivalent to

13 injection near the less ischemic epicardial

14 surface, or is a minimally dispersed bolus of cells

15 at each injection site equivalent to wider

16 dispersion of cells at each injection site?

17 We currently suspect that catheter design,

18 cell suspension characteristics, and injection

19 speed can all affect injection depth and spread.

20 If a clinical study is performed using a

21 specific injection catheter and a specific cell

22 suspension, will the same therapy then be delivered

23 if a different injection catheter is used to

24 deliver that same cell therapy?

25 Of note, you can use animal studies, and

306

1 this is where the large animals become important,

2 to characterize the depth and spread of the cell

3 suspensions produced using a specific catheter.

4 Finally, and this is a question I think

5 that unites all the questions listed above on this

6 slide, when an investigational therapy is studied,

7 how important is it that the therapy delivered be

8 characterized? When an investigational therapy is

9 poorly understood, is the characterization of

10 therapy more or less important?

11 Today\'s meeting is focused on scientific

12 discussion of cell therapies for cardiac disease,

13 and because of both the focus of this meeting and

14 the time constraints, this is not a good forum for

15 discussion of regulatory concerns related to

16 cardiac catheters, however, if you would like to

17 discuss cardiac catheters intended for delivery of

18 cell therapies, you may contact either myself or my

19 branch chief, Mr. Elias Mallis.

20 I have listed our contact information on

21 this slide. Again, it will be posted on the web

22 site.

23 Finally, because this is one of the final

24 presentations today, I have one final slide. My

25 manager has repeatedly asked the following

307

1 question: Whether there are earlier or predicate

2 devices that have been used to provide catheter

3 delivery of biologicals.

4 He has also asked what we can learn from

5 any earlier devices. Initially, I was unable to

6 define a useful predicate for catheter delivery of

7 biologicals. Then, I did find a useful example,

8 and it was among photos from the Minnesota State

9 Fair.

10 The obvious lesson from this photo, I

11 would say is that intense concentration may be

12 required during catheter manipulation.

13 \[Laughter.\]

14 DR. JENSEN: Thank you.

15 \[Applause.\]

16 DR. RAO: Thank you, Dr. Jensen.

17 I am going to suggest that we wait and

18 hold off questions until we hear from Dr. Lederman,

19 as well, since he may be perhaps answering some of

20 those questions, and then direct questions to both

21 people at the end of that talk.

22 Transcatheter Myocardial Cell Delivery: Questions

23 and Considerations from the Trenches

24 DR. LEDERMAN: I am going to be quick. I

25 am grateful for the opportunity to speak before

308

1 this audience and this committee. Thank you for

2 your service.

3 I am speaking to you as a clinical

4 cardiologist, so I like to think of myself as Joe

5 six pack of clinicians. I will be talking about the

6 considerations, the frustration that many

7 investigators feel when we would like to talk with

8 the agency to get some guidance about how to start

9 bringing these interesting therapeutics to clinic

10 assuming we have determined that the timing is

11 right to bring therapeutics to clinic.

12 I am sorry there will be a bit of

13 repetition. I will try to go quickly through

14 repetitive slides.

15 We are dealing with integrated therapies

16 and unfortunately, we are also dealing with a

17 morass of regulatory purviews that don\'t

18 necessarily intersect. You have seen already that

19 we have considerations of delivery devices, as well

20 as cellular agents, to combine in therapy, and we

21 haven\'t discussed much in this room combinations of

22 novel mobilization agents should we choose to use

23 that route to drive our cells, and it becomes

24 difficult when we have proof of concept in some

25 animal models to find an appropriate proof of

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1 concept to support our clinical trial and to

2 support our safety considerations when some of our

3 colleagues outside the U.S. have kind of moved

4 ahead.

5 So, let\'s see if I can generate some

6 interesting questions for the committee, and that

7 is really what I hope to end on.

8 You have seen this slide from Strauer. I

9 will review again just the different approaches to

10 cell delivery, into coronary cell delivery is

11 attractive because it is very easy, there is a wide

12 dispersion into the target territory, and there are

13 a lot of available devices to be used although they

14 must be used off label.

15 The disadvantages have been mentioned that

16 there is a potential coronary artery injury. One

17 of the real clever innovations by Strauer\'s group

18 and by Sawyer\'s group and the Hanover group is that

19 they have chosen to deliver cells through an

20 occlusive balloon deployed at the site of a

21 recently deployed stent, so there is really almost

22 no possibility of coronary injury from the delivery

23 device, however, there is a possibility of a

24 coronary microembolism, and when you test this is

25 the setting of a recent acute myocardial

310

1 infarction, that coronary embolism may be difficult

2 to detect, so clinicians have gotten away with it,

3 or at least I should say their patients have gotten

4 away with it.

5 Certainly, there is a great potential for

6 direct washout of injected cells. Very few people

7 have actually measured this or reported this, but

8 there is some evidence that there is a low

9 fractional retention of delivered cells, and really

10 this intracoronary cell delivery is yet another way

11 to expose the entire patient to the therapeutic

12 agent.

13 This kind of approach is probably

14 unsuitable to certain patient populations,

15 especially those with chronic myocardial ischemia

16 when the inflow arteries are occluded. Most

17 investigators have taken advantage of transient

18 coronary flow interruption ostensibly to improve

19 local retention, but it is just not clear the value

20 or importance of this transient coronary flow

21 interruption. Certainly, a lot of patients can\'t

22 tolerate prolonged coronary flow interruption

23 without incremental myocardial injury.

24 There has been discussion about both

25 surgical and transcatheter cell injection.

311

1 Certainly, most of us will recognize that

2 catheter-based injection is less morbid than

3 surgical epicardial injection. Primary surgery has

4 been unattractive in investigational studies when

5 the surgery is offered only for the sake of cell

6 delivery.

7 The problem is in small studies, combining

8 cell delivery with an effective therapy, I think

9 has been mentioned by several people before,

10 doesn\'t really generate meaningful safety or

11 efficacy data, because the assessment of toxicity

12 events is confounded by the concomitant surgical

13 procedure, and the assessment of efficacy events is

14 very easy to ascribe to the concomitant effect of

15 therapy.

16 So, I think this kind of approach should

17 probably be discouraged in small, single-center

18 studies. I hope some investigators in the room

19 have already overcome this in moving to larger

20 studies.

21 Direct catheter injection of the

22 myocardium is attractive because we can achieve a

23 high local cell density and probably a high total

24 dose. It is certainly very easy also, with the

25 variety of catheters that I will describe, and the

312

1 entire myocardium is for the most part accessible

2 irrespective of the patient\'s individual coronary

3 anatomy.

4 But these devices are disadvantageous in

5 the U.S. because there are no approved devices,

6 although a few are available through

7 investigational device exemption.

8 I will show you some data that there also

9 is low retention of injected cells and that direct

10 myocardial injection is yet another means of

11 systemic exposure of the patient to the cellular

12 agent. We are left even in the best situation with

13 multifocal cell accumulation, meaning a

14 heterogeneous dispersion of the cellular agent.

15 Whether or not that is important isn\'t

16 clear, and there is the potential for damage to the

17 myocardium or to the chordal structures or the

18 valve structures, however, this potential has not

19 been supported by experience with comparably

20 aggressive or more aggressive intramyocardial

21 catheters especially in the fairly large laser

22 myocardial \"revascularization\" experience or

23 angiogenic gene transfer.

24 There are a bunch of variants of

25 myocardial injection catheters. There are

313

1 techniques to access the pericardium and bathe the

2 epicardial surfaces of the heart with the cell

3 preparation of interest.

4 Patients with chronic myocardial ischemia

5 who have undergone coronary bypass surgery are, for

6 the most part, not eligible, so this is a difficult

7 approach in early clinical trials.

8 There are investigators who have

9 demonstrated satisfactory delivery of genes and

10 cells by retrograde coronary venous approach. I

11 will show you a picture to show what that means. A

12 company has commercialized tangential transvenous

13 intramyocardial injection.

14 Then, there are a bunch of endocavitary

15 catheters that go across the aortic valve

16 retrograde and are pretty successful in delivering

17 cellular agents. There are an array of what I call

18 \"dumb\" catheters that we use just under x-ray

19 guidance that are very attractive because they are

20 quick and easy. Two examples are Boston Scientific

21 Stilletto and Biocardia Device.

22 There a couple of smarter devices, it is

23 not clear that they are better, but they employ a

24 Static Roadmap like the Cordis Biosense

25 electromagnetic guidance system with some

314

1 non-fluoroscopic guidance. There are also devices

2 that have an integrated ultrasound, and the

3 smartest devices, I will show you an example, in

4 research mode only, of instantaneous imaging both

5 of the tissue and the device.

6 We should all open our hearts to the

7 possibility that surgical videothoracoscopic, a

8 minimally invasive surgical procedure may

9 accomplish cell delivery with very little morbidity

10 even in a primary surgical procedure.

11 This is just a demonstration of one of the

12 so-called \"dumb\" catheters. This is a Boston

13 Scientific catheter going from a femoral artery of

14 a pig, across the aortic valve, and can

15 successfully guide whatever agent you want with

16 centimeter, not millimeter, precision to any aspect

17 of the endocardial surface.

18 Medtronic has recently bought

19 Transvascular, which is an interesting device that

20 has an integrated ultrasound to guide the

21 deployment of a needle through a coronary vein and

22 can access target myocardium through any of the

23 coronary veins in a tangential fashion. This has

24 been tested in clinic in a very small number of

25 patients, and so far there haven\'t been any safety

315

1 disasters.

2 A retrograde transvenous approach has been

3 demonstrated I believe only in animals. The

4 Stanford group, Keith Marsh\'s group, have been

5 interested in this for delivery of dyes or gene

6 agents or even some cellular agents. I am sorry

7 the pictures aren\'t very attractive. It is not

8 clear to me how this can possibly work, but the

9 proof of principle has been shown.

10 It is also attractive in that the inflow

11 coronary artery anatomy is not a problem since

12 coronary vein patency is maintained in virtually

13 all patients. Dr. Perin is an expert in the use of

14 the Cordis electromagnetic guidance system, the

15 Biosense system that has its advantages in that it

16 has been widely used and tested for a variety of

17 investigational approaches.

18 It is disadvantageous in that it is a

19 prior roadmap of the heart that may vary over time,

20 and so it is not clear to me that you accomplish

21 millimeter scale precision of your injections, but

22 it is also not clear that that is very important.

23 I think Dr. Epstein\'s group also has great

24 expertise in the use of the device for cell

25 therapy.

316

1 Just to brag about some work in my lab for

2 a moment, we have used real-time MRI to guide cell

3 injection to very small targets with great

4 precision and great ease. This is, as you see, a

5 multi-slice real-time movie of the heart. You can

6 see it in long axis and short axis, of a pig in

7 which we have caused a tiny, little

8 microinfarction, shown in white.

9 The catheter is shown in red and green,

10 and we can see with great 3-dimensional sense where

11 we are steering our catheter, and if we like, we

12 can label cells, say, mesenchymal stromal cells,

13 very easily.

14 Here, the mesenchymal stromal cells are

15 showing up in black, so you can actually see

16 interactively, as you deliver the cells, that at

17 least some are attained in the target myocardium,

18 and this is almost ready for clinical application.

19 I want to show a little bit of data. This

20 is from my youth when I was just out of fellowship,

21 a study that was funded by Boston Scientific while

22 I was at still at University of Michigan.

23 We injected neutron-activated microspheres

24 into the heart with an endocavitary catheter,

25 direct surgical approach or postmortem, and what

317

1 was interesting is that we inject and then kill the

2 animal within minutes, and most of what we inject,

3 both surgically or transcatheter approach, is lost

4 immediately.

5 There is some effect of volume. Smaller

6 volumes were associated with slightly greater

7 retention in tissue. This is 10 microliters. That

8 is a tiny, tiny injection compared with 20 or 100.

9 A better study was published by Smits from

10 the Thoraxcenter group in Rotterdam using

11 scintigraphy and radiolabeled albumin, either plain

12 radiolabeled albumin or a colloid, and they showed

13 also loss of the majority of injectate after just a

14 minute by scintigraphy.

15 Their colloidal preparation had great

16 retention, which is interesting, and that there

17 might, of course, be some interaction between

18 biological agents and the myocardial interstitium,

19 so conceivably, cells won\'t necessarily be lost.

20 These are studies that can be done in the lab in

21 healthy animals.

22 But I think it is easy to say that local

23 myocardial injection is at best an exaggeration,

24 that most injectate is lost rapidly and exits

25 either by backflow directly into the myocardial

318

1 cavity, which we can see directly, or with

2 intracardiac ultrasound or even with

3 high-resolution x-ray, that there is also a clear,

4 what I call \"intravasation\" or return to the

5 coronary circulation or coronary lymphatics.

6 When we inject too deeply and directly

7 into the pericardium, that is another mode of exit,

8 but clearly, the interstitial myocardial target

9 retains only a fraction of what we intend to

10 deliver there.

11 So, where does this material go and is

12 that really important? I think it is important at

13 least that we assume that what we think we are

14 injecting by any route goes everywhere, and I think

15 that means that conventional toxicology or

16 biodistribution experiments can be conducted in

17 uninfarcted animals without the needle of interest

18 just by modeling it as a left atrial or left

19 ventricular cavitary injection that is not device

20 specific.

21 I think also it is interesting to talk

22 about open- label autologous unfractionated bone

23 marrow data, what is the incremental value of

24 animal safety or tox experiments in light of the

25 fact that provisional safety has been shown in

319

1 open-label studies if we are convinced that the

2 safety reporting has been complete, and I am pretty

3 comfortable that it has been.

4 For autologous leukapheresis products, say

5 we apherese CD34 cells for a direct myocardial

6 injection, those cells are circulating already. It

7 is not clear to me what is the value of incremental

8 biodistribution experiments regarding systemic

9 exposure.

10 I will qualify that by saying that

11 allogeneic material perhaps should be treated

12 differently, but for autologous material, it is not

13 clear to me that these animal data are valuable,

14 and to require it of investigators before going to

15 clinic sounds dubious in my opinion.

16 Also, it is worth noting that the

17 importance of targeting is just not established.

18 While I am very interested in precise anatomic

19 targeting, it is not clear why we need it.

20 Delivery targets certainly vary by

21 application. We may want to target infarct

22 borders. Doris Taylor had some data today that

23 infarct borders may be unattractive for certain

24 therapies.

25 Do we want to target ischemic zones, or do

320

1 we want to avoid ischemic zones, do we want to

2 avoid thin myocardium? Is roadmap data worse than

3 blind data or worse than instantaneous real-time

4 MRI data? It is just not clear.

5 Certainly, good targeting is attractive in

6 that it may reduce overlapping injections and waste

7 of injections, and overlapping injections may

8 increase systemic loss. It is hard to imagine that

9 we might be exceeding some therapeutic index.

10 So, in other words, the value of targeting

11 is just not clear to me, and if we are able to show

12 some efficacy, it is not clear how much we must ask

13 of investigators to establish these catheter-based

14 information before going to clinic.

15 A point that has not been mentioned, but

16 that I have encountered in animal studies is that

17 operators need feedback regarding delivery of their

18 therapeutic agent, and I would like to encourage

19 the committee and the regulatory agency to consider

20 contrast labeling at the time of cell delivery. It

21 is certainly clearly more important than needle

22 stability measures.

23 There are lots of ways to label injection

24 mixtures. You can admix contrast into your

25 injection cocktail. Iodinated radiocontrast is

321

1 clearly tolerated in myocardium. We inject

2 high-dose, full-strength intracoronary

3 radiocontrast, replacing blood inflow for many

4 seconds in patients in all settings, acute MI,

5 acute and chronic ischemia, and healthy myocardium.

6 It is certainly very well tolerated. It

7 is hard to believe that iodinated radiocontrast

8 injected into the myocardial interstitium is not

9 tolerable. It is certainly well tolerated in

10 animal experiments, but this kind of feedback under

11 x-ray guidance, for example, is critical in knowing

12 that we are delivering the cells into the target

13 that we think we are.

14 If we are doing injections under MRI,

15 then, certainly we can admix gadolinium-based MRI

16 contrast agents in very dilute form just like the

17 agent that reaches the myocardium after systemic

18 exposure, and these are very easy to be tested

19 biocompatible in vitro.

20 Certainly, we can do test injections of

21 contrast to test the purchase of our needle in the

22 myocardium before injecting the cell of interest,

23 but the problem with these test injections of

24 radiocontrast is that the catheter and hub dead

25 space often exceeds the volume of the desired cell

322

1 injection, so that is a problem that is difficult

2 to overcome.

3 Alternatively, we can label our cells.

4 That is certainly easy if we want to deliver our

5 cells under MR or under echo, but it is not clear

6 what options we have under x-ray, so to the members

7 of this committee, please facilitate solutions to

8 this clinical need.

9 Some engineering concerns have been

10 mentioned in the very excellent guidance materials

11 supplied to members of the committee. I just want

12 to speak to some of them.

13 The issues of biocompatibility of lumens

14 and potential clogging of lumens, this is easy to

15 test on benchtop and doesn\'t require animal

16 experiments.

17 The issues of balloon injury of target

18 coronary arteries is an important one. You have

19 seen creative solutions by investigators in Europe

20 by protecting the target coronary artery, inflating

21 their occlusion balloon inside a recently deployed

22 stent.

23 Certainly, there are noninjurious

24 compliant occlusion balloons that are clinically

25 approved for a variety of peripheral artery

323

1 applications, that are used widely in the cerebral

2 circulation, that are also used for coronary

3 protection and substantial equivalence data are

4 already widely available.

5 We have also heard discussion of

6 considerations of the pressure capacity of balloon

7 wire lumens. The European investigators, for

8 example, are administering their cells via the wire

9 lumen of an inflated coronary balloon.

10 As a practicing interventional

11 cardiologist, I don\'t consider that an important

12 concern because every balloon, every over-the-wire

13 design balloon that I put into a patient, I expect

14 to use for intracoronary angiography, and I use for

15 intracoronary angiography with a fairly high

16 pressure system.

17 There are many times I need to know that

18 my balloon is in the right place, so I pull out the

19 wire and I inject contrast at a fairly high

20 pressure directly through the balloon lumen.

21 That is not, of course, an indicated use,

22 but it is a wide use by all operators of coronary

23 artery balloons, and I think the test of time has

24 already been past, but if you like simple benchtop

25 pressure data, they are easy to acquire.

324

1 Regarding endomyocardial injection

2 catheter engineering concerns, the same

3 biocompatibility and clogging concerns are easy to

4 answer on benchtop tests. This issue of variable

5 needle extension is probably an important one if

6 injection depth proves to be important, and it is

7 not clear that it is, but this can be addressed in

8 benchtop testing.

9 It has been mentioned that purchase

10 stability is important to assure injectate reaching

11 the target tissue. My assertion about marking or

12 labeling injection cocktails with contrast might

13 address that concern.

14 The report from UC/SF from Jonathan

15 Coleman using an old intracardiac echo device

16 reporting instability is actually a spurious

17 observation because of through-plane motion of the

18 target that the UC/SF group was inspecting with a

19 fixed vena caval or a right atrial intracardiac

20 echo device. In other words, I don\'t think it has

21 been shown that the contact of EP catheters or

22 myocardial injection catheters cannot be

23 maintained, in fact, just the opposite, especially

24 from the Biosense device which has a local cardiac

25 electrogram capability to assure contact stability.

325

1 So, I think this is not really an

2 important problem for us to worry about.

3 The issue of potential myocardial

4 perforation is often raised when we discuss the

5 possibility of delivering cells directly into

6 patients after a recent large myocardial

7 infarction, and I think that is an important one.

8 It is interesting that you refer to myocardial

9 biopsy devices as predicate devices, because as I

10 view the biotomes as some of the most dangerous

11 devices we ever laid hands on, they are so

12 incredibly stiff and indeed perforations do

13 sometimes occur.

14 Fortunately, there is an animal experience

15 from my lab. I guess we should probably get it out

16 there, of a large number of injections directly

17 into freshly infarcted myocardium, and I think this

18 kind of data is easy to obtain.

19 But the bigger issue is not that freshly

20 infarcted myocardium can be safety injected, it is

21 that the device companies can\'t really control the

22 operators. I have seen this so many times. An

23 engineer walks into a lab and cringes as the

24 interventional cardiologist effectively abuses the

25 device. How do you model operator misbehavior? It

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1 is kind of difficult.

2 In reality, proof of principle has been

3 established.

4 The issue of inadvertent pericardial

5 injection probably has little or no clinical

6 importance especially when compared with the loss

7 of injectate via other routes, and its only value

8 is that you are not delivering what you think

9 directly into target tissue again, the value of

10 instantaneous visualization of injections.

11 The issue of distribution of injected

12 material within the target myocardium, I think it

13 may be reasonable to assume that this distribution

14 is different in normal myocardium versus fresh

15 infarct versus chronic scar, but the value of these

16 data are just not clear compared with the efficacy

17 data in support of preclinical or early clinical

18 experiments, so having this information of how many

19 cubic centimeters of myocardium are exposed to

20 target cell based on a given volume or dose of

21 cells, it is just not clear why we need that

22 information. This kind of information ultimately

23 can only be valuable in patients.

24 Are endomyocardial injection catheters

25 generic? In my opinion, assuming benchtop

327

1 biocompatibility has been determined, and assuming

2 that mechanical performances are satisfactory

3 compared with predicate devices, I think that a

4 myocardial injection catheter is pretty much the

5 same from one to another.

6 One needle device should be translatable

7 to another, and the scientific and regulatory value

8 of additional data from large mammals, healthy

9 ischemic infarcted, is really pretty small and hard

10 to justify, in my opinion. So, I keep giving this

11 message, nihilistic message about the large animal

12 models investigators have been asked to provide.

13 To summarize my opinions, I think

14 engineering and biocompatibility concerns can be

15 addressed with benchtop data. I think that animal

16 model safety experiments matching a given catheter

17 device with a given putative therapeutic agent

18 don\'t meaningfully contribute to patient safety and

19 are, in fact, potentially misleading.

20 I wish that there were a way to get a

21 screening IDE or IND capability to support testing

22 new cell preparations without repeating unnecessary

23 preclinical experiments as we switch from device to

24 device, and ultimately, careful human

25 experimentation is what is most important.

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1 Let me just make a few more points before

2 I turn to some questions and step off the podium,

3 so people can go home.

4 I want to reiterate some points made by

5 other speakers today, and I want to reiterate it

6 especially to the regulatory officers here. I

7 think that blinded placebo groups are mandatory

8 even in first experiments.

9 Why would you conduct an experiment

10 without a suitable matched control in the name of

11 safety? That is just bad science. Interestingly,

12 there has not been a single open-label or \"Phase I\"

13 safety trial that fails to make an efficacy claim

14 without a suitable matched control.

15 Unfortunately, the agency is inadvertently

16 discouraging blinded controls, for example, when

17 they ask for a delay in between exposing a given

18 subject within a given group of patients, asking

19 for seven days or four-week delay between patients

20 to look for safety of individual patients. This

21 often frightens investigators away and makes them

22 drop placebo groups.

23 I think that in cardiology, we rarely

24 conduct classic Phase I studies in end-stage

25 subjects in spite of the conversation in Doris

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1 Taylor\'s speech.

2 So, I would like to encourage people in

3 the committee and encourage regulatory agencies to

4 facilitate inclusion of blinded placebos in early,

5 first in man even, experiments.

6 I was also asked to talk a little bit

7 about the safety of direct myocardial injection,

8 and unfortunately, there are no large series of

9 direct myocardial injection of cells or any other

10 agents, however, a related catheter has been tested

11 in a few hundred patients.

12 Cordis had a myocardial\--they called it a

13 DMR, direct myocardial revascularization procedure,

14 but it was a way to burn the myocardium from a

15 transcatheter approach. In this Cordis-sponsored

16 study presented by Martin Leon and Ron Kornowski a

17 few years ago, a Cordis Biosense derivative, a

18 device much like the myocardial injection catheter

19 shown today, was used to steer into the myocardium

20 of 300 patients with refractory ischemia, mild or

21 moderate left ventricular dysfunction, and preserve

22 wall thickness.

23 One hundred patients underwent sham

24 procedures, placebo burns of the myocardium, and

25 200 more received laser in two different doses.

330

1 This is just the clinical complications. I will

2 point you to left ventricular perforation. There

3 were none in the 100 placebo patients, there was 1

4 out of 100 in the highest laser dose, which is

5 comparable to some other laser trials.

6 I think this establishes a relative safety

7 base from a perforation perspective. These other

8 events unfortunately weren\'t very well described,

9 and this study unfortunately has not been

10 published, and I am not sure it ever will be

11 submitted for publication, but the acute safety of

12 this device, I think is relatively self-evident.

13 So, is placebo and the myocardial

14 injection safe in principle? I think yes, and it

15 is not a reason to discourage these placebo groups

16 in early first clinical studies.

17 So, again, we are trying to bring

18 therapies to clinical testing, and we are trapped

19 between delivery devices and cellular agents that

20 we would like to use together.

21 I want to just ask a few hypothetical case

22 questions to the committee before I step down. I

23 certainly don\'t have answers, but I hope you find

24 it provocative.

25 Let us say that an investigator identifies

331

1 a novel marker, an HL321, let\'s call it, that

2 identifies some kind of progenitor cell for which

3 there is no clear animal homolog.

4 There are some limited preclinical

5 efficacy that when an enriched human HL321

6 population is injected to intrinsically

7 immunocompromised rat infarct, it causes functional

8 recovery compared with a population of known cells

9 that are relatively depleted, so this is not the

10 most robust type of experiment, but based on

11 experiments like this, and based on patients who

12 are clamoring for therapy of their massive

13 myocardial infarction, investigators may want to

14 bring some test of this therapy to clinic even now.

15 How could we test local autologous HL321

16 cells assuming we had a feasible way to mobilize

17 and recover these cells? How could we test that

18 for safety and efficacy?

19 In the example I want to give you, there

20 already is a commercial cell system available,

21 marked with a CE in Europe, and in Europe, hundreds

22 of patients have successfully undergone bone marrow

23 transplantation with a population positive

24 selection for this marker, and also in Europe,

25 dozens of patient underwent local cardiac delivery

332

1 trials in a variety of applications, and Phase II

2 trials have been offered.

3 Is it unreasonable to permit U.S.

4 investigators to conduct similar experiments now

5 that provisional safety has been tested in Europe,

6 if incompletely reported in Europe?

7 For this kind of clinical experiment, are

8 additional animal data really necessary when human

9 studies have already been conducted, and when there

10 is no animal homolog of that positive selection

11 marker? What animal model is really adequate?

12 Can we use the experience of investigators

13 like Dr. Epstein, investigators like Dr. Perin,

14 using undifferentiated bone marrow to support the

15 local delivery of other autologous cells, and are

16 individual cell preps, autologous cell preps

17 substantially equivalent in this case when they

18 have just been derived directly from the patient

19 irrespective of sources? Is bone marrow that much

20 different from apheresis product, from a

21 mobilization product?

22 So, how can we apply non-U.S.A. human

23 safety data to support U.S. clinical trial

24 proposals, or should we continue the way we are now

25 and just sit and wait for others to do their

333

1 experiments without us?

2 Thank you very much for your attention,

3 and I hope you found these questions provocative.

4 \[Applause.\]

5 DR. RAO: We have time for a few

6 questions.

7 Q&A

8 DR. NOGUCHI: I think that was a terrific

9 representation of the tension between belief and

10 what is published or not published, and what we

11 might call the paradigm for FDA, which is absence

12 of evidence is not evidence of absence.

13 I will just challenge you a little bit.

14 When we see safety data whether it is from

15 another country or here, one of the critical

16 questions for an adverse event is, well, is that

17 really showing that if you give a product that you

18 have a lack of an adverse event, or could it be

19 that, in fact, you didn\'t give a product, which by

20 your own arguments you would say most of the time,

21 you lose most of the product all over the place.

22 So, some of it falls into the category of

23 our experience is that adverse events actually

24 other than from the actual injections of others for

25 biological products don\'t occur unless you actually

334

1 also have bioactivity, and sometimes if you don\'t

2 have any cells, you may not have any bioactivity.

3 So, I think that you have a number of very

4 genuine points very well worth arguing, but I would

5 just caution that it is very simple to say

6 something is safe. We rarely say look at all the

7 published data and it\'s safe, because we don\'t

8 really know if a product was actually being used

9 there, and perhaps that is one of the points you

10 might want to just think about.

11 DR. LEDERMAN: I, of course, can\'t even

12 answer your question, and I want to point out I

13 want to thank the regulatory officers and the FDA

14 for trying to protect our patients, and to try to

15 protect the American public. You are in a very

16 difficult position that is often a thankless

17 position, but I just hope you are open to this kind

18 of conversation.

19 DR. NOGUCHI: Absolutely. That is why we

20 would like to have you end it here, because I am

21 sure it will provide the focus of discussion for

22 tomorrow.

23 DR. RAO: Any other questions?

24 DR. EPSTEIN: Bob, I really enjoyed that

25 presentation, it was really great. I would just

335

1 like to raise one point. For example, to my

2 knowledge, no one has really tested in depth the

3 safety of the transvascular administration of an

4 angiogenic agent or its cells.

5 The reason I feel that might be different,

6 for example, than a transendocardial or a

7 transepicardial injection is because you are

8 injecting it right around a large artery. It is

9 conceivable that there can be pro-atherosclerotic

10 or pro-restenotic effects.

11 DR. LEDERMAN: I am sorry. Let me

12 interrupt your question. Do you mean intracoronary

13 approach or the Medtronic transvascular approach?

14 DR. EPSTEIN: The Medtronic approach, so

15 where you are injecting it, not downstream at the

16 small vessel level, but at the large vessel level,

17 so if you are injecting it, for example, through

18 the venous system, it is contiguous with the

19 arteries that have atherosclerosis in it.

20 So, I would think that for that special

21 case, the FDA would require that you have to show

22 that there is no deleterious effect in terms of a

23 pro-atherosclerotic effect. I would be interested

24 in your thoughts about that.

25 DR. LEDERMAN: I think that point is well

336

1 taken and every such safety request or demand is

2 interesting and valuable, but how do we answer that

3 kind of question satisfactorily.

4 Let\'s take the question you just asked, a

5 tangential myocardial needle or perhaps a

6 retrograde venous administration of agent X, and

7 the problem of an unrecognized atherogenic effect,

8 how on earth do we test that? Are apoE knockout

9 mouse experiments satisfactory?

10 DR. EPSTEIN: For this particular, I would

11 injure a vessel in a pig, and then inject whatever

12 agent you are interested in transvenously in the

13 area of that injured vessel and just see whether

14 there is an increase in the neointimal response.

15 I don\'t know how you carry\--I mean your

16 question would be so what, whatever you see, and

17 that would be a good question.

18 DR. LEDERMAN: But that is exactly right,

19 that might reassure us, but that is also not

20 atherosclerosis.

21 DR. EPSTEIN: That\'s right, but the AMI

22 studies, you know, you are doing angioplasty, so if

23 you were to increase the incidence of restenosis in

24 the pig, you know, quite predictively, it would

25 certain add a major cautionary note to approval of

337

1 such a protocol.

2 DR. TAYLOR: I would ask two questions.

3 One, very short, but one is you said that you would

4 argue that exogenous delivery of any given cell

5 population is equivalent essentially, but I would

6 think that the GCSF, or if you have given one bone

7 marrow cell population, is it right to go ahead

8 with all the others without necessarily more safety

9 data?

10 I would argue that the GCSF data that just

11 came out would actually argue the converse, that

12 the only difference there was mobilization of cells

13 that would otherwise be endogenous to that same

14 patient, and yet an increased number of those cells

15 clearly caused some negative effect.

16 DR. LEDERMAN: I wasn\'t actually making

17 that assertion. I was making the assertion that

18 needle injection catheters are ultimately very

19 similar.

20 DR. KURTZBERG: But the difference, there

21 have been, I don\'t know how many tens of thousands

22 of patients have had GCSF, we have gotten bone

23 marrow in their right atrium, so I mean the

24 dissemination of those cells is not the issue, it

25 is combining that with a local technical injection

338

1 and trauma to that site that is different.

2 I mean there is experience with these

3 cells disseminated through the human body for two

4 decades, so that is not the issue. The issue is

5 what do the cells do in the setting of a local

6 technical injection into an artery or other part of

7 the heart that is sick.

8 DR. TAYLOR: I think that is sort of the

9 point I am trying to make, that exogenous delivery

10 is not necessarily the same thing as mobilization

11 of cells, and that having more cells there, that we

12 don\'t understand, we can\'t just interpolate from

13 other data.

14 I want to ask a very short question. What

15 do you think about clinicians moving forward who

16 don\'t have experience with preclinical studies? I

17 mean one of the things that probably enabled

18 Philippe to do the studies he did is he had that

19 six years of preclinical experience making mistakes

20 or whatever.

21 What do you think about any clinician

22 moving forward in a trial without having previous

23 preclinical experience?

24 DR. LEDERMAN: My short answer is who

25 cares what I think, and we all operate as parts of

339

1 teams with expertise in our respective areas. In

2 cell therapy, for example, it would be outrageous

3 for me to do an early clinical study without the

4 close collaboration of cell therapy experts like

5 some that are fortunately in the room.

6 And would we need to have our local

7 on-site preclinical experiments? It is not clear

8 to me how important that is. It is more important

9 to me that our agents be well characterized, that

10 the studies be well conducted, and that they be

11 designed in a way that the data can be interpreted

12 rather than open-label, early clinical experiments

13 that are very difficult to interpret.

14 DR. RAO: I really agree with on the

15 emphasis you made about the fact that you should

16 have a placebo-controlled trial, but then on the

17 same token, you know, you also said that one

18 catheter is much like the other, but I don\'t think

19 that we can extrapolate that from saying one is

20 much like the other, you know.

21 I mean we worry about drugs when we say

22 whether it is a generic formulation or whether it

23 is a formulation which contains the same active

24 ingredient, and to me, when you are looking at the

25 device, you are making it with cells, you have to

340

1 worry about how clearly or how similar the device

2 is to any other device.

3 We can\'t simply say, well, you know, the

4 benchtop pressure was the same. You know, I can

5 take syringes and I can show you that the benchtop

6 pressure on that cell agent injection is exactly

7 the same because of how I do it, but, you know, I

8 can put cells through it, and I can guarantee you

9 that there would be a difference.

10 I mean Dr. Menasch showed in his data you

11 use a 27-gauge needle, and it is very different

12 from using a 29-gauge needle. It doesn\'t matter

13 whether you have got the same pressure or not.

14 So, I think that it would be hard put at

15 least for me to be convinced that most catheters,

16 even if they are giving delivery externally in much

17 the same way, that one can logically extend it and

18 say that it will probably be the same.

19 DR. LEDERMAN: So you are telling me\--and

20 I don\'t mean to belabor the point\--but if you have

21 two catheters by two different vendors, that have

22 satisfactory benchtop testing for biocompatibility,

23 have satisfactory hydraulic characteristics, that

24 what you inject at one end comes out the other end,

25 and one such catheter has satisfactory efficacy

341

1 data in some kind of preclinical model, that you

2 would require a repetition of that preclinical

3 model for another catheter that is virtually the

4 same?

5 DR. RAO: I am saying that right now we

6 can\'t make the assumption that it will be the same,

7 and the reason I say that is that we know that when

8 we make minor manipulations to cells which we are

9 delivering, for example, if we take CD34 cells,

10 which have been kept in culture for 48 hours as

11 opposed to 12 hours, we have a very different

12 endpoint result. We know that.

13 We don\'t know what the interaction will be

14 with the catheter, and we can\'t make the assumption

15 that because we know five parameters, that those

16 will be adequate in making a reasonable prediction,

17 so until I have a lot more data, I will be very

18 surprised that one could make that statement or

19 anybody would agree that that is okey.

20 DR. LEDERMAN: The end result is that we

21 have an unmanageable number of permutations, an

22 unmanageable number of permutations that makes it

23 hard to make progress.

24 DR. RAO: But again I think this is to

25 reemphasize what Phil said, it is not that we can\'t

342

1 do it, so that means it shouldn\'t be done. It is

2 to try and identify what is critical, so that you

3 make sure that the critical points are done, so

4 that is the critical issue to me.

5 DR. RIEVES: Dr. Lederman\'s presentation

6 was excellent, and I think it raised some excellent

7 points. I think the important part will be to

8 discuss them tomorrow, and can give one example,

9 because for every point that was raised in that

10 discussion, there is always the other hand. It is

11 like the two-armed economist. I will give you one

12 example right now.

13 It is true that one study with what is a

14 laser TMR system, there were very few perforations.

15 There was another blinded, randomized study,

16 completed in the U.S., published in JACC, 140

17 patients. Only the treated patients, 70 treated

18 patients actually were catheterized.

19 Now, in that study, 5 of them had

20 perforations, so it is often difficult to\--just as

21 one example, there is always another side to this,

22 and the important thing is I think you have raised

23 some excellent points. We have left the tough

24 questions for our committee members to address.

25 DR. SIMONS: To come back to the point

343

1 that catheters are different, not only are they

2 different from each other, they are different from

3 the cells depending on the cell type used. I

4 absolutely do not think there could be universal

5 device.

6 DR. LEDERMAN: And these questions were

7 not answered in benchtop testing?

8 DR. SIMONS: No.

9 DR. RAO: I think your point is well made,

10 though, that I think the way devices need to be

11 regulated is somewhat different from cells, because

12 of the number of variables one might have to

13 consider are somewhat different, and I think that

14 is a very valid point.

15 If there is no more questions, we will go

16 to the open part of the question and answer

17 session.

18 Open Public Hearing

19 Before we can have the open public

20 hearing, by law, I am required to read a statement.

21 I will do that right now.

22 Both the Food and Drug Administration and

23 the public believe in a transparent process for

24 information gathering and decisionmaking. To

25 ensure such transparency at the open public hearing

344

1 session of the advisory committee meeting, FDA

2 believes that it is important to understand the

3 context of an individual\'s presentation.

4 For this reason, FDA encourages you, the

5 open public hearing speaker, at the beginning of

6 your written or oral statement, to advise the

7 committee of any financial relationship that you

8 may have with any company or any group that is

9 likely to be impacted by the topic of this meeting.

10 For example, the financial information may

11 include the company\'s or a group\'s payment of your

12 travel, lodging, or other expenses in connection

13 with your attendance at the meeting. Likewise, FDA

14 encourages you at the beginning of your statement

15 to advise the committee if you do not have any such

16 financial relationship.

17 If you choose not to address this issue of

18 financial relationships at the beginning of your

19 statement, it will not preclude you from speaking.

20 There were two people who had asked to be

21 recognized before the meeting started. The first

22 person is Dr. Vulliet.

23 DR. VULLIET: Thank you for the

24 opportunity to come and present some data to you.

25 Am I supposed to make a statement I have no

345

1 financial interest in this?

2 DR. RAO: Yes.

3 DR. VULLIET: Okay. I have no financial

4 interest in this.

5 This is an example of some studies that

6 were done very recently where we have been

7 investigating, using my research team, which is

8 myself, a cell biologist/pharmacologist, Dr.

9 Greeley is a pathologist, Mitch Halloran is a cell

10 biologist, Kristin McDonald and Mark Kittelson are

11 both board-certified cardiologists, so we have a

12 very interdisciplinary team.

13 We are at a vet school, which is probably

14 novel for this group, and we are very specifically

15 interested in animal models. I was very pleased to

16 hear quite the discussion of animal models. I

17 disagree with almost everything every one of the

18 speakers complained about, not being suitable

19 animal models.

20 I guarantee we see animal models that

21 definitely are real patients, that have real

22 disease. It is not induced, it is a real disease.

23 It is there, it needs to be treated. For that

24 reason, we have decided to investigate the

25 possibility of using cytotherapeutics to see if we

346

1 can produce a beneficial effect in animals.

2 A good example of our animal model\--and

3 this slide is probably Peter\'s example of what they

4 think we do with animals\--but this is Oscar, and I

5 guarantee you he will grow up to have somewhere

6 later in life, lumbar disease, lumbar disk disease,

7 either at the L2/3 or the L3/4, and he is a great

8 model if you are into disk disease, but that is a

9 different committee we talk to about that.

10 Steps for successful cytotherapeutics.

11 This is my perception, not the committee\'s

12 guideline, the first step is safety studies. This

13 is about where we are at. In fact, most of the

14 stuff we are squabbling about right now is whether

15 these things can be done safely or not, if you

16 think about it.

17 Very little good data on dose response,

18 nothing on time course that I am aware of. Nothing

19 or very little on clinical endpoint. What do I

20 mean by clinical endpoint? When I am giving an

21 antibiotic, I can tell you I need to hit serum

22 concentrations of 1 microgram per ml.

23 Okay. I can design a pharmacokinetic

24 regimen, I can hit 1 microgram per ml. If I am

25 treating dilated cardiomyopathy, what is my

347

1 endpoint? If I see something four to six weeks

2 later, I will be lucky.

3 So, when I am administering cells, I

4 really don\'t have a defined clinical endpoint at

5 this point other than the lack of adverse reaction.

6 Think about it. It is a very interesting point of

7 view.

8 Anyway, because we started off with safety

9 studies, that is what we did. We asked a very

10 simple hypothesis, and we started with can a half a

11 million cells\--we are using mesenchymal stromal

12 cells, we call them stromal cells rather than stem

13 cells because we are not convinced that primordial

14 germ layers have been demonstrated coming from MSC,

15 so we refer to them as stromal cells, you can call

16 them stem cells if you like.

17 We also use a terminology I don\'t think

18 anybody else has used in this room yet, is we are

19 using MICs \[ph\]. That is a million cells per

20 kilogram. We are very interested in a

21 dose-response relationship. Doses are a key in any

22 therapeutics.

23 So, we are giving half a million MSCs per

24 kilogram of body weight, and can they be safely

25 injected into the coronary arteries of the

348

1 anesthetized dog?

2 Simple experimental design, they are

3 autologous. We collect bone marrow somewhere about

4 a month later, we inject 10 million cells. These

5 are 20 kilogram dogs, half-million cells per

6 kilogram.

7 Seven days later, the dogs were

8 anesthetized, physical exam, CV exam, necropsy and

9 histo and immunocytochemistry.

10 I should also point out everywhere in this

11 study, all of these dogs, after recovering from

12 anesthesia, passed the cold nose test. They were

13 perfectly normal, you would not be able to tell.

14 They jumped, their tails wagged, they licked your

15 hand. They were nice dogs.

16 So, in that regard, on physical exam, they

17 looked good.

18 Abbreviated methods. Autologous, four

19 sites, collect them. Freshly dispersed, and this

20 will be a key point at the end. These are

21 autologous cells, freshly dispersed MSCs into the

22 circumflex artery. Catheter placement verified

23 before injection, after injection, with

24 fluoroscopy, physical exam. All dogs basically

25 appeared to be normal once the effects of

349

1 anesthesia had worn away.

2 This is the first dog we did. This is the

3 highest dose we did, and we did 1 million cells per

4 kilogram, injected in the coronary. At 2 hours,

5 took a section, you can see the catheter placement

6 there, took a section of ventricular myocardium.

7 It\'s a lightly stained hematoxylin section. These

8 2 cells, basophilic cells here, translate to the

9 CMFDA-labeled cells or fluorescent cells there.

10 This is one of the things we are looking

11 for. Our research team, probably different than

12 many of the people in this room, feel that the only

13 way we will get effective therapeutics is to have

14 intimate contact between these cells and the dead

15 and dying cardiomyocytes that they are going to

16 replace. We don\'t believe in direct injection, and

17 we can talk about that later.

18 So, that was our first dog, 1 million

19 cells per kilogram. The reason he went\--it was at

20 two hours\--was he went into V fib and died. Two

21 hours, so it is fairly easy to do your necropsy in

22 that. So, we then bacted those off to a

23 half-million cells per kilogram and injected them,

24 and the injections are 5 injections of 2 ml each

25 and 1 million cells per ml, and this was the

350

1 control, the anesthetized dog, this is the ECG.

2 Dogs normally have inverted T waves, I

3 don\'t know how many people know that, and it has to

4 do with chest dimensions and chest geometry more

5 than anything else. This is a normal ECG for a

6 dog.

7 After the fifth injection, the T wave has

8 converted to a normal position, but more

9 importantly, what you see here is ST elevation,

10 very profound ST elevation. This increase with each

11 dose and at the fifth injection it was the most

12 severe.

13 Twenty-four later, post-injection, you can

14 see you have got back to an inverted T wave, but

15 you have got bizarre complexes here. One-week

16 post-injection you have got normal ECG, and this

17 was published recently in Lancet, so I am not going

18 to go through a lot of this published thing two

19 weeks ago.

20 ST elevation made us think of troponin.

21 We measured troponin at various times after

22 injection. You can see it goes up, increases to

23 about 45 nanograms per ml. If you ligate an LAD in

24 a dog, you normally see ranges in the order of 150

25 to 200, so we had subnormal, if you will, levels of

351

1 troponin, but the time course it would be

2 consistent with some sort of myocardial ischemia.

3 One of the things you also don\'t see in

4 many of these preparations that you are seeing,

5 which is my personal\--I will give you guys my

6 personal things that sort of irk me a little bit

7 about science\--is we are using H&E histology.

8 This is the gold standard of pathology. I

9 don\'t care how many immunocytochemistry studies you

10 see, H&E is what medical pathologists use to

11 evaluate an outcome of a case. It is essential, it

12 should be included, I don\'t know why people don\'t

13 use it, but it is a great technique, I like it

14 although I didn\'t do very well in pathology.

15 Here is a good example of a section of the

16 ventricle of one of the injected dogs. What you

17 see is three areas here of hypercellularity. This

18 is the normal myocardium here as you zoom in. This

19 is at 4X or 40X, this is 100X and 400X, and we are

20 zooming in on this area right in here.

21 You see it is more hypercellular. What

22 you see in here is you see mononuclear cells,

23 rounded nucleus. You see some elongated nucleus.

24 You see fibrosis. You see some lytic lesions. I

25 looked at that as a cell biologist, and I said,

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1 great, that\'s where my stem cells are, right?

2 Wrong. My pathologist looked at it and said, \"Rick,

3 you have got a problem. Those are macrophages.

4 You have just produced a heart attack in this dog.\"

5 And I said, \"Oh.\"

6 So, to verify that, these were CM dye/I

7 labeled cells zooming in. You can see that the CM

8 dye/I label is in the vicinity of this

9 hypercellularity, so both the MSCs and these

10 macrophages are here, but how do we know they are

11 macrophages?

12 Again, using canine-specific antibodies

13 that we have available in the vet teaching

14 hospital, this is an H&E section, this is a CD18

15 monoclonal antibody specifically raised against

16 canine macrophages. You can see you have punctate

17 lesions, it is very characteristic, and, indeed, to

18 confirm this, 7 days later, what we also see

19 characteristic of myocardial infarctions is

20 increased fibrosis and collagen deposition.

21 As you can see here, this is normal

22 myocardium, it stains very red in Masson\'s, and

23 what you see here is you see the blue here is

24 collagen fiber deposition. This is very

25 characteristic of myocardial infarction.

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1 Five cardinal signs of MI are ECG changes,

2 we showed that; proteins released from damaged

3 myocardium, we showed that; decreased wall motion,

4 we did not see that. We did ultrasound, but I

5 don\'t know that we could. We have the

6 sophistication to measure wall motion. Myocardial

7 infarction is just not a common veterinary disease

8 that we see. Characteristic cellular infiltrates,

9 we saw that. Collagen deposition.

10 Basically, our conclusion is that at 0.5

11 million cells per MSC, will produce myocardial

12 ischemia, microinfarctions in these dogs.

13 Our original interpretation of this was

14 that this was a dose/rate of delivery problem. So,

15 our feeling was, because there really is not much,

16 is you critically look at the clinical studies out

17 there in humans, they have got doses all over the

18 place. It is very hard to extrapolate what the

19 dose is. That is why use milligrams per kilogram.

20 I would encourage anybody doing these studies to

21 use some sort of a normalization like that.

22 I would encourage the committee to require

23 it, so that you can start comparing, but more

24 importantly, what we did, just to give you an in

25 idea, is possibly post-injection cell clumping. We

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1 didn\'t consider this as a possibility. We have

2 taken the holding media the cells were in, and the

3 injection media, to inject them into the cells.

4 At the end of 2 hours of holding them in

5 the holding media, I personally inspected most of

6 the dogs. We inspected the cells. None of the

7 cells are clumped, so we assumed clumping was not a

8 problem.

9 Just to give you guys a little bit more

10 gray hair in terms of your job as far as making

11 decisions, what we didn\'t do is we didn\'t do the

12 right control, and the right control was to put

13 these cells in 100 percent serum, because that is

14 what you are doing, you are injecting them into the

15 artery. From the time they leave that artery, they

16 are going into 100 percent serum.

17 We did that and the cells started

18 clumping. I don\'t know why. They didn\'t clump

19 when we pulled them out of the bone marrow because

20 they would have been clumped when we put them in

21 tissue culture dish.

22 They didn\'t clump then, but during the

23 process to 2 weeks of preparing them, sticking to

24 plastic, their adhesive properties had changed.

25 One dog, and I have heard several people talk about

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1 fetal bovine serum, I don\'t know if it is known to

2 this group or not, Darlen Procoff \[ph\] last year

3 had an abstract, I don\'t think he has published it

4 yet, had an abstract where he looked at fetal

5 bovine serum in cells. It carries over, I believe

6 it was about a milligram per million cells if you

7 grow cells in FBS, at least with the MSCs, will

8 pinocytose and hold about a milligram per million

9 cells of fetal bovine serum.

10 It is not released by washing. These

11 cells were rinsed three times in Hanks\' balanced

12 salt before they were injected. If you want to

13 remove the fetal bovine serum, you have to grow

14 them for at least 48 hours to get rid of it.

15 This may explain some of the early dieoff

16 of the myoblasts that you are seeing, if you have

17 got FBS in there, because you will get a reaction

18 from it.

19 Basically, this research team does not

20 feel that we are in a position to perform these

21 studies on client-owned patients at this time,

22 although that is our long-term goal.

23 What do we think is happening? What we

24 think is happening is, we think the cells are

25 coming in, we think they are clogging, producing

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1 microinfarctions by clogging either a second or

2 third order arterial, and causing areas of ischemia

3 and microinfarctions.

4 Are there clinical techniques to detect

5 this? That is one of those things. Would you see

6 that, or does all the old imaging, does the modern

7 world ultrasound pick up something like that? I

8 don\'t think so. So, that is why I am saying

9 histology is pretty important.

10 How do our cell preparations compare?

11 DR. RAO: Dr. Vulliet, we would like try

12 and make sure that we stay on time.

13 DR. VULLIET: Sure. Let me finish this

14 because that is actually what I think we are more

15 interested in.

16 This is bone marrow from canine bone

17 marrow. As you can see, you have got metas and

18 milas and bands, and that kind of stuff. This is

19 canine MSCs. Our cell size is about 19 microns, 20

20 microns. Mean cell size on this population,

21 because they are much smaller, is 10 to 12 microns.

22 More importantly, look at this. The range

23 on these, again, these are characteristic things.

24 These are done in dog, they don\'t publish them in

25 humans. Range is 7 to 50 microns, okay, and it

357

1 could go even go higher, go as high as 80.

2 Plasticity in this population of cells,

3 this is probably being generous, saying it is about

4 0.01 percent. If you take the canine population of

5 cells, and you go through a CFU selection process,

6 you can get plasticity on the order of 40 to 60

7 percent.

8 If you are starting to compare potency,

9 potential potency between these two preparations

10 of cells, this preparation of cells, after it went

11 through a CFU selection process, would be about

12 4,000 to 40,000 times more potent than just crude

13 bone marrow in terms of plasticity potential, if

14 you will.

15 Intrinsic properties of these cells, I

16 don\'t know. These cells have a tendency to lay down

17 collagen if you just leave them sitting in a tissue

18 culture dish.

19 Successes for safety studies,

20 therapeutics, efficacy studies need to be done in

21 well characterized model diseases and patient

22 diseases.

23 What I would like to do is leave you with

24 a couple of philosophies. We believe that bone

25 marrow stem cells have potential to treat many cell

358

1 loss diseases, especially the myocardium, and we

2 will continue using these even in spite of the

3 negative report. We believe this is a technical

4 problem. We believe we will solve it by adjusting

5 the dose and a few other things.

6 However, strict scientific disciplines are

7 necessary to avoid a train wreck. As Doris Taylor

8 said, we do not want to repeat the gene therapy

9 trial.

10 The other thing I can give you is those of

11 us who used to have gray in our hair, and now I

12 don\'t have hair, is the comment, when clinicians

13 read their own press clippings, patients are going

14 to suffer.

15 Thank you for your attention.

16 \[Applause.\]

17 DR. RAO: Thank you, Dr. Vulliet.

18 Our next speaker is from Genzyme. Let\'s

19 try and restrict the time to about 10 minutes.

20 MR. DU MOULIN: It is very tough being the

21 last speaker of the day, but it is absolutely worse

22 being the last speaker of the day and following a

23 vet who talks about puppies.

24 \[Slide.\]

25 Good afternoon. My name is Gary C. du

359

1 Moulin. I am vice president of Quality Systems for

2 the cell therapy operations at Genzyme Corporation

3 in Cambridge, Massachusetts.

4 Genzyme Corporation is collaborating with

5 Professor Philippe Menasch of Paris, France, in a

6 multicenter, Phase II/III clinical study autologous

7 skeletal myoblast implantation in Europe.

8 \[Slide.\]

9 Ensuring the therapeutic success of

10 cardiac cell therapy is predicated on a rigorous

11 scalable autologous cell culture program based upon

12 the principles and practices of good manufacturing.

13 Our long experience with the scale-up and

14 delivery of cartilage and keratinocyte-based cell

15 therapy products and services to thousands of

16 patients has confirmed that each element of good

17 manufacturing practices contributes an essential

18 part of an overall program that optimizes chances

19 of providing cell therapy products expressing the

20 attributes of safety and consistent quality for

21 patients.

22 Controls required for the manufacturing

23 process begin at the collection site of the muscle

24 biopsy and ends approximately three weeks later as

25 the suspension of cells exhibiting quality and

360

1 safety characteristics that once implanted can

2 consistent initiate a robust repair process.

3 Maintaining the sterility of the cell

4 culture system and ensuring lot segregation are

5 critical attributes of success.

6 \[Slide.\]

7 All the elements of the GMP-based

8 manufacturing program are essential in order to

9 control the inherent variability representative of

10 autologous cell culture. Briefly, these controls

11 must be established based upon the following

12 aspects of GMP.

13 These include, and they are listed here, a

14 process that is validated, personnel who are

15 trained and certified to manipulate cell safely, an

16 appropriate facility expressing stringent

17 environmental controls, records and documentation

18 of all processes conducted, equipment that is

19 calibrated and validated, raw materials that have

20 been tested for their quality, accepted formally,

21 and released into the manufacturing stream. Unique

22 to an autologous cell process is to maintain

23 stringent patient lot segregation.

24 \[Slide.\]

25 This is a photograph of our sole

361

1 manufacturing facility. It is approximately 10,000

2 square feet, contains about 70 biosafety cabinets

3 in which individual patient\'s tissues are

4 manipulated, but other organizational requirements

5 necessary beyond the GMPs include these elements

6 here, beyond the manufacturing, a purchasing

7 element, materials handling element, logistics for

8 the shipment of cells, customer care for

9 communicating with the surgeon and the patient,

10 engineering and facilities to maintain your

11 facility, manufacturing technical services

12 responsible for the training of personnel, process

13 development, and clinical manufacturing, a

14 formalized quality assurance, quality control, and

15 validation services program.

16 \[Slide.\]

17 In order to scale up manufacturing

18 activities for a clinical development paradigm, an

19 organization must be created to effectively manage

20 a myriad of direct and ancillary responsibilities,

21 but here, quality controls including environmental

22 monitoring for the manufacturing facility are

23 critical components of the operational elements of

24 cell therapy productions.

25 Robust testing programs ensures\--and I

362

1 have listed them here\--that cell therapy products

2 meet the highest standards of safety,

3 effectiveness, and reliability as a therapeutic

4 modality, that transmission of communicable

5 diseases is prevented, that one ensure that all

6 manufacturing and processing controls are in place

7 and consistently followed, that there is compliance

8 with existing and anticipated regulatory

9 requirements, that validated assays are performed

10 which support lot release and performance

11 monitoring of materials and components, and,

12 finally, encouraging the development of new assays

13 which enhance product safety, and, finally,

14 generating and analyzing that data, the

15 quantitative data to support continuous

16 improvements to your process.

17 \[Slide.\]

18 Putting these concepts together, a

19 manufacturing process whose key manufacturing

20 events from biopsy receipt through final product

21 fill finish are well understood, can optimize the

22 ex vivo cell culture process, and supported by

23 validated quality controls can ensure safety and

24 product consistency.

25 \[Slide.\]

363

1 We believe that call product

2 characterization is possible based upon validatable

3 measures of viability, purity, identity, and yield,

4 with safety indicators of sterility, endotoxin in

5 the absence of mycoplasma.

6 Here are shown the percent viability and

7 percent CD56 expression of three cell therapy

8 products prepared during process validation studies

9 from cadaveric skeletal muscle tissues.

10 Included in these data is evidence of cell

11 product stability over a 72-hour time frame, one

12 reasonably to be expected if transportation of the

13 cells over a long distance to the patient is

14 required. Note that the lot release parameters

15 remain stable over 72 hours.

16 Viability and CD56 expression, both flow

17 cytometric and validatable measures of cell product

18 identity can be consistently maintained above the

19 90 percent range.

20 \[Slide.\]

21 Measures of sterility and potency are

22 shown in this slide. It is crucial to ensure that

23 sterility is maintained throughout the

24 manufacturing process. In the case of short shelf

25 life cell therapy products utilizing automated

364

1 microbial detection systems can provide benefits to

2 improve time to detection should microbial

3 contaminants be present.

4 Finally, in order to demonstrate the

5 potential for a therapeutic effect, the presence of

6 myotubule formation can be used as a measure of

7 identification and perhaps potency. Myoblasts are

8 undifferentiated muscle precursor cells which, when

9 fused, become the differentiated myotubules. The

10 presence of multinucleated muscle cells is a strong

11 visual indicator that muscle differentiation has

12 occurred and may be an important predictor of cell

13 function.

14 In these photographs, one can readily see

15 multinucleated myotubules indicated by the yellow

16 arrows from freshly prepared samples and in cells

17 after a simulated 72-hour shipment period.

18 \[Slide.\]

19 In conclusion, quality, safety, and

20 effectiveness must be designed into cell therapy

21 products. Quality cannot be inspected in or tested

22 into cell therapy products. Despite the fact that

23 we may, at this time, not know every aspect of cell

24 product characterization, institution and

25 maintenance of stringent manufacturing controls

365

1 through rigorous observance of GMPs can contribute

2 to the safety and consistency necessary for the

3 production of cell therapy products intended for

4 cardiac cell therapy.

5 Developers of cell therapy products must

6 consider, understand, and incorporate quality

7 requirements at the earliest possible stages of the

8 clinical development program, and in so doing, can

9 optimize the therapeutic potential of these

10 promising technologies.

11 Thank you.

12 \[Applause.\]

13 DR. RAO: Thank you.

14 If there are no questions, I would like to

15 ask if anybody else from the audience wishes to

16 make any comment at this time. I am going to ask

17 that they limit their comments and be brief.

18 \[No response.\]

19 DR. RAO: If there are no more comments,

20 then, I can declare the meeting adjourned.

21 \[Whereupon, at 5:46 p.m., the proceedings

22 were recessed, to reconvene at 8:00 a.m., Friday,

23 March 19, 2004.\]

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