> 1 DEPARTMENT OF HEALTH AND HUMAN SERVICES

FOOD AND DRUG ADMINISTRATION

CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

> BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE

MEETING #31 - VOLUME II

Thursday, October 25, 2001

8:00 a.m.

> Holiday Inn Gaithersburg Two Montgomery Village Avenue
>
> Gaithersburg, Maryland

2

PARTICIPANTS

Daniel R. Salomon, M.D., Chairperson

Gail Dapolito, Executive Secretary

MEMBERS

> Richard E. Champlin, M.D. Katherine A. High, M.D.

Alison F. Lawton (Industry Representative) Richard C. Mulligan, Ph.D.

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

Edward A. Sausville, M.D., Ph.D.

CONSULTANTS

Jonathan S. Allan, D.V.M.

> Kenneth Cornetta, M.D. (by telephone) Michael Emerman, Ph.D. (by
> telephone) David W. Gaylor, Ph.D.

Katherine E. Knowles (Consumer Representative)

> Jeffrey H. Kordower, Ph.D. Bruce E. Torbett, Ph.D.

John A. Zaia, M.D.

GUESTS/GUEST SPEAKERS

Dale G. Ando, M.D.

Yvette Delph, M.D. (Patient Representative)

John C. Kappes, Ph.D.

> Susan Kingsman, Ph.D. Marina O\'Reilly, Ph.D. Amy Patterson, M.D.
> Gabor Veres, Ph.D. Inder Verma, Ph.D.

FDA PARTICIPANTS

> Jay P. Siegel, M.D. Philip D. Noguchi, M.D. Daniel Takefman, Ph.D.
> Carolyn Wilson, Ph.D.

3

C O N T E N T S

Opening Remarks

Dr. Daniel Salomon, Chair 4

Conflict of Interest Statement

Gail Dapolito, Executive Secretary 7

Introduction of Committee 8

OPEN COMMITTEE DISCUSSION

Topic 1 - Lentivirus Vectors in Gene Transfer

Clinical Trials

Lentiviral Vectors

Dr. Inder Varma 11

Questions & Answers 50

Lentiviral Vectors: Safety Issues

Dr. Daniel Takefman 75

Questions & Answers 90

LentiKat Vectors Overview

Dr. Gabor Veres 105

Lentiviral Gene Therapy

Dr. Dale Ando 122

Questions & Answers 130

Lentivirally Delivered GDNF for Parkinson\'s Disease

Dr. Jeffrey Kordower 152

Questions & Answers 185

Lentiviral Vectors for the Treatment of Cancer, Neurodegenerative
Diseases and AIDS

Dr. Susan Kingsman 209

Questions & Answers 251

Predicting Lentiviral Vector Safety in Vivo

Dr. John Kappes 295

Open Public Hearing 326

Committee Discussion of Questions 326

> 4 1 P R O C E E D I N G S

2 Opening Remarks

3 DR. SALOMON: Good morning to Meeting No.

4 31. We still haven\'t got titles. One of my big

5 disappointments with the FDA is that we have never

6 had any kind of sexy titles for these meetings, and

7 I am not in a position to make them up on the fly,

8 so I apologize.

9 So, this is Meeting No. 31 of the

10 Biological Response Modifiers Advisory Committee.

11 Today is I think the beginning of a very important

12 two days in which discussions of a new vector class

13 for gene transfer and gene delivery, that of

14 lentiviral vectors will be discussed.

15 There is just a number of organizational

16 things. There is a lot of new people around the

17 table and I welcome everyone from yesterday, the

18 table has expanded somewhat.

19 One thing for those of you who have not

20 been at these conferences, but this button in front

21 of you, if you speak, you push down, the button

22 turns on red, and when you are done speaking\--that

23 way you don\'t get pickup from everyone, and the

24 transcribers and the audience will be a lot

25 happier, so I appreciate that.

> 5 1 There will be two days here. Today, we

2 are going to be talking and being educated in some

3 context by some experts in the field along the

4 lines of lentiviral vectors, and there are a series

5 of questions that the FDA staff has generated.

6 That doesn\'t mean that we can\'t generate other

7 questions.

8 I think this is really, particularly in

9 looking a new gene delivery class, is an excellent

10 opportunity for everyone to interact in the context

11 of trying to identify what sorts of issues are

12 important in FDA\'s approach to developing,

13 regulating, and providing appropriate IND guidance

14 to sponsors in this new field.

15 So, this is our chance to input this kind

16 of data. With that said, a couple quick things. I

17 am going to try, one of my jobs is to come up with

18 consensus. That doesn\'t mean that, number one,

19 consensus is always possible or even appropriate,

20 so there will be times when the committee has every

21 right to say no, I don\'t agree with that, that is

22 not consensus.

23 There will be other times in which

24 consensus might come in terms of, you know,

25 majority opinion, but I would very strongly

> 6 1 encourage anyone with a well articulated and

2 defended minority position to take it, and that is

3 I think very appropriate and not to feel any

4 pressure from me to be in consensus. If we can

5 obtain consensus, that is excellent, however.

6 Today, we are also going to hear from a

7 series of sponsors who have special expertise and

8 experience in developing lentiviral vectors for

9 gene delivery. Today, I think it is very important

10 to point out a distinction here. These sponsors

11 are here at the request of the FDA, and they have

12 stepped forward voluntarily to provide us with

13 information that gives us specifics and gives us a

14 chance to look at their experience and sharing

15 their experience.

16 We are not here to judge their protocols.

17 Many of them are not ready to put them forward for

18 formal INDs. So, it is very different than what is

19 going to go on tomorrow where we have a sponsor who

20 has very seriously stepped up to the plate and

21 proposed a real clinical study now.

22 I think there, then, the committee has a

23 different charge. So, I just want to explain to

24 everyone that these sponsors are coming up and we

25 really, really appreciate their participation. It

> 7 1 is very important just to cut them some slack.

2 The idea here is to share their experience

3 and none of them are saying they are ready for a

4 clinical trial tomorrow. They all realize that

5 every strategy has some limitations and some future

6 for it.

7 So, with that, I think we are going to

8 read the Conflict of Interest Statement or at least

9 an abbreviated form of it, and then we will go

10 around and introduce everybody, and then we will

11 get started.

12 Thank you.

13 Conflict of Interest Statement

14 MS. DAPOLITO: A Conflict of Interest

15 Statement was read for the record yesterday. I

16 don\'t need to go through the whole entire thing

17 again. I will just repeat that the FDA has

18 appointed Ms. Katherine Knowles, Dr. Gaylor, Drs.

19 Allan, Cornetta, Emerman, Kordower, Lane, Torbett,

20 and Zaia as Temporary Voting Members for the

21 Committee discussions today and tomorrow.

22 The following participants were issued

23 waivers to participate in the meeting: Drs.

24 Champlin, High, Mulligan, Lane, and Kordower.

25 I think that is all that needs to be said

> 8 1 today.

2 DR. SALOMON: Again, just because I don\'t

3 even know everybody on the Committee, I would like

4 to go around, starting with Dr. Zaia, and introduce

5 yourself, where you are from, and a brief idea of

6 where your areas of interest and expertise are.

7 Introduction of Committee

8 DR. ZAIA: My name is John Zaia. I am the

9 Director of Virology at the Beckman Research

10 Institute at City of Hope. I am also interested in

11 clinical research, and I am the Director of the

12 General Clinical Research Center and have an

13 interest in gene transfer studies.

14 DR. TORBETT: I am Bruce Torbett from the

15 Scripps Research Institute, Department of Molecular

16 and Experimental Medicine. I am interested in gene

17 delivery, myeloid development, and protection from

18 HIV via gene delivery.

19 DR. HIGH: I am Kathy High. I am the

20 Director of Research in the Hematology Division at

21 the Children\'s Hospital of Philadelphia, and I have

22 an interest in gene transfer for hemophilia.

23 DR. ALLAN: I am Jon Allan from Southwest

24 Foundation in San Antonio. My area of study is

25 natural host resistance to SIV, so I study

> 9 1 pathogenesis of AIDS viruses.

2 DR. GAYLOR: I am David Gaylor of Sciences

3 International. My area is biostatistics and risk

4 assessment.

5 DR. CHAMPLIN: I am Richard Champlin from

6 the M.D. Anderson Cancer Center. I am the Chairman

7 of the Department of Blood and Marrow

8 Transplantation.

9 DR. SAUSVILLE: I am Ed Sausville from

10 National Cancer Institute. I am from the

11 Developmental Therapeutics Program, which evaluates

12 and manufactures drugs and biologicals for cancer

13 and AIDS.

14 MS. LAWTON: I am Alison Lawton. I am the

15 industry rep on the Committee. I chair the Cell

16 and Gene Therapy Committee for the Pharmaceutical

17 Association, PhRMA, and I work for Genzyme.

18 DR. SALOMON: I am Dan Salomon. I am at

19 the Scripps Research Institute in Molecular and

20 Experimental Medicine. My interests are in cell

21 and organ transplantation, tissue engineering, and

22 gene delivery.

23 Before we go further, we have through the

24 miracles of modern technology Dr. Michael Emerman,

25 who we are going to have him introduce himself and

> 10 1 test this whole system out.

2 Dr. Emerman?

3 DR. EMERMAN: My name is Mike Emerman. I

4 am at the Fred Hutchinson Cancer Research Center.

5 My expertise is in HIV, molecular biology, and

6 replication.

7 DR. SALOMON: Thank you.

8 MS. DAPOLITO: Gail Dapolito, Executive

9 Secretary.

10 DR. RAO: Mahendra Rao. I am the Stem

11 Cell Chief in the Laboratory of Neurosciences at

12 the National Institute on Aging.

13 MS. KNOWLES: I am Kathy Knowles. I am

14 the consumer representative on the Blood Products

15 Advisory Committee and I am serving in the consumer

16 role here today at this committee.

17 DR. DELPH: Good morning. I am Yvette

18 Delph. I am with the Treatment Action Group, which

19 is an HIV/AIDS treatment activist organization.

20 DR. VERMA: I am Inder Verma from the Salk

21 Institute in La Jolla. I am interested in signal

22 transduction and also vectors for gene delivery.

23 DR. PATTERSON: I am Amy Patterson,

24 Director of the Office of Biotechnology Activities

25 in the Office of Science Policy at NIH.

> 11 1 DR. TAKEFMAN: Dan Takefman. I am a

2 product reviewer in the Division of Cellular and

3 Gene Therapies at the FDA.

4 DR. WILSON: Carolyn Wilson. I am also a

5 member of the Division of Cellular and Gene

6 Therapies, FDA/CBER.

7 DR. NOGUCHI: I am Phil Noguchi, Director,

8 Cellular and Gene Therapies at FDA.

9 DR. SIEGEL: Jay Siegel, Director, Office

10 of Therapeutics Research and Review, FDA/CBER.

11 DR. SALOMON: I welcome everyone and we

12 might as well just go and get started.

13 Dr. Verma.

14 TOPIC 1: LENTIVIRUS VECTORS IN

15 GENE TRANSFER CLINICAL TRIALS

16 Lentiviral Vectors

17 DR. VERMA: Thank you very much. Thank

18 you very much for the invitation and to the members

19 of the committee and the audience.

20 This morning, when I came in, I ran into

21 Luigi Naldini and suddenly realized it was only

22 about five years ago that it was an academic

23 exercise we had whether we can convert HIV into a

24 useful vector system. I am delighted to see that

25 today we are here discussing the possibility that

> 12 1 it might actually have an application in the

2 clinic.

3 \[Slide.\]

4 So my job is really today to give you a

5 general introduction of the vectors. I realize

6 there are many, many experts here in the field. In

7 fact, some of the founders, some of the people who

8 discovered the original. So I apologize to them if

9 this is something very simple, but I would like to

10 bring everybody to the same level so that the rest

11 of the day will be easy for you.

12 Gene therapy is a form of molecular

13 medicine which will have a major effect on human

14 health in the coming centuries. I think the

15 concept of gene therapy is disarmingly simple;

16 introduce the gene, and its product should have the

17 ability\--I am a little confused because a guy is

18 going like this all the time. Okay; I am going to

19 igonre the guy. In any event, I was telling you

20 about gene therapy. It is is a relatively simple

21 concept.

22 But the fact of the matter is it hasn\'t

23 been really as successful as we had anticipated and

24 part of it has to do with the fact that the matters

25 of delivery haven\'t been really quite as exquisite

> 13 1 as we would have liked to have them.

2 In fact, there are many, many ways to

3 introduce the gene. What I would like to do is

4 really give you one. But just to give you the

5 background of it, as I said, there are many

6 different ways to introduce the genes. Some have

7 generally divided the physical method by which you

8 can directly introduce the gene and there are

9 wonderful ways of introducing the gene but it

10 depends on what you want to do.

11 The bottom line for all the vectors is

12 whether it is physical or biological, it really

13 depends what you want to do. If you simply wanted

14 to make a vaccine against a small amount of the

15 protein, sufficient amounts of this can be done by

16 direct DNA injection.

17 \[Slide.\]

18 But if you wanted to make sustained

19 amounts of a foreign protein for a sustained period

20 of time, by and large, most of us have concentrated

21 on the biological method. Again, the important

22 point here is not to be exhaustive about the number

23 of the vectors because there are many missing. The

24 important point is that each one of them has a

25 major limitation.

> 14 1 So, again it comes back to what we want to

2 do. For the two people in this audience who don\'t

3 think about these vector systems, the principle of

4 making these vectors is again disarmingly simple.

5 All the viruses do, in their life, is to replicate.

6 They have really no interest to kill you. Their

7 main job is to simply replicate.

8 But, occasionally, they do acquire

9 sequences which have the ability to cause disease.

10 So all that we are trying to do, everyone who makes

11 vectors, is to eliminate the disease-causing

12 component, substitute with the therapeutic gene of

13 interest, reconstitute the virus which is no

14 different than the starting one essentially.

15 So the idea is simply to replace the

16 therapeutic component, in this case removing the

17 pathological sequences and recreate the virus

18 which, hopefully, will have the same sets of

19 function, by and large, that you started out with.

20 So that really is the principle of making most of

21 the biological viral vector systems.

22 So what we would like to do, really, is to

23 create a vector\--as I said, there is no ideal

24 vector, but we would like to set up some parameters

25 which we believe will be useful in the long run for

> 15 1 making, or at least thinking about vectors which

2 can perform many of the functions we are desirous

3 of.

4 What we would like to do is a vector which

5 we can generate at fairly substantially high

6 amounts. Again, this is for the aficionados in the

7 field, 10 8, 109. It

depends upon how many virus

8 particles you can introduce, so you need to make

9 substantial amounts.

10 Particularly for this audience, it is

11 important to have reproducibility and the

12 convenience to make them. We would like to

13 introduce the gene in any cell type regardless of

14 the fact that it is the tumor cell which is

15 dividing or it is a brain cell which is not

16 dividing, we would like to have the ability to

17 introduce genes in a wide variety of cell types.

18 Since many of the vectors we discuss have

19 the ability to become part and parcel of your

20 chromosome, it would be nice to know where they

21 went. It would be really nice to know with the 3.2

22 billion basis of the genome where did the vector

23 actually go so we have some idea. That will be a

24 very desirable property.

25 It would be nice to control the amount of

> 16 1 the protein or the gene transcription from outside;

2 that is to say, you can regulate how much protein

3 when and where you want to make, a sort of a review

4 of turning on and off.

5 It would be nice to infect any cell type,

6 hopefully liver, lung, brain, kidney, all the cell

7 types. And, of course, we want to have no

8 undesirable immunological consequences. So we are

9 asking a lot. We are asking it to behave like a

10 typical retroviral vector to integrate and yet have

11 the ability to infect nondividing cells. At the

12 same time, we are asking it to behave like an

13 adenovirus, to behave like an episome, and yet have

14 not immunological consequences.

15 \[Slide.\]

16 So we are asking a lot. But, fortunately

17 for us, a completely unexpected ally came over at

18 this time in the form of the HIV. HIV, as many of

19 you know, is a member of the Retroviridae and has a

20 number of properties similar to them but has also

21 the unusual property of the ability to introduce

22 itself into nondividing cells.

23 Some of the reasons that we got started on

24 was the idea that they can infect neurons, that

25 they can infect nondividing resting T-lymphocytes.

> 17 1 They can infect monocytes. So we were interested

2 to see if that property of HIV could be utilized

3 and we could convert them into a useful vector by

4 which they can introduce their gene, integrate in

5 the chromosome in a manner analogous to the typical

6 Moloney leukemia virus which is a prototype of

7 other retroviral vectors and yet have the ability

8 to be able to produce the foreign protein.

9 \[Slide.\]

10 With that in mind, then, we constituted a

11 team at the Salk Institute with the following idea

12 behind it. This is an idea which really came from

13 the work of Michael Emerman and colleagues, Mario

14 Stevenson and colleagues and also Didier Trono who

15 has been at the Salk Institute, now is in Geneva.

16 The idea was the following, that a typical

17 retrovirus, when it makes its DNA, is much too

18 large to be able to cross the nuclear membrane and,

19 therefore, the cells have to divide. The nuclear

20 membrane has to break down. The chromosome has to

21 become available. Only then the viral DNA can

22 become part and parcel of the chromosome.

23 Therefore, retroviruses only infect cells

24 which are dividing because they need for them to go

25 through the nuclear membrane whereas the

> 18 1 lentiviruses, of which HIV is the prime example,

2 have this unusual property and we really don\'t know

3 the precise mechanism and we can talk about it

4 sometime that they have this karyophilic

5 properties; that is to say, their viral DNA can

6 cross the nuclear membrane and thereby integrate

7 into the chromosome and thereby relieving itself of

8 the restriction that the cells must divide.

9 So that is the principle on which we based

10 our basic idea and began to ask the question, can

11 we convert an HIV into a vector.

12 \[Slide.\]

13 I apologize for the number of colors and

14 perhaps you can\'t see from the back, but you will

15 see many renditions of this slide today, I am sure,

16 through the rest of the day. But the bottom line

17 is the following. A typical HIV virus, in

18 addition to getting the prototypic three proteins,

19 the gag, reverse transcriptase and envelope, which

20 is necessary to make the virus which is really

21 common to all prototypic retroviruses of the

22 Lentiviridae or Retroviridae family.

23 The argument was the following. First and

24 foremost, we want to avoid the envelope of HIV

25 because it has a restriction to a very specific

> 19 1 receptor, the CD4 receptor. We had no intention to

2 limit our vector strictly for those cells which

3 have only that type of receptor.

4 So the first modification that we

5 performed was to take this envelope gene and

6 separate it out and substitute it from a vesicular

7 stomatitis glycoprotein which is really a cattle

8 virus but it has a glycoprotein which has been

9 shown almost 30 years ago by Alice Wong and Ian

10 Sabosa and Robin Weiss that it can actually

11 phenotypically exchange itself for the envelope of

12 a retrovirus.

13 Now, the VSAG protein, of course, allowed

14 it to be pantropic meaning it has now the ability

15 to infect a wide variety of cell types and,

16 therefore, eliminate the restriction so restrictive

17 to the CD4-positive cells because of the HIV

18 envelope protein.

19 In addition, we began to manipulate

20 sequences around it and that will be much of the

21 emphasis today to begin to make this vector such

22 that it has the least amount of dependence on its

23 own sequences but, in fact, uses autologous

24 sequences.

25 Just to cite an example, the LTR, which is

> 20 1 very common to all retroviruses, can be replaced by

2 other promotors so as to eliminate or reduce the

3 chances of recombination. We will talk about this

4 as we go along.

5 So, the idea was, then, to make a vector

6 where some of these genes are eliminated,

7 glycoproteins to substitute for the envelope and

8 ask the question can we now create a virus which

9 has the formal ability to infect nondividing cells

10 by virtue of the fact it has acquired that property

11 of HIV which allows it to infect nondividing cells

12 and yet has no ability to make an infectious virus

13 particle.

14 \[Slide.\]

15 This is, again, an old experiment done by

16 Luigi Naldini when he was in the lab along with

17 Didier Trono and Rusty Gage. The very first

18 experiments, we asked the question, A, can you make

19 high titers. The answer is yes, you can easily

20 make 10 6, 107 virus particles

which, by virtue of

21 the fact it is a glycoprotein from G as shown by

22 Ted Friedman and colleagues, can be concentrated

23 which means, again, for the cognoscenti of the

24 field, that we can make up to 10

9 to 1010 virus

25 particles per ml which, of course, is an enormous

> 21 1 titer for these kind of viruses.

2 Again, for those who do not think about

3 it, meaning you can take a billion cells, put a ml

4 of this virus and all those cells should now be

5 transduced to the foreign gene product.

6 This experiment simply shows that at least

7 we have with these vectors to infect macrophages.

8 There are our typical Moloney leukemia viral

9 vectors, the vectors we are traditionally using as

10 retrovirus vectors, do not have the ability to make

11 the foreign protein.

12 So that was the first evidence we had

13 formally that we had a vector which has the formal

14 ability to at least infect cells in vitro which are

15 not normally dividing and, as such, can be

16 transduced.

17 Encouraged by this, they began to ask the

18 question, what happens if we use these genes in

19 vivo. What I will do today is to give you a bunch

20 of those examples and then tell you a little bit

21 more about the safety modification.

22 \[Slide.\]

23 I know you can\'t see it, but this is an

24 experiment where we directly injected the virus

25 into the brain of a rat. We asked the formal

> 22 1 question, can you have the production of the

2 foreign protein in the brain and how long we can

3 have the production.

4 So here is an injection, one side with the

5 HIV vector making brown GFP protein, which is a

6 green fluorescent protein and, on this side, we

7 have the Moloney leukemia viral vector. Again,

8 after about six months period of time, infection of

9 the brain. You have the expression of the protein

10 here and none in the case of MLV.

11 I can\'t escape but to again tell you how

12 sad this slide makes me because fifteen years of my

13 career were made on MLV vector and all it can now

14 do is really a control. But that\'s the way it is.

15 \[Slide.\]

16 But more important, really, is to ask the

17 question how efficient is actually transduction.

18 Here, again, is a single injection, 2 microliters

19 of the virus, about 30 million virus particles

20 directly injected into the hippocampus. Again, you

21 can\'t see it, but 90 percent of the cells at the

22 site of injection\--that is to say, within 2 to 2.5

23 millimeters at the site of injection, 90 percent of

24 the cells are not transduced. This is about eleven

25 months period of time.

> 23 1 So three things happened for us. One; we

2 can make a vector which can infect nondividing

3 cells. Two; it can be directly introduced in vivo

4 into nondividing cells. Three; there was a

5 substantially efficient transduction at the site of

6 injection and there was a sustained production of

7 the foreign proteins.

8 So, armed with this, we began to ask the

9 question, what other tissues where we can introduce

10 the gene.

11 \[Slide.\]

12 Muscle. Muscle is a very interesting

13 tissue because 40 percent of the body weight is

14 muscle and it is a good system to secret the

15 protein. For example, hemophilia, the proteins can

16 be secreted if you can introduce the gene in the

17 muscle.

18 Shown here is again direct injection into

19 the muscle. These are the long fibers which are

20 not dividing. Again, you can see the production of

21 the foreign protein for eight months period of time

22 and nothing in the case of the Moloney leukemia

23 virus, our traditional vectors which do not infect

24 nondividing cells.

25 \[Slide.\]

> 24 1 Another example I give you is the eye. A

2 number of diseases are involved in the deficiency

3 of genes in the vision. A lot of our work in the

4 lab is concentrated in the areas which are largely

5 animal-model systems, in a number of mice-model

6 systems in which there is a defect in the vision

7 system, either of the rhodopsin or of the different

8 kinds of other proteins.

9 So we asked the question, can we directly

10 introduce the gene in the subretinal pigmented

11 epithelium to the specific example of retinitis

12 pigmentosa which is a blindness due to the

13 deficiency of many genes, one of them including the

14 phosphodiasphase gene.

15 So we asked the question, can you

16 introduce the gene. The answer is yes. If you use

17 our traditional CMV promoter\--CMV is a promoter

18 which sort of expresses in every cell type. It

19 allows the expression at the site of injection

20 whereas if we now introduce the gene with the

21 rhodopsin promotor, a promotor especially for the

22 rod cells and the cone cells, now you see the

23 expression largely in the rod cells and the cone

24 cells.

25 \[Slide.\]

> 25 1 More importantly, if you now take a mouse

2 which has a deficiency of phosphodiasphase beta,

3 which is required for dephosphorylation of CGMP,

4 removal of which causes blindness in these mice,

5 and, in fact, if you make a section of the eye, it

6 has all the right components except it is missing

7 all the rod cells and the cone cells by virtue of

8 the fact that they have not this enzyme. Thereby,

9 there is apoptosis and thereby there is blindness.

10 So we argued, can you introduce the gene

11 phosphodiasphase beta and restore at least some of

12 the retinal cells. There are about eight or nine

13 layers and the have none. Can we restore some of

14 those layers.

15 \[Slide.\]

16 The answer is at least\--this is difficult

17 from the back\--but at least four new layers of

18 opsin which are now found in these animals which

19 contain the phosphodiasphase gene introduced by

20 direct injection in the eye and none in the case of

21 the controls.

22 These mice haven\'t really lost their

23 blindness. They are still partly blind, but it at

24 least gives you the hope that you can begin to ask

25 the question to directly introduce these genes in

> 26 1 retinal cells where there is a deficiency of a

2 certain gene.

3 \[Slide.\]

4 Let me give you one other example which

5 will be talked about here quite extensively, and

6 also has the dream of most gene-therapy folks, to

7 be able to infect hematopoietic stem cells because

8 then you will have a continuous production of the

9 foreign protein because these cells continuously

10 produce the foreign protein for the rest of our

11 lives.

12 So, in collaboration with Bruce Talbert,

13 who is here today, and Hiro Mioshi from the lab in

14 Bruce\'s lab, we asked a very simple question; can

15 we take human cord blood cells and purify the stem

16 cells from them. This experiment has also been

17 done with many other viruses, particularly the

18 retroviruses.

19 The difficulty is the following. The stem

20 cells are very few. If I take 1 million

21 bone-marrow cells at any given time from anyone,

22 there may be 100 to 1,000 of these guys. They are

23 not easy to find. The worst of their life is that

24 they don\'t divide.

25 Therefore, most traditional vectors have

> 27 1 been difficult to be used although people have very

2 cleverly manipulated them now by using appropriate

3 growth factors. But, by and large, they are

4 difficult to introduce foreign genes because they

5 are not dividing.

6 Lentiviruses, because of their opportunity

7 to infect nondividing cells, have this unique

8 ability, then, the hope, that they will infect

9 these nondividing, noncycling stem cells and,

10 therefore, allow you the opportunity to have much

11 better transfection than with the traditional

12 vectors.

13 So, with that aspect and that hope, we

14 took the CD34 cells, transduced them in the virus.

15 Transduction, in this case, you take the cells, put

16 the virus, no growth factors, no lymphokines, no

17 cytokines, and simply introduce directly into the

18 tail vein of the SCID/NOD mouse\--we need SCID/NOD

19 because are using human cells\--and ask the question

20 do they now make the foreign protein in the

21 peripheral blood, spleen and the bone marrow.

22 So a simple experiment; take the stem

23 cells, infect them with the virus, put them back

24 into the animal and hope for the best.

25 \[Slide.\]

> 28 1 This, I think, is probably what I find one

2 of the most exciting experiments in our lab and

3 that is that you can now produce the foreign

4 protein in the peripheral blood lymphocytes. You

5 can see the peripheral blood lymphocytes now

6 producing the foreign protein, in this case, 18

7 weeks were infected for the rest of the life of

8 this mouse.

9 About 15 percent of the cells are

10 producing the foreign protein. You and I make 10

11 billion of these cells a day. A billion of them

12 now can make the foreign protein for the rest of

13 your life which really encourages and gives you

14 great hope in terms of proteins that you want to

15 produce when there is a deficiency of a given

16 product.

17 \[Slide.\]

18 More importantly, nearly all myeloid

19 colonies\--remember, these don\'t have T-cells

20 because these are SCID mouse, the myeloid colonies

21 are positive for ritchard colonies, venocytic

22 macrophage colonies and even early progenitor

23 cells. So this we think is really one of the most

24 interesting aspects of lentivectors is their formal

25 ability to infect nondividing cells, in this case

> 29 1 the stem cells and, as shown by their virtues, and

2 repopulate, nearly all kinds of the foreign cell

3 types, at least in the myeloid lineage.

4 \[Slide.\]

5 Again, for the true cognoscenti in the

6 field, they only believe these stem cells are

7 really transduced if they can do a second

8 retransplant meaning that if you now take the bone

9 marrow of the first mouse that you transduced, can

10 you take their bone marrow and put in the secondary

11 mouse, and that done again by Bruce Talbert and

12 Hiro Mioshi. You can see, even in the second

13 recipient, nearly all cells are positive what they

14 started out once again suggesting that it is very

15 likely we truly transduced the stem cells.

16 \[Slide.\]

17 So I think I have given you a number of

18 examples of the generality and the wide spectrum

19 which could be utilized by these vectors for a wide

20 variety of different tissues. The question really

21 now is how useful these vectors are in terms of the

22 formal clinical setting and that will really call

23 for how safe are these vectors.

24 \[Slide.\]

25 So what are the HIV vectors? Well, we all

> 30 1 know the HIV has this unique structure of

2 inhibition to these three replication-competent

3 necessary proteins, the gag, pol and envelope. It

4 has this array of six additional genes which are

5 referred to as the vif, vpu, vpr, net, tat and rev.

6 These are all essential for the replication of the

7 pathogenicity of the HIV which are not present in a

8 traditional Moloney leukemia viral vector or the

9 other kind of retroviral vectors.

10 So the argument was very simple. All we

11 are really interested is to have these vectors

12 introduce their gene in nondividing cells. We have

13 no interest in any of these genes if they do not

14 contribute to that function. So a number of

15 people, Luigi Naldini\'s lab, Didier Trono\'s lab, my

16 own lab, they come to a cell genesis. A number of

17 other people have started to ask the question, can

18 we begin to eliminate these genes and asked the

19 question, do we still have a structural prototype

20 which will introduce the gene into a nondividing

21 cell without the baggage of these unwanted genes.

22 So a number of these have been eliminated.

23 Unfortunately, I don\'t have the next slide. Can

24 you just put that on for me for a moment? It is

25 left in the United Airlines, that slide.

> 31 1 \[Overhead.\]

2 Again, you will see many renditions of

3 this. This is now a vector which is a composite of

4 vectors for a number of labs, our lab, other labs

5 here, just to give you an idea what is now

6 currently considered a third-generation vector from

7 the HIV.

8 There are other vectors you will hear

9 from, I am sure, Dr. Kingsman and other people

10 later, different species of lentiviral vectors but

11 we are concentrating only on the HIV here today.

12 The vector is the following. We have

13 essentially eliminated all the fixed genes, the

14 vif, vpr, vpu, tet and rev as well as the envelope.

15 VSV-G is provided separately and the rev is

16 provided separately. In addition, the long-term

17 repeats which are necessary for the replication of

18 this virus for integration have been deleted to

19 what is called SIN vectors, meaning only those

20 residues are kept which are necessary for

21 integration. All the other components which are

22 involved in its ability to cause the transcription,

23 the transcription element and enhancement element

24 in the LTR, have been deleted.

25 So the vector now constitutes a cell which

> 32 1 will eventually integrate is basically

2 rev-responsive element deleted LTRs and\--may I have

3 the next slide. You can shut that off.

4 \[Slide.\]

5 So what we have now in this vector is the

6 following. Altogether, now, only about 10 percent

7 of the viral genome is left in this vector. So

8 this is the starting HIV. This is the vector we

9 have. And these that I have listed here, the base

10 fields that are left from the main genome. They

11 are about 10 percent of the genome left.

12 So, of the 9,000 or 10,000 nucleotides,

13 there are about 900 to 1000 that are left at

14 various junctures. In fact, most of these genes

15 are gone. Some of these genes are gone. The

16 reason I am showing this to you is to show, first,

17 how debilitated it is, and, second, that the

18 probability that you have recombination with a

19 full-length HIV either following infection or

20 trying to coinfect with it is not zero but it is

21 extremely low because you have at least six or

22 seven new genes to introduce, LTR to introduce and

23 many other sequences, a number of replications, a

24 number of recombination events.

25 So I think that is currently the favorite

> 33 1 one where almost all genes are deleted and there

2 are a few other bells and whistles over here. But,

3 by and large, this is the vector I think what you

4 will hear a lot today discussed in terms of the

5 utility for introducing in the clinic.

6 \[Slide.\]

7 How good is this vector? Well, it is true

8 we can eliminate everything but does it work? I

9 wouldn\'t be showing you all this if it didn\'t work.

10 The answer is yes, it works just as efficiently as

11 the first generation of HIV vectors where we simply

12 eliminated the envelope gene and some other small

13 things. But, by and large, other genes are still

14 present.

15 It infects, for example, HIV, the stem

16 cells, just as efficiently as we had our first

17 generation vector. Again, in our hands, the

18 Moloney does not do so.

19 \[Slide.\]

20 It can infect even the peripheral blood

21 lymphocytes which have been mobilized with GCSF

22 which I think will be eventually how, in the

23 clinic, a lot of things will be used. They will

24 also transduce, albeit only for a six-week period

25 of time. That is the time point, but they can

> 34 1 actually be transduced with these vectors.

2 \[Slide.\]

3 What about bone-marrow transplantation

4 from the mouse? That does just as well again. You

5 can get the bone-marrow transduction, peripheral

6 blood lymphocyte transduction, just equally well

7 with these third-generation vectors meaning that,

8 regardless of the effect whether we have eliminated

9 all these genes or not, the basic ability of these

10 viruses to transduce foreign cells is still intact.

11 \[Slide.\]

12 Just to expand to it a little bit more, if

13 you now take, in collaboration with Marcus Grompi

14 at the University of Oregon, we have taken\--so, if

15 you now have a mouse, which we have fanconi-C and

16 fanconi-A-deficient mice. If you now introduce

17 their stem cells, bone-marrow cells, directly

18 infect them with the virus, in this case the HIV

19 containing the fanconi-C or the fanconi-A, put them

20 back into the animal, they are all phenotypically

21 recovered.

22 We make the FANCC, if we make the FANCA.

23 But, more importantly\--this is a slide given to me

24 by Minoxchi Nole from Marcus Grompi\'s

25 lab\--normally, what happens to them, if you give

> 35 1 them cytotoxin, they become extremely sensitive to

2 them but these mice now all behave like the normal

3 mice.

4 The yellow here shows you that a mouse

5 which is deficient eventually will die. The

6 heterozygous here shows they all survived. Those

7 which got the bone-marrow transduced with the FANCC

8 or FANCA, in this case, completely behave like the

9 normal. So, for all practical purposes, these mice

10 have now phenotypically the same characteristics as

11 if they had a bone-marrow transplant from a sibling

12 or, in this case, the heterozygous bone marrow.

13 \[Slide.\]

14 What about liver? If we use these

15 third-generation vectors, directly introducing them

16 into the liver, I can show you here is the direct

17 introduction into the liver. We can use the CCD

18 camera to take a light imaging of this liver to ask

19 the question if the cells are transduced.

20 \[Slide.\]

21 The answer, again, is here is directly the

22 liver and here is the autopsy of the liver. You

23 can see lots of cells are transduced. About 4 to

24 10 percent of the hepatocytes are transduced in

25 these transductions.

> 36 1 \[Slide.\]

2 We don\'t see any liver toxicity, because

3 there was some question whether lentiviral vectors

4 with VSV-G cause liver toxicity. We do not see any

5 liver toxicity regardless of the fact that we have

6 TDS or lentiviruses. So we think these vectors

7 have the added ability to introduce genes not only

8 to the tissue as I have described before but also,

9 again, to the hepatocytes and they do not need to

10 be dividing.

11 \[Slide.\]

12 Let me give you now a few examples of how

13 we have also used these vectors, not just for gene

14 therapy because much of the interest in my lab

15 really is gene-transfer vectors for many biological

16 basic questions. So I want to give you a few

17 examples, just to give you the breadth of these

18 vectors in addition to the safety issues that we

19 shall discuss.

20 \[Slide.\]

21 In an experiment done by Yoshika Azawa in

22 the lab, he basically asks the question which many

23 people are asking, can you convert certain cells,

24 stem cells, into different types of a cell.

25 He here took bone marrow from a male

> 37 1 mouse, transduced them with the lenti-GRP. Again,

2 the lenti can transduce bone-marrow cells, in this

3 case the stem cells, but them back into a female

4 mouse and asked the question, can he convert some

5 of these bone-marrow cells into hepatocytes because

6 we cause injury in the liver by using the anti-FAS

7 antibody which causes damage to the liver. The

8 argument is can these blood cells now be transduced

9 into the liver cells, and can be they be

10 transdifferentiated.

11 \[Slide.\]

12 I don\'t know if you can see, again, in the

13 front. About 1 percent of the cells are non-liver

14 cells which we started out\--these are the green

15 cells which were marked and they were put back into

16 the animal, so we can actually begin to ask the

17 question, can you do transdifferentiation by

18 introducing genes into non-dividing cells.

19 This will be particularly useful when you

20 begin to ask if you have pancreatic-specific genes,

21 or liver-specific genes, that you can convert any

22 cell directly into a transdifferentiated cell type.

23 \[Slide.\]

24 Let me give you another example. Many of

25 us in biology these days are very interested in

> 38 1 making knock-out animals. Many of us are

2 interested in making knock-out animals which are

3 conditional, meaning that the animals are born, but

4 the gene can only be deleted post-birth.

5 Otherwise, these genes are lethal to the animals.

6 So, what we do now is we have a system

7 which is used\--to use specific sequences called lac

8 sequences which block the transcription of the gene

9 until you remove these lac sequences from there,

10 which can be done by an enzyme called CRE.

11 Normally, you cross these animals, which is a long

12 process. But now these vectors, lentivectors, can

13 be directly introduced into the tissue where we are

14 interested to remove the gene.

15 So you make a mouse with these specific

16 sequences, introduce the gene CRE directly by

17 lentiviruses and you can begin to see, at least

18 this is now in vitro, these cells have no

19 expression following the introduction of the CRE by

20 lenti, you have the cell all blue.

21 \[Slide.\]

22 Can you do that in vivo? Here is an

23 example. If you take, now, a gene where it is

24 blocked by lac sites to make the foreign gene in

25 the liver, so this is conditional for the

> 39 1 production of this gene in the liver. If you now

2 introduce directly into this the lentiviruses, you

3 can\--now the cell becomes blue within the range the

4 gene was introduced.

5 So, again, a very useful utility of these

6 vectors is that you can introduce the gene directly

7 in vivo.

8 \[Slide.\]

9 Here is directly in the striatum in the

10 brain. You see the genes are again expressed. So

11 it is really a very useful tool, particularly for

12 those who are interested in tumor genesis. You

13 have a conditional mutant, introduce the gene in

14 the prostate, liver, lung, wherever you want,

15 eliminate the gene and ask the question, what

16 happens to the animal subsequently.

17 \[Slide.\]

18 Let me give you another example. This

19 also refers a little bit to the safety of the

20 issue. We were interested to know can you use

21 lentiviruses for two purposes for transgenesis.

22 So, Matha, in the lab, did the following

23 experiment. He asked the question, what happens if

24 you directly introduce the lentiviruses in the

25 testes and then asked the question, can they be,

> 40 1 then, used to create a transgenic mouse.

2 His hope was that, by introducing in the

3 testis, he will have the expression eventually in

4 the sperm.

5 \[Slide.\]

6 What he found was that in the control\--so

7 here is the lacZ which is nuclear localized and

8 here is the lacZ direct injection into the testis.

9 When he analyzed them, what he found was\--this is,

10 again, for those people like me who don\'t

11 understand too much biology\--the point is the

12 following.

13 You have here the sertoli cells and,

14 eventually, the sperm cells are right in the middle

15 here. All these other cells are really the

16 supporting cells. So the gene was introduced

17 directly here, and we are now asking the question,

18 can you make mature sperm which contain the foreign

19 genes?

20 \[Slide.\]

21 The answer is no. Almost all the

22 expression is in the sertoli cells and none in the

23 case where, in the middle, where the sperm are.

24 So, even if you put billions of virus particles

25 directly into the testis, while you can get the

> 41 1 transduction of all supporting cells, you do not

2 transduce sperm directly.

3 In fact, if you make pups from these

4 animals which have been directly introduced, you

5 don\'t see a single pup which is positive for the

6 PCR. The pups are born, but they are not positive

7 for this. So the argument is at least directly

8 injection. We are unable to directly introduce the

9 gene into the mature sperm cells.

10 \[Slide.\]

11 Here is the direct injection again.

12 \[Slide.\]

13 Let me finish my talk by giving you one

14 other very exciting piece. Here, can we actually

15 begin to use these viruses for transgenesis. The

16 standard way of making transgenic animals these

17 days is you take the egg, you introduce the gene

18 directly into the nucleus, fertilize it and then

19 put it back into the animal.

20 This has been very successful in the case

21 of the mouse. But it has been more difficult in

22 the case of other animals because the nuclease is

23 often pigmented.

24 So we did a very simple experiment to ask

25 the following question; can we introduce genes

> 42 1 directly into either ES cell by infection with

2 virus of lentiviruses, the formal viruses, the

3 traditional MLV viruses, are unable to do so and

4 can we create a mouse which is transgenic or a rat

5 which is transgenic.

6 The way to do that is these days you take

7 embryonic stem cells, you infect them with the

8 virus and you get the expression of the foreign

9 genes for over a six-week period of time easily,

10 which is a big distinction from traditional

11 retroviral vectors. They shut off their

12 transcription.

13 The HIV-based vectors, for some reason, do

14 not shut off the transcription and, therefore, you

15 can have the ES cell. You can also have

16 preimplantation embryo, but you have to remove the

17 zona pellucida. If you don\'t remove the zona

18 pellucida, you cannot get the infection. But if

19 you remove the zona pellucida, then put the virus,

20 you get the infection.

21 \[Slide.\]

22 The most interesting is here now. This is

23 a litter of four. Many animals are not chimeric

24 for the foreign gene.

25 \[Slide.\]

> 43 1 You cannot see this. This is a really

2 beautiful picture. These are live births of

3 animals. You are taking simply ES cells, directly

4 introducing the virus and now you are making\--most

5 of these animals are containing the foreign gene.

6 David Baltimore\'s lab has also done very

7 similar sorts of experiments. This is another idea

8 to explain to you that the idea of using these

9 lentiviral vectors that, because they have the

10 ability to infect many of these cells, because the

11 transcription is not shut off, they can actually be

12 used for additional purposes like transgenesis. I

13 suspect this will be the method used for making

14 transgenesis from monkeys and many other different

15 kinds of species because you don\'t have to do

16 nuclear injection, just infect the cells.

17 But you can\'t infect the sperm. You can\'t

18 infect the eggs until you remove the zona

19 pellucida.

20 \[Slide.\]

21 So far, then, all I have told you is the

22 lentiviral vectors can be made easily, large

23 titers. Most of the genes which we think have the

24 pathogenic consequences can be eliminated and it

25 really has a wide utility not only for different

> 44 1 kinds of tissues, for gene therapy, but also for

2 other scientific purposes like transgenesis, knock

3 outs as well as for transdifferentiation of stem

4 cells.

5 The last part of the few minutes I have, I

6 will tell you a little bit about how we can do

7 regulation. There are number of ways to regulate

8 the transcription of foreign genes. Those of you

9 who are in the field know there is tetracycline,

10 there is the dimerized formation and there are

11 also ectosome receptors.

12 The first thing we wanted to use is a

13 method which is using tetracycline and, again, no

14 details are necessary except to say this is an

15 antibiotic in the absence of which the gene is

16 turned on in the presence of which the gene is

17 turned off.

18 The first question we asked was, A, can

19 you use this kind of methodology to make cell

20 lines. That is to say, the way I have described to

21 you so far is we take three or four plasmids, mix

22 them together and we have the virus out. But

23 people who really want to make a very specific gene

24 that they are interested in, they would like to

25 make cell lines where they can produce the protein

> 45 1 continuously\--the virus from those cell lines.

2 \[Slide.\]

3 So a post-doc in our lab created the idea

4 that he can actually make a cell line where you can

5 continuously

6 produce the virus rather than having the four

7 plasmids or three plasmids cotransfected into the

8 cell.

9 What he basically did was to use the

10 tetracycline as a regulatable element. The

11 interesting thing to show you here is that if you

12 now take these viruses which contain the

13 tetracycline-regulatable element, he can generate

14 titers not very different from those vectors where

15 we have cotransfected four plasmids.

16 In other words, you can make cell lines

17 from these plasmids rather than having always the

18 four plasmids together. It really depends on what

19 you want to do. If you are interested to use

20 different sets of promotors, you may want to do one

21 thing. If you are interested in making only one

22 type of a virus, you might want to make a cell

23 line.

24 \[Slide.\]

25 For the purpose of showing this, these

> 46 1 viruses are equally good in infecting the neuronal

2 cells. In other words, you can make cell lines

3 rather than just using the plasmids.

4 \[Slide.\]

5 These are the third generation which has

6 the same vectors.

7 \[Slide.\]

8 Finally, because we have these vectors

9 where the LTRs have been manipulated and have been

10 deleted, largely, we can now substitute regular TC

11 culture and ask the question can you turn on the

12 gene and turn off the gene at will. So here we

13 introduce tetracycline elements directly inject it

14 in the brain.

15 In the presence of tetracycline, there is

16 hardly any expression. If you remove tetracycline

17 from the water, there is expression, the work of

18 Karl Kaffree. More importantly, you can turn the

19 gene on, turn the gene off, turn the gene on, turn

20 the gene off, at will for over a six-month period

21 of time.

22 This is not perfect, but it is a

23 reasonably good way to start thinking that you can

24 actually regulate the sequences just as well.

25 \[Slide.\]

> 47 1 So, let me come to the final slides.

2 Where are we now? Remember, we started out by

3 asking what is an ideal vector we would like to

4 have. We would like to have a vector which has the

5 ability to do many of these things. We think\--and,

6 again, this is specific for lentis. There are many

7 other vectors which can do many other different

8 things. So this is not a competition or a

9 comparison. It is simply to say what we planned

10 out and this is what we have come out with.

11 The vectors certainly have the ability to

12 make large amounts of particles. That is not

13 difficult. Convenience of reproduction, at least

14 in the lab, is not a problem. I don\'t know when

15 you have to make 100,000 liters. That is the

16 business of the people who do it in the biotech

17 companies.

18 It has the ability to infect nondividing

19 cells and dividing cells. I haven\'t told you about

20 tumor cells, but you can do that\--to integrate in a

21 site-specific manner. We don\'t know that. We have

22 not learned anything how to control the

23 integration. In fact, that has been a very

24 difficult task. So that part, I don\'t even know

25 actually how to approach at this point, to have a

> 48 1 site-specific integration.

2 Fortunately, there is considerable

3 experience in the clinic from the previous vectors,

4 like Moloney leukemia virus, that we have not see

5 any untoward effect of integration. But that is

6 something we have not been able to achieve as yet.

7 I think we have the rudiments of a

8 regulatory system, that we can turn the gene on or

9 turn the gene off. We can infect a wide variety of

10 cell types. I don\'t know if every cell type can be

11 but a large number that I have shown you. We have

12 not had any immunological consequences, at least

13 not at the moment, particularly not with the

14 viruses because part of the reason is a lot of

15 the\--4 percent of the human genome really is

16 retrotransposon and has sequences much like the gag

17 and the pol kinds of sequences, all the broken

18 ones.

19 We certainly have antibodies against

20 VSV-G. If you take the dogs, infect them with the

21 virus, we have titers, antibodies; not a surprise,

22 because VSV-G has fallen. But we have not seen any

23 inflammation at the site of injection. But, then,

24 again, we can\'t compare them with adenoviruses

25 where the titers can be trillions of virus

> 49 1 particles. But, within the constraints of 100

2 million particles that we can inject, we do not see

3 any inflammation and immunological consequences.

4 So we believe, at this point, these

5 vectors do offer the opportunity of the ability to

6 infect nondividing cells and a number of people

7 have made strong attempts to try to make them safe,

8 safety in terms of their inability to, perhaps,

9 make a replication-competent virus.

10 The deletion of LTR offered the

11 opportunity to not allow mobilization of the virus

12 and the fact that we have been able to manipulate

13 the genome such that you have the ability to infect

14 a wide variety of cell types offers many

15 possibilities that these viral vectors have the

16 ability to perform many of the things you would

17 like them to do in terms of the production of the

18 foreign protein and eventually into the patients.

19 \[Slide.\]

20 Finally, I would like to thank a number of

21 individuals. Not all of them are listed here, but

22 I would particularly like to thank Luigi, who

23 happens to be in the audience, who started this

24 along with Didier Trono and Rusty Gage with whose

25 lab I collaborate very extensively. Bruce Talbert

> 50 1 with whom we do a lot of our work on hematopoiesis,

2 and a number of other individuals, and finally a

3 audience for your indulgence.

4 Thank you very much.

5 \[Applause.\]

6 Questions & Answers

7 DR. SALOMON: Thank you, Inder.

8 It is generally our policy to generate

9 some questions and discussion. There is no agenda

10 to this part of the meeting. It is just to get

11 some issues out on the table. So I am very

12 flexible about what kinds of things you want to

13 raise.

14 Just to start, Inder, one question. You

15 started off by pointing out that one of the first

16 things you established was that you could make 10

9

17 viral particles per ml. To me, that raises the

18 question, in the context of the FDA thinking about

19 setting some kind of standards for this as a

20 product, exactly what do you think is the best way

21 to express the efficacy of an expression system,

22 transient or stable; in particles per ml? I mean,

23 in retroviruses, that would not necessarily be the

24 best way to describe something; right\--that we

25 would talk about infectious titers.

51

1 DR. VERMA: I think, first of all, I,

2 perhaps, misspoke if I gave the impression you make

3 10 9 virus particles per ml. I might

have wanted to

4 say that we made about 10

6 or 107. Then we can

5 concentrate them by virtue of the fact that the

6 glycoprotein has reached the titers of 10

9, and

7 some people can claim titers of 1010.

8 So that is the general\--now, you are

9 asking what is the way\--each lab, I think\--there

10 are no standard ways to do that. Most of use p24

11 as a marker to see how many p24 antigen amount will

12 be equal to infectious units, and use that as a

13 major\--that is what we do in our lab.

14 Some people use reverse transcriptase.

15 Some people actually do the count of the particles.

16 So I think this is something which people who are

17 more familiar with these kinds of things, in terms

18 of measurements, they will have to make a decision

19 what is the best for their cause.

20 The second question regarding whether you

21 should use plasmids combined together to make the

22 virus or do you make a cell line. That was the

23 implication. I think again it really depends on

24 the individual.

25 We have used almost always plasmid

> 52 1 transfection so far, three or four plasmids. We

2 have seen, whatever we do in the lab\--and,

3 remember, we do these things in 1 liter, 2 liters,

4 3 liters at best. So you have to take that into

5 constraint that when you go to thousands of liters,

6 what might happen.

7 We have not seen any recombination. We

8 have not seen and PCL-positive tat in the usual

9 tests we do regardless of the fact whether you use

10 plasmids or\--so I have really a fairly open mind.

11 I personally think there is no reason if people

12 want to use three, four, plasmids together, that is

13 perfectly fine. So I have really no preconceived

14 notions in my mind on this issue.

15 DR. ALLAN: Just a point of interest. The

16 VSV-G envelope is very good especially ex vivo

17 where you take the cells out and infect them and

18 get about 90-something percent. What about if you

19 are going to treat someone by injecting the virus.

20 Obviously, you are showing that you can target

21 expression with the tat oppressor genes but I am

22 wondering whether you can do tissue-specific

23 expression and whether you are actually

24 manipulating the envelopes that target specific

25 cell types like, say, hepatocytes.

> 53 1 DR. VERMA: So the question is twofold.

2 One, can you manipulate the glycoprotein so as to

3 allow it to go to a very specific cell. For

4 example, VSV-G being very general, unfortunately we

5 don\'t know the receptor of the VSV-G, so we it is

6 difficult.

7 One area we have failed miserably in the

8 lab in the last ten years and that is the area of

9 targeting. If we chose even a single nucleotide in

10 VSV-G, it either refuses to bind and, if it binds,

11 it doesn\'t fuse. I think the viruses have billions

12 of years of evolution to really make themselves

13 perfect.

14 There are viruses\--Jim Wilson had a paper

15 on philoviruses lately in which he found\--now, I am

16 using them philo because if I said they were ebola,

17 it sounds even worse than that. That day he found

18 their G-protein to be very specific for the apical

19 parts of the lungs. So there are specific types of

20 viruses you can use. We haven\'t really had much

21 success but I am sure those in the audience have

22 done better experiments.

23 The second is to control it by

24 transcription regulation of a promoter. The only

25 experiments we have really some experience with is

> 54 1 in the case of probasin which is prostate-specific,

2 where, again, you can directly introduce the gene.

3 We haven\'t really succeeded much in making

4 intravenous delivery and hope the expression will

5 be only in the tissue where it goes. But we don\'t

6 have enough virus. There is a lot of biological

7 loss of the virus by the time you go, so we haven\'t

8 really much experience on that.

9 DR. RAO: I had a question. Is it clear

10 that when you do lentivirus infections, you have

11 single-site insertion at the concentrations you use

12 it at?

13 DR. VERMA: So the question is if you want

14 to use a multiplicity infection of 1, 10, 100, 200,

15 2000. I can\'t answer the question because we have

16 never systematically done that. But we have rarely

17 seen more than 2 to 3 viral integrations. Rarely.

18 It is not unusual with the retroviruses, very

19 often, that you have very few integrations. The

20 best one I know is the XE cells. We have about

21 twenty integrations when the Rous sarcoma virus was

22 introduced.

23 But, by and large, we haven\'t seen many.

24 But, again, I have never really known to do any

25 systematic experiments. Maybe somebody did it. I

> 55 1 just don\'t know.

2 DR. DELPH: You showed that when you

3 injected the vector into the testis that there was

4 no transduction of the sperm cells. Have you

5 looked at all to see what has happened to the

6 offspring of transduced animals?

7 DR. VERMA: Yes. We got perfectly fine

8 animals. There was no a single transgenic-positive

9 animal. That is what I showed in one slide.

10 Perhaps I went too fast. The PCRs are all

11 negative.

12 DR. DELPH: That is both male and female?

13 DR. VERMA: Yes. It was like five and

14 five.

15 DR. SALOMON: Dr. Zaia?

16 DR. ZAIA: When you are packaging the

17 final virus, I normally think that viruses in

18 nature make mistakes and there are defective

19 particles. In your system that is constrained, is

20 there less likelihood of this or is there more

21 likelihood of having defective or incomplete

22 particles and will that have a biological effect,

23 do you think, when you are injecting vectors into

24 muscle or liver?

25 DR. VERMA: It is a good question. We

> 56 1 don\'t really know how many defective interfering

2 parts. I know if you have VSV alone, those kinds

3 of stay in, so there you do make. In cytoplasmic

4 viruses, they often make defective interfering

5 there. I don\'t know, really. We have looked at

6 the homogeneity of these viruses. By and large,

7 they seem to be the same size, but if there was 1

8 percent, 10 percent\--they might interfere, but it

9 can\'t be a tremendous interference unless there are

10 a very large number of them to interfere with it.

11 But no systematic study is done.

12 Incidently, I can\'t have the opportunity,

13 Dr. Zaia. As I was coming on the plane, I was

14 looking at all the papers of FDA. The guy sitting

15 next to me said, \"Ah; that is my sister\'s husband,

16 Dr. Zaia. Do you know him?\" The probability that

17 I should sit next to him in the plane, who knows

18 you so well, I was astounded. So there is always a

19 chance.

20 DR. SALOMON: I am not sure what chance

21 you are referring to.

22 DR. VERMA: Very low.

23 DR. SALOMON: One of the questions I think

24 you are uniquely suited to answer is this

25 terminology of generation, as one of the people who

> 57 1 basically started this. Now, we are talking about

2 first generation and second generation and third

3 generation. So these kind of terminologies tend to

4 become something we are comfortable with.

5 But, from time to time, as the field

6 evolves, they can also lose their specificity. So

7 one of the things I was struck, and we are going to

8 get into this tomorrow but just to put this into

9 context, was that part of the discussion at the RAC

10 of the VIRxSYS protocol was an argument about

11 whether this was a first generation or a second

12 generation.

13 When you really looked at the details of

14 what VIRxSYS had done, I am not sure whether it

15 fits your concept of a generation because what you

16 have done in generations here is continually split

17 further apart, whereas VIRxSYS took a very

18 different approach.

19 So are we kind of done with this

20 generation thing or can you suggest a new way to

21 define generations of lentiviral vectors that will

22 be more useful?

23 DR. VERMA: I think it is a good question

24 and it really hasn\'t crossed my mind at all to

25 think about it, really. We do generations that

> 58 1 really actually defines the post-doctoral era of my

2 lab, the first generation post-doc, the second

3 generation. The fact of the matter is the first

4 generation vectors, by and large, are defined by

5 most of us and I think these are may perhaps be

6 different in the system of the simian or the feline

7 ones, as those where really largely the envelopes

8 have changed and some of the long-term repeats have

9 changed except most of the accessory genes would

10 concept. So that is really the first generation of

11 vectors because we really didn\'t know what genes

12 are required for integration. If you look at the

13 history of what we required for integration in

14 nondividing cells, at least six people will tell

15 you six different genes. So we didn\'t want to

16 eliminate them.

17 The second-generation vectors have been

18 defined, at least, and I don\'t think many people

19 will use that very much, where the tat and nef were

20 still present, or tat and rev were still present

21 where the other genes were eliminated. Again, I

22 haven\'t read the VIRxSYS thing. It is such a big

23 document to read, but I think they are using the

24 second generation of that type\--I think.

25 But there LTR are not deleted. So you

> 59 1 don\'t have the SIN vector. So the third generation

2 vectors are the SIN vectors where the LTRs have

3 been truncated, where most of the accessory genes

4 have been eliminated. That is how, really, we are

5 defining them.

6 But I think in the future, one has to

7 define them as probably fully deleted vectors or

8 something like that. I just haven\'t thought about

9 how to nomenclature them.

10 DR. KINGSMAN: Is it okay protocolwise for

11 me to make a comment?

12 DR. SALOMON: Yes. In fact I would

13 say\--you need to identify. I guess I am remiss and

14 I apologize to everyone. I would encourage both

15 the invited speakers, as you are doing, as well as

16 the audience to stand up and come to the mike since

17 there is no attempt to restrict the discussion

18 here.

19 DR. KINGSMAN: Thank you. I am Sue

20 Kingsman from Oxford BioMedica. As Inder mentioned

21 my name, I felt dutybound to stand up. I don\'t

22 think that the word \"generation\" is a useful

23 concept in a regulatory framework. I think it is a

24 laboratory-specific statement to say we are

25 gradually beginning to understand our system and

> 60 1 that people observing our system should realize

2 that we are defining issues, solving them, moving

3 forward.

4 I think the take-home message is that all

5 of us are making progress towards understanding our

6 system and what we should seek to do is to define

7 some general concepts and parameters that most

8 lentivectors can fit in with because I think the

9 word \"generation\" will mean different things to

10 different laboratories.

11 So I think it is the substance of the

12 vectors that we would focus on, not this overall

13 terminology. That would be my viewpoint.

14 DR. MULLIGAN: Since I think we are going

15 to talk about the relative virtues of the transient

16 transfection versus the packaging cells, I had two

17 issues. The first is, as we have talked in the

18 past, you can make stable packaging cells.

19 Everyone could make these. What seems to be the

20 difficulty is actually transfecting the vector and

21 getting high enough RNAs to make high virus titers.

22 There are definitely reports by cross

23 infection or reinfection that you can get enough

24 proviral copies to get very good virus titers. But

25 I think the last time we talked there weren\'t a lot

> 61 1 of people that had actually, with SIN vectors, been

2 able to do transfections and get good virus titers.

3 So, in your own experience, has that been

4 possible, so when we get to the issue of the

5 relative virtues, if no one can really make good

6 stable producer cells, that will be very important.

7 The second question just is a more

8 philosophical question which I think we will end up

9 getting to which is the issue of there is a

10 difference between theoretical safety and

11 detectable safety. I want to pin you down on the

12 transient versus stable packaging.

13 My impression is that whether you use a

14 first-generation, second-generation,

15 third-generation transient-transfection system,

16 people will report that there is no difficulty,

17 there is no helper virus functions, et cetera, et

18 cetera.

19 We had a meeting here many months ago

20 about good old-fashioned retrovirus packaging cells

21 and the merits of PA317 which you know very well

22 and other more advanced cells. I think the FDA at

23 one point was asking for our guidance as to should

24 they ever legislate against a less sophisticated

25 packaging cell.

> 62 1 The discussion was somewhat controversial

2 and I guess I came down to the fact that, well, if

3 you can\'t prove, by experimental means, that there

4 is a difficulty, then you have a real difficulty

5 preventing people from moving ahead.

6 On the other hand, there is no doubt that

7 there are theoretical, good, sound theoretical,

8 reasons to think that the split packaging cells

9 would be a safer product than the transient

10 transfection. So I am curious where you come down

11 on that point.

12 DR. VERMA: I tried to mention it quickly.

13 What Richard is asking\--it is a long question;

14 right? But I think I get the gist of what you are

15 asking. This is also again the question we are

16 often asked in the past. Richard is asking the

17 question\--two questions, mainly\--if you really can

18 make a cell line from all these systems that you

19 have, you still would like to continue using, for

20 example, the transient transfection.

21 My experience in the lab largely has been

22 on the transient transfection, so I can only speak

23 very little. The only stable cell line we have is

24 the one that I just described which Karl Kaffree

25 made in the lab prior to his departure. We have

> 63 1 really seen no big difference, again in terms of

2 infectivity, in terms of production of our usual

3 safety efforts of the tat production and so on.

4 I think it is very hard, really, to say at

5 this point. Theoretically, if you think you might

6 conceive the idea that if you have four plasmids

7 together, that you may be causing real

8 recombination when they are growing up and you are

9 adding to that. It is a theoretical possibility.

10 I have had no really direct evidence for

11 that. You asked me for a recommendation I will

12 have. I think, personally, if you can make a cell

13 line, and I think we have shown that you can make

14 the third-generation cell line, and the titers are

15 not really compromised because there are ways to do

16 that, I would say that if I were the one doing it,

17 I would take a cell line just because of the

18 convenience of it and that you know the

19 reproducibility of it and you know that you know

20 exactly what you started with.

21 But I really can\'t definitively answer

22 your question to say the other is the wrong way of

23 doing it because I really have no experience on

24 that.

25 DR. MULLIGAN: Can you make one with the

> 64 1 SIN vector?

2 DR. VERMA: That is the one with the SIN

3 vector. We made it with the SIN vector. That is

4 the paper Karl just published.

5 DR. SAUSVILLE: You alluded to the

6 karyophilic nature of the virus as being a key

7 advantage. I think that really came through as a

8 real leap with this vector generation. Yet it

9 would seem, from the standpoint of the product

10 definition, that could also be a point in

11 variability in how much expression you get. Could

12 you expand on whether or not there is a concept of

13 how to standardize\--is it a function of the gene

14 you are trying to make? Is it a function of

15 sequences that are in the vector that determines

16 that property?

17 DR. VERMA: Implicit in your question is

18 that we understand the mechanism by which the viral

19 DNA actually crosses the nuclear membrane. In

20 fact, that is really, still in my mind a fairly big

21 black box. There have been proteins identified

22 that Didier Trono showed the PL10 protein which

23 binds to it.

24 At one time, there were different sets of

25 proteins. Once it was the gag protein. Once it

> 65 1 was the VPR protein. They are all involved. We

2 don\'t know the mechanism of that. So think, at

3 present, to use that any kind of way is probably

4 not a good one. On top of that, you may have seen

5 some of my slides, once again from the French group

6 and from Luigi\'s group, there are polybrene checks

7 called the cPPT\--some call them flaps\--which seem

8 to allow a better transduction into the nucleus.

9 So we don\'t know if the presence of that

10 will make a difference or not on how efficient is

11 that process. That still remains to be done. So I

12 think that is not going to be a very easy way at

13 this point to use as a mechanism to define that as

14 a late property.

15 DR. KINGSMAN: I think that question needs

16 to be answered on a case-by-case basis, that when

17 you are doing your efficacy studies, you will

18 design a vector that will transfer genes into the

19 cells that you are targeting and will give the

20 effect that you want. Sometimes, you may have to

21 alter the properties by adding the cPPT in and

22 other times you won\'t. But you will have defined

23 the potency of your product with the specific

24 endpoint in mind.

25 So I think you will be able to get a

> 66 1 product definition for your particular product but

2 I agree with Inder. I don\'t think you will be able

3 to come up with a generic specification for all

4 lentivectors to perform similarly under all

5 circumstances. I think if we try to go down that

6 route, it will be a very long tortuous path.

7 DR. SAUSVILLE: I certainly agree that

8 that is an area that is of great theoretical

9 interest to figure out and also, in a particular

10 case, to define. Yet, it seems to me, that would

11 ultimately influence the number of particles that

12 would result in an efficacious outcome and,

13 therefore, this issue of background safety issues

14 then becomes potentially influenced by this

15 sufficiency issue.

16 DR. VERMA: I really can\'t answer any

17 better. I just don\'t know enough about the actual

18 mechanism of transfection.

19 DR. SALOMON: I guess one question I think

20 Inder has already answered it for his experience,

21 but one of the key issues for me when I look at the

22 safety of a transient versus a stable line is the

23 question that I don\'t know the answer to, so I want

24 to pose it to the group. The answer may be, as

25 Inder has already said, that he doesn\'t know. But

> 67 1 the question would be if you have a situation in

2 which you have a transient-transfection system in

3 which up to four purified plasmids are transfected

4 at the same time.

5 So, forgive me, but my image is of all

6 this semipurified DNA in very high concentrations

7 at various points in the cell and in the cell cycle

8 versus a stable cell line, packaging cell. We are

9 thinking about relative safety now, not efficacy or

10 production, not that those aren\'t very important.

11 The question would be is there any data

12 out there suggesting that such a multi-transfection

13 system leads to higher rates of recombination? I

14 am not saying that you can\'t make alterations in

15 the vectors and lack of homology, et cetera, all of

16 which have cleverly been done and proposed by

17 different people, but is there just any evidence

18 that there would be more recombination in such a

19 multi-plasmid system? Does anybody have an answer

20 to that?

21 DR. VERMA: Theoretically, you might

22 imagine, because there are all these thing and

23 maybe recombination\--I think a lot of work has been

24 done in the past on one or two plasmids,

25 particularly with the recombination of the

> 68 1 endogenous genes. That has been extremely low.

2 But to actually have high amounts, because

3 we have been asking the question what amounts, I

4 think it really comes down in the end to

5 individuals, how they want to proceed with it. I

6 don\'t believe there is any strong evidence at this

7 point whether three plasmids versus four plasmids

8 versus two plasmids gives you any worse result if

9 you have a cell line, if there is any greater

10 recombination. I don\'t think there is any direct

11 evidence.

12 DR. KINGSMAN: Could I just add to that.

13 In the early days of plasmid-based gene transfer,

14 if you go back and read the papers in the early

15 \'80\'s, people addressed those questions about what

16 happened to plasmids when they went into cells.

17 What happens is they do recombine and concatenate

18 and rearrange.

19 So, a priori, you might expect that there

20 would be some DNA-DNA interactions when you put

21 large amounts of DNA in a cell. But whether

22 anybody has then studied retroviral vectors coming

23 out of that and done some of the studies like

24 Howard Temin did to ask what are the nature of

25 retroviral recombinants, I don\'t think they have.

> 69 1 But, a priori, there will be DNA-DNA interactions.

2 DR. VERMA: But it is the final product

3 you are really interested in in the end.

4 DR. KINGSMAN: Yes.

5 DR. MULLIGAN: I would echo Sue\'s point

6 that there is no question that the DNA that is the

7 template for making the RNA in a transiently

8 transfected cell is a very complicated DNA. So

9 there is no doubt that there is recombination at

10 very, very high efficiency, probably near unit

11 efficiency. So I don\'t know if anyone has actually

12 looked at the RNA transcript in a transiently

13 transfected cell but I would bet you that you would

14 undoubtedly see very funny things.

15 Now, Inder\'s point is, all that being

16 said, what gets selected to be packaged and

17 transferred and so forth appears to be no

18 different. What I would think I would really

19 strongly emphasize that this is not the optimal way

20 to generate RNA to be packaged.

21 If people were to look, if we thought it

22 was important to look at this process, I would

23 think we would undoubtedly see the effects of that.

24 So that is a fundamental difference between having

25 integrated templates for helper functions and

> 70 1 vector functions in the transient system.

2 DR. SALOMON: Yes. I just wanted to point

3 out that that is sort of the point. My point is

4 that I think one of the questions that the

5 committee has in front of it, and we are not going

6 to answer it immediately, but as we consider

7 safety, if we agree that these are important

8 scientific questions and the data is not out there,

9 it may be important to solve these issues before

10 you say, we are going to defend the use of one or

11 another type of strategy.

12 If it turns out to be a wash by the time

13 you package the vector, then great. Then you could

14 do it any way you want.

15 DR. NALDINI: Luigi Naldini from Torino.

16 I apologize for my voice. One point, in terms of

17 the packaging cell line versus transient

18 transfection which has to be made, I think we have

19 to be careful in really using experience with

20 retroviral vector into the lentiviral field.

21 The lentiviral vector that we have

22 discussing until now uses the VSV envelope making a

23 packaging cell line, the VSV envelope poses

24 challenges not only in terms of regulating that

25 envelope because it is toxic but also because it

> 71 1 allows superinfection of your cells, quite

2 extensively. Even if you have an inducible system,

3 you may not completely suppress that.

4 I think, overall, that means that, in the

5 long time in which you grow your cell, there is

6 actually more changes for recombination to take

7 place and for recombinants to spread in the system

8 and to accumulate the multiple steps required to

9 build a virus as compared to the short window of

10 time of transient transfection.

11 So I think it is obvious that a stable

12 cell line has an advantage in terms of

13 manufacturing, standardization. I would doubt that

14 actually, at the moment, we can think it is

15 actually safer. Transient transfection, as long as

16 you use multiple plasmids in a very short window of

17 time, makes it very unlikely, even if there is

18 recombination going on and there is no question,

19 that you rebuild a complete genome.

20 In a stable cell line which grows for a

21 long time, we may allow a certain level of

22 infection going on even by partial recombinant,

23 this may happen. So I think we have to be very

24 careful with that.

25 DR. VERMA: You can be careful with that

> 72 1 but the bottom line is, in the end, it is the final

2 product whether you made it with one system or the

3 other system. That is the one we want to really

4 need to know, whether that has recombinants in it

5 or not.

6 DR. KAPPES: John Kappes. I am from the

7 University of Alabama at Birmingham, UAB. We took

8 a very careful hard look using highly selective

9 pressures to address whether RNAs were incorporated

10 into vector particles that could recombine during

11 reverse transcription.

12 Specifically, we were looking for

13 recombinants that could generate something that

14 would be produced from the cells. So, minimally,

15 you would have to generate a recombinant which had

16 the capability to produce a retroviral particle

17 because we were providing envelope in trans. So

18 this would be an envelope minus recombinant, to say

19 the least.

20 But my point is, in that context, in that

21 examination, we did find DNA recombinants that had

22 properties that, when envelope was provided in

23 trans, by transfection of those cells that received

24 supernatants from vector-generated stock which

25 contained DNA, that that, too, could, as our

> 73 1 endpoint, mobilize retroviral DNA or marker genes

2 which we had introduced into the cell.

3 So the point is, in rare cases, we did

4 identify DNA recombination.

5 DR. SALOMON: Dr. Mulligan and then Dr.

6 Jolly.

7 DR. MULLIGAN: Just on Luigi\'s point, we

8 have actually, with a MLE VSV-G packaging cell,

9 looked at the transient-transfection issue. I

10 think if you were to look in your system, you would

11 see probably that the same thing happens in the

12 transient transfection. It somewhat depends on how

13 you do your harvests, but we have seen with

14 intron-containing constructs that, even in

15 transient transfections into the stable packaging

16 cells, that you can detect intron incision and

17 remobilization suggesting that what you say can

18 occur in both the transient transfection and the

19 stable cells.

20 DR. JOLLY: My name is Doug Jolly. I work

21 for Biomedica, Incorporated. Just I guess the

22 first thing I would say is there is almost no data

23 about this. It is pretty early to make any choices

24 without the data. I think part of the problem is,

25 drawing on the experience from murine retroviral

> 74 1 vectors, we had a packaging cell line which

2 retained some homology although it was split into

3 three pieces.

4 Really, the way we gathered data on that

5 was to do 60 200 liter preps. Then three of those

6 had RCR positivity. So that is only assay for the

7 very rare events that we are worrying about is to

8 do something like that. You can\'t see it often

9 unless it is an acute event in the scale

10 experiments that we are talking about now.

11 So I think it is too early to close any

12 doors with respect to the lentiviral vectors.

13 DR. SALOMON: Yes. I agree with that. I

14 guess I would just also point out to broaden the

15 context that this is not\--I don\'t think the only

16 safety issue for any sort of vector delivery is

17 replication-competent lentivirus or

18 replication-competent retrovirus albeit, obviously,

19 that is front and center, particularly with this

20 class.

21 But it is also if recombinations occur

22 that alter the integrity or the structure of the

23 trans gene could also be very potentially dangerous

24 in terms of autoimmunity and other effects. It

25 certain would affect efficacy.

> 75 1 DR. VERMA: I agree. I think it is a

2 point worth thinking about. I certainly too

3 thinking about how to actually how you can do the

4 experiments. I was thinking about it.

5 DR. SALOMON: I think that was excellent,

6 Dr. Verma.

7 We had a discussion yesterday that, having

8 grown up on the East Coast, born in Boston and now

9 have been out in Southern California, I am having

10 this conflict about referring to people by their

11 first name or referring them as Doctor. So I am

12 going to try and go with the East Coast formal

13 until we can finally get the FDA to have one of our

14 meeting out on the West Coast.

15 DR. NOGUCHI: As long as you host it.

16 DR. SALOMON: I think I can say that

17 Scripps would be happy to host the next FDA-BRMAC

18 meeting. I don\'t think that is going to really

19 happen though.

20 It is my pleasure to announce the second

21 speaker which is Dan Takefman from the Office of

22 Therapeutics Research. He is going to talk

23 specifically about lentiviral vectors and continue

24 our discussion of potential safety issues.

25 Lentiviral Vectors: Safety Issues

> 76 1 Dr. Daniel Takefman

2 DR. TAKEFMAN: That was really a great

3 introductory talk by Dr. Verma. I am very

4 encouraged by the excellent discussion thus far.

5 \[Slide.\]

6 Today, I will be speaking about safety

7 issues associated with the use of lentiviral

8 vectors in the clinic. As many of you know, the

9 first patient participating in a gene transfer

10 clinical trial received cells that were exposed to

11 a murine gammaretroviral vector. Since that time,

12 murine gammaretroviral vectors continue to be

13 tested in clinical trials, one long-term gene

14 expression is desired.

15 \[Slide.\]

16 This is a figure you are going to see a

17 number of times today. Lentiviruses, like

18 gammaretroviruses, belong to the Retroviridae

19 family. Gammaretroviruses have encode for three

20 open reading frames - gag, pol, and env.

21 Additionally, the genome is surrounded in both ends

22 by long terminal repeats

23 Lentiviruses, such as HIV, depicted here,

24 have a more complex genome. In addition to gag,

25 pol, and env, there are two regulatory proteins,

> 77 1 tat and rev, which promote viral gene expression

2 through transcriptional and posttranscriptional

3 mechanisms respectively.

4 There are also four accessory genes, vif,

5 vpr, vpu, and nef, which are involved in viral

6 replication and pathogenesis.

7 \[Slide.\]

8 The complexity of the lentivirus genome

9 has made adaptation of this virus family to a

10 vector system challenging, but a worthy goal, as

11 Dr. Verma mentioned, a major advantage to the use

12 of lentiviral vectors is that they transduce

13 non-dividing cells.

14 Interestingly, in lentiviral systems, you

15 see efficient adaption to SIN technology, or

16 self-inactivating technology, and this is in

17 contrast to what you see with gammaretroviral SIN,

18 and I will elaborate on this point later on in my

19 talk.

20 In both systems, you have the advantage of

21 integration to host chromosome potentially

22 resulting in long term gene expression of the

23 transduced cells and the progeny cells.

24 Additionally, in both systems, there are no viral

25 genes expressed in target cells.

> 78 1 Both systems have the disadvantage for

2 potential of recombination events occurring,

3 resulting in replicating virus with potential

4 pathogenicity.

5 \[Slide.\]

6 There are a number of lentiviral vector

7 systems currently under development, two that are

8 based on primary lentiviruses, such as HIV and

9 simian immunodeficiency virus, and two systems

10 based on non-primate lentiviruses, such as FIV and

11 equine infectious anemia virus.

12 \[Slide.\]

13 So, what are the safety concerns specific

14 to the use of lentiviral vectors?

15 Recombination during manufacturing may

16 generate a replication-competent lentivirus, an

17 RCL. Of course, I should mention in my talk. I am

18 primarily going to focus on the use of HIV-based

19 vectors.

20 In terms of generating an RCL, of course,

21 this is of particular concern with HIV-based

22 vectors, since HIV is a known human pathogen.

23 Additionally, since lentiviral vectors are commonly

24 pseudotyped with G glycoprotein, a VSV, a broadened

25 tropism may potentially result in increased

> 79 1 pathogenicity of an RCL.

2 Additional concerns are associated with

3 the use of HIV-based vectors in HIV-positive

4 subjects. Recombination of vector with wild-type

5 virus in HIV-positive subjects is a concern and has

6 the potential to lead to a more pathogenic

7 wild-type virus.

8 Additionally, mobilization of vector by

9 wild-type virus is a concern, and I am going to

10 touch upon this point again later on in my talk.

11 \[Slide.\]

12 In terms of recombination events, we

13 certainly have learned a lot from the

14 gammaretroviral vector field as from basic research

15 done in the gammaretroviral basic research areas.

16 It is known that homologous recombination

17 can occur when two different RNAs are packaged into

18 one virion. This is the result of reverse

19 transcriptase template switching or undergoing a

20 process of strand transfer.

21 This same mechanism has been shown to

22 occur with HIV RT, as well, in in vitro systems.

23 \[Slide.\]

24 In terms of a recombination event leading

25 to a replication-competent retrovirus, or an RCR,

> 80 1 we know that this is a safety concern from the

2 well-known study in which immune-suppressed rhesus

3 monkeys were exposed to bone marrow cells

4 transduced with a preparation of RCR-positive

5 vector.

6 In that study, 3 out of the 10 animals

7 treated developed lymphomas and died within 200

8 days. Follow-up analysis revealed that these

9 animals had sequences identified as recombinants

10 between vector and helper, and vector and

11 endogenous sequences. I should point out that in

12 the system, the investigator was using a murine

13 leukemia virus-based vector and murine cell lines

14 for production.

15 \[Slide.\]

16 So, how do we use these lessons learned

17 for the manufacturing of gammaretroviral vectors?

18 It is known that homologous recombination occurs at

19 a rate approximately 100 to 1,000-fold lower than

20 non-homologous recombination. Therefore, reduction

21 in homology between vector and helper sequences

22 will lower the likelihood of a recombination event

23 occurring.

24 I should point out that in a study by Otto

25 and co-workers, it was shown that as little as 10

> 81 1 base pairs of nucleotide identity between packaging

2 and vector sequences were sufficient to allow for

3 RCR generation.

4 \[Slide.\]

5 Additionally, splitting helper sequences

6 into more than one plasmid, for example, splitting

7 env and gag-pol open reading frames, is likely to

8 decrease the incidence of RCR generation by

9 increasing the number of recombination events

10 necessary to generate an RCR.

11 \[Slide.\]

12 Vector mobilization. This is an

13 additional concern with the use of lentiviral

14 vectors in HIV-positive subjects. Mobilization

15 occurs when a vector genome is packaged by a

16 wild-type HIV present in the same cell.

17 Mobilization occurs by the same mechanisms

18 that allow for helper sequences to package vector

19 genomes.

20 \[Slide.\]

21 So, there are potential advantages and

22 disadvantages to vector mobilization. Mobilization

23 of a vector designed to inhibit or prevent HIV

24 replication or pathogenesis has been argued to

25 enhance the therapeutic effect by allowing for

> 82 1 spread of the therapeutic transgene.

2 In terms of disadvantages, vector spread

3 beyond the intended target tissue may have safety

4 consequences. Additionally, co-packaging of

5 wild-type HIV RNA and vector RNA may result in

6 recombination.

7 \[Slide.\]

8 How to address these safety concerns. I

9 list here four approaches - vector design, safety

10 testing during manufacturing, preclinical safety

11 studies, and clinical monitoring.

12 In terms of vector design, one can

13 incorporate features intended to decrease the

14 likelihood of recombination and mobilization, and

15 again, lentiviral vectors benefited from the

16 beginning from lessons learned from gammaretroviral

17 vectors.

18 \[Slide.\]

19 I very briefly want to highlight some of

20 the features in what has been called first, second,

21 and third generation vectors with, of course, the

22 caveat that these definitions may be outdated in

23 the future.

24 Very brief, as an example of producing a

25 first-generation vector, one might perform

> 83 1 transient transfection of three plasmids. Again

2 the packaging plasmid would contain all HIV viral

3 genes except for env. The envelope plasmid

4 contains VSV-G for broadened tropism or your

5 vector, and in the case of a HIV-based vector, the

6 HIV transfer vector, would contain the gene or cDNA

7 of interest and the minimal cis-acting elements of

8 HIV.

9 \[Slide.\]

10 Just a few of the highlights of

11 first-generation vectors include limited homology

12 between vector and helper sequences, separation of

13 helper plasmids. Again, these two are benefited

14 from the use of gammaretroviral vectors.

15 Additionally, in first-generation vectors, we see

16 the retention of all the accessory genes in the

17 packaging plasmid, which is in contrast to

18 second-generation vectors where we see the

19 elimination of accessory genes from the packaging

20 plasmid.

21 \[Slide.\]

22 Interestingly, this seems to have no

23 effect on vector titer. These vectors still retain

24 the property of transduction of many dividing and

25 non-dividing cells, and it could be argued that

> 84 1 there is an increased safety margin with these

2 vectors since there is fewer wild-type HIV genes

3 involved in the manufacturing process.

4 \[Slide.\]

5 Third-generation vectors certainly have a

6 number of interesting features, but I just want to

7 describe in detail the use of a self-inactivating,

8 or SIN, vector.

9 This involves a deletion in the enhancer

10 region of the 3-prime U3 of the long terminal

11 repeat. During the process of reverse

12 transcription, this 3-prime deletion is transferred

13 to the 5-prime LTR and results in a

14 transcriptionally inactive vector that cannot be

15 converted into a full length RNA in the target

16 cell.

17 We also see a reduced likelihood of RCL

18 generation and SIN seems to hamper mobilization by

19 wild-type HIV.

20 Additionally, the use of SINs may reduce

21 the risk of tumorigenesis via promoter insertion.

22 \[Slide.\]

23 There is certainly many other

24 developments, and I just wanted to give a brief

25 outline, but other developments include the use of

> 85 1 a four-plasmid system in which one would split

2 helper sequences into three separate plasmids. As

3 an example, rev can be split on a separate plasmid,

4 or gag-pol coding regions can be split in two

5 separate plasmids.

6 There has been development of stable

7 packaging cell lines based on third-generation

8 technology, and there has also been development of

9 non-HIV-based vectors, such as the EIAV, SIV, and

10 FIV, which are not known human pathogens.

11 \[Slide.\]

12 So, even with the incorporation of all

13 these safety features, one cannot reduce the risk

14 of a recombination event occurring to zero, and

15 therefore, it is important to have appropriate and

16 sensitive assays in place that will detect a

17 recombination event during the manufacturing

18 process.

19 \[Slide.\]

20 It is certainly going to be very important

21 to have an assay in place that will detect an RCL.

22 RCL assays are typically done by an infectivity

23 type assay which would involve several passages on

24 a permissive cell line or cell lines. Then, one

25 can perform endpoint assay for viral or transgene

> 86 1 sequences by a PCR-based assay.

2 The use of a positive control might be

3 problematic since the generation of a

4 replication-competent VSV-G pseudotype lentiviral

5 vector may not be desirable.

6 \[Slide.\]

7 One can also detect for helper sequences,

8 and this could be done by functional assay. The

9 transfer assay is an assay that has been used in

10 the HIV field for a number of years. This assay

11 tests for the generation of a recombinant that

12 expresses a functional tat protein. The assay

13 relies on the ability of tat to transactivate an

14 LTR reporter gene construct in the target cell.

15 In the absence of tat or in the absence of

16 a tat recombinant, no LTR-driven reporter gene

17 expressed in the SIN.

18 One can also test for recombination

19 intermediates, and we are fortunate to have Dr.

20 Kappes in to talk about this concept in the

21 afternoon session.

22 \[Slide.\]

23 Certainly, one can directly test for

24 helper sequences in a vector production lot or in

25 transduced cells by a PCR-based assay. While this

> 87 1 can be a very sensitive assay, perhaps it is not

2 the most biologically relevant assay to perform

3 especially in terms of detecting an RCL.

4 Perhaps this assay would have usefulness

5 for VSV-G detection when you are treating

6 HIV-positive subjects with an HIV-based vector

7 since transfer of the VSV-G gene into a

8 HIV-positive subject is highly unwanted.

9 \[Slide.\]

10 Finally, in terms of addressing safety

11 concerns, I wanted to briefly outline how one might

12 go about performing preclinical safety studies and

13 clinical monitoring. I mostly want to emphasize

14 some concerns especially with the use of HIV-based

15 vectors in HIV-positive subjects.

16 A lot of these concerns are going to be

17 addressed to the Committee in the form of

18 questions, both in this afternoon\'s session and in

19 tomorrow\'s session.

20 \[Slide.\]

21 In terms of the use of animal models to

22 assess safety, studies to assess mobilization and

23 recombination with wild-type HIV are difficult.

24 This has been learned in the HIV vaccine field.

25 It is difficult to find an animal model

> 88 1 that can examine the replication and pathogenicity

2 of HIV. In terms of non-human primates, it is

3 known that HIV replicates, but is non-pathogenic in

4 chimpanzees. Perhaps the macaque model might be

5 appropriate for SIV-based vectors. Unfortunately,

6 the murine model is very limited due to the fact

7 that HIV does not replicate in murine cells.

8 Along the same lines, a SCID mouse model

9 will also be limited, perhaps can serve as a \"in

10 vivo test tube,\" but any replication of your vector

11 seen will still be limited to the human cells that

12 are added in.

13 \[Slide.\]

14 In terms of clinical monitoring, it

15 certainly will be important to have an assay in

16 place to detect for RCL in gene transfer

17 recipients, and this is analogous to current

18 recommendations with gammaretroviral vectors. How

19 best to perform this assay in an HIV-positive

20 subject is a question. There is certainly a number

21 of ways one can go about this.

22 Additional concerns again are in terms of

23 recombination events of your HIV-based vector with

24 wild-type HIV. It is difficult to predict the

25 outcome of this recombination event and therefore

> 89 1 consideration should be given to have an

2 appropriate assay in place.

3 Likewise, one might want to assay for

4 changes in wild-type HIV following administration

5 of a lentiviral vector. For example, if your

6 vector was targeting a specific HIV gene, one might

7 want to assay.

8 \[Slide.\]

9 So, in conclusion, recombination during

10 manufacturing is a safety concern, one that perhaps

11 can be adequately addressed through incorporating

12 safety features in the design of your vector.

13 Additionally, it will be important to have

14 appropriate and sensitive assays in place to

15 monitor for recombination events occurring during

16 the manufacturing process.

17 In terms of recombination of vector with

18 wild-type virus in each of the positive subjects,

19 it is worth considering having appropriate assays

20 in place to monitor for recombination events in

21 subjects.

22 \[Slide.\]

23 In terms of mobilization by wild-type

24 virus, certainly a lot could be shown through in

25 vitro assays as to the potential for vector to be

> 90 1 mobilized. Unfortunately, preclinical animal

2 models will be difficult and perhaps consideration

3 should be given to having appropriate assays in

4 place to perform clinical monitoring.

5 I will end there.

6 DR. SALOMON: Thank you, Dr. Takefman.

7 \[Applause.\]

8 Questions & Answers

9 DR. SALOMON: So, this discussion is

10 obviously now kind of beginning the FDA staff\'s

11 leading us towards some questions that we are going

12 to discuss this afternoon, but we already began

13 some of this discussion of safety issues, and I

14 encourage some discussion now. After that, we will

15 take a break, so just to give you kind of an idea

16 how the morning will flow.

17 One question that I have, again, it may

18 not be totally answerable right now, is we keep

19 talking about the VSV-G protein, and that seems at

20 the moment, I think partly through the first

21 generation of vectors to use this, but is that a

22 safe envelope to be using? Is that an issue that

23 we ought to be dealing with at some point as a

24 direct safety issue?

25 The molecule itself is toxic, right, when

> 91 1 it is expressed, if it\'s expressed at high levels,

2 it can even kill the target cells? We don\'t know

3 its receptor.

4 Certainly, in vivo we understand that it

5 is targeting, at least brush borders an intestinal

6 epithelium, but the question is, if injected, if

7 it\'s present and injected in cells, so in terms of

8 in vivo gene therapy, we really have no idea

9 whether it is even functional.

10 DR. TAKEFMAN: Those are good questions.

11 I would welcome the Committee\'s opinions.

12 Certainly, in my mind, a major issue is potential

13 transfer of VSV-G gene to an HIV-positive subject

14 and resulting recombinant.

15 DR. SALOMON: I guess the point that I was

16 making here, though, is given how little we know

17 about VSV-G\'s function, some of its features would

18 certainly make one think that it was a major safety

19 concern in the sense that it can be toxic.

20 On the other hand, given that it is

21 unclear to me at least, and again I defer to an

22 expert audience here, about what its function would

23 be in vivo. If it has no or little function in

24 vivo, then, its expression on an HIV particle would

25 be pretty meaningless from a safety point of view.

> 92 1 I guess these unknowns bother me in the

2 context of the safety discussion.

3 Dr. Sausville.

4 DR. SAUSVILLE: I was going to say, on the

5 other hand, though, there are certain features of

6 it that could actually be construed as quite

7 beneficial. I mean this field has had a problem

8 with efficiency of transduction in many of its

9 aspects, so to me, the question really comes, I

10 mean as was alluded to, there is a marked problem

11 with recombination with HIV.

12 We may have to consider different safety

13 issues in a non-HIV infected population, because I

14 think the potential safety ramifications are quite

15 different actually, and you might reach different

16 conclusions.

17 DR. CHAMPLIN: Of course, the non-HIV

18 population can become HIV-positive two days after

19 the gene therapy is administered, so one has to

20 think of these things and then think of the truly

21 rare event if one in a million event occurs once to

22 develop a highly pathogenic virus, that can

23 obviously have major public health implications.

24 The preclinical studies in primates, has

25 there been much experience there in terms of

> 93 1 looking at safety and stability in animals?

2 DR. SALOMON: Dr. Verma.

3 DR. VERMA: I think there is some

4 experiments, but very little. I think Dalcone is

5 doing some stuff. It is relatively recent. There

6 is not very much known, but nothing untoward that I

7 know of at the moment.

8 Regarding the VSV-G, it is not that it is

9 not a human pathogen, there have been outbreaks of

10 VSV infection. It is a cattle wild normally. I

11 know it because when I was post doc in David

12 Baltimore\'s lab, we worked on VSV, and we mouse

13 popped everything in those days. We don\'t do that,

14 but it\'s fact we used to do that.

15 So, it is known to be human pathogen, but

16 it is something in terms of toxicity, there is I

17 think enough data on it, it is a just a matter of

18 somebody to mine it, because there have been

19 periodic epidemics of it, of VSV.

20 DR. CHAMPLIN: If you put VSV and SIV,

21 would it have any increased pathogenicity in the

22 monkey?

23 DR. VERMA: I think the argument really

24 there is the testing of it. There is no reason why

25 there should be any VSV gene that should come

> 94 1 through in the mouse, there is no reason for that.

2 So, I think that is a moot point really.

3 DR. SALOMON: I guess the point I was

4 making was I know we are tending to take this

5 default that everything that we raise as a safety

6 issue means it will make it less safe, and I was

7 actually raising the point that it could cut both

8 ways.

9 If you could demonstrate that VSV-G,

10 having cut past the mucosal surface, which is its

11 natural target, as you just pointed out, from the

12 known zoonotic disease and from its disease in the

13 cattle, if you could demonstrate that it had very

14 little, if any, targeting effect when released into

15 the circulation, you could then use it to say even

16 if our strategy allowed a VSV-G recombination, it

17 would have little\--I mean we are making this

18 assumption that oh, my gosh, if VSV-G got onto the

19 lentiviral vector, we would suddenly have this

20 horrible new pathogen.

21 I am okay with that concept, but where is

22 the data for it?

23 DR. KINGSMAN: I am Sue Kingsman. There

24 is some evidence that VSV-G is quite pretty rapidly

25 inactivated by human complement, which may be a

> 95 1 point in its favor.

2 DR. VERMA: I think the whole work on the

3 VSV, the Moloney vector VSF-G, there is

4 considerable experience on that, that could be used

5 as relevant experience in this case, in terms of at

6 least the G toxicity.

7 DR. MULLIGAN: I would separate the

8 toxicity from the mobilization question, and we

9 will get into that, I am sure, so I think any

10 mobilization context where you could pick up a gal

11 VM or any other envelope, I think would be a real

12 issue. That is a real safety issue.

13 I would view the gene no more dangerous

14 and safe than other kinds of envelopes. One

15 argument is that G is toxic and therefore you would

16 maybe be better with that because you will kill the

17 cell effect, and not propagate it, but work with

18 the packaging cells, suggest even at a low level

19 where you don\'t have toxicity, you can get virus

20 particles that are infectious. So, it suggests

21 there is a potential for a level G that gives you

22 infection without having F.

23 DR. NOGUCHI: The discussion is superb and

24 we really appreciate it, but there are just a

25 couple of cautions I will continue to try to put

> 96 1 out. One is, for example, the fact that there

2 should be no VSV gene in the final product is, of

3 course, what we expect, but what we expect is not

4 what we always get.

5 It is sort of going beyond what the data

6 are or have been generated. We need to be able to

7 consider some of the further ramifications of what

8 could happen, so even if there is no data, that

9 doesn\'t necessarily mean we discard the concern.

10 Regarding human complement inactivation of

11 the VSV envelope, the same argument had been made

12 for murine retroviruses, as a matter of fact. You

13 may inhibit or you may deactivate a certain number

14 of viruses, but as many virologists have told us,

15 well, it just takes one to get an infection, and

16 you may have 10 7

clearance, but if you are putting

17 in 10 9, it then becomes a moot

point as to whether

18 or not it is inactivated by complement to whatever

19 extent.

20 So, as you are going along, there are

21 certain data-driven declarations that can be made,

22 there are certain speculative things that will be

23 made, but the fact of the matter is in all these

24 things, there really are no advantages or

25 disadvantages, it is just the best we can do at the

> 97 1 time.

2 DR. SALOMON: Dr. Allan.

3 DR. ALLAN: One of the points you made

4 was, you know, if you got a recombination event and

5 you had VSV-G on either HIV or SIV, and maybe

6 that\'s not any worse than anything else.

7 I graduated from vet school too long ago

8 to remember how VSV is limited in terms of its

9 infection. I mean if you get a limited infection,

10 does that necessitate that it is VSV-G or not, is

11 it the immune response, is the immune response to

12 VSV-G highly protective, in other words, it limits

13 the infection soon after, or is it at a level of

14 the cell tropism? I don\'t know that in terms of

15 how that particular virus replicates.

16 But I think those are some of the

17 questions you can ask, too, in terms of if you did

18 get a recombinational event and you get VSV-G

19 expressed with HIV, would it replicate less well

20 than an HIV wild-type virus because of the immune

21 response to the envelope, so that would be

22 something that I would ask, and I don\'t know the

23 answer to that.

24 DR. MULLIGAN: I think that is a great

25 question. The tropism issue is obviously much more

> 98 1 than the envelope protein, and I guess, again, I

2 think when we move to the mobilization issues, the

3 issues are can you protect the biological effects

4 of a recombinant HIV that has some different

5 characteristics, and I think that would be a grave

6 concern about whether or not you could possibly

7 model what would be the tropism characteristics of

8 something.

9 I read somewhere in one of the voluminous

10 IND packages or heard a comment that, well, you

11 can\'t make anything worse than HIV, you know, the

12 worse that could happen is you will get back what

13 you already have, the patients, I think that is

14 very ridiculous and I think the issue with

15 mobilization is definitely whether or not, not only

16 are you picking up VSV-G, but you are putting it

17 into something, let\'s say, that has codon-optimized

18 gag-pol sequences or has something, or has non-HIV

19 long terminal repeats, and all of those elements

20 would give you a very good chance of different

21 tropism characteristics.

22 DR. VERMA: But the VSV biology, by

23 itself, is really well understood. It\'s a negative

24 stranded virus which replicates in the cytoplasm.

25 So, there is considerable biochemistry and

> 99 1 molecular biology of the gene, if not in terms of

2 now introducing directly what it will do in terms

3 of genicity, that is less known, but the biology of

4 the virus itself is very well established.

5 DR. ALLAN: So, do you know its cell

6 tropism?

7 DR. VERMA: In fact, it\'s all cell types,

8 very broad cell type, but initially, the infection

9 in the mucosa initially, a lot of intestinal

10 infection.

11 DR. SALOMON: But the one correction,

12 again, if I am wrong, please correct me, but the

13 statement it affects all cell types is largely

14 based on in vitro cell culture infections, not

15 specifically on in vivo infections.

16 DR. VERMA: Right. In vivo, the only data

17 that I know is really largely in the case of

18 cattle, because that is really the VSV-G is a

19 cattle virus, is largely the infection of the

20 mucosa in the intestine.

21 DR. SAUSVILLE: But if you were to

22 parenterally introduce it beyond the sanctuary, you

23 would expect replication, correct?

24 DR. VERMA: By itself, I don\'t know the

25 answer.

> 100 1 DR. SALOMON: I don\'t think we know the

2 answer to that question. That was the question I

3 was asking.

4 I guess all I am trying to do here is play

5 maybe a devil\'s advocate, but the question is that

6 what we are doing here is taking all these

7 different elements from different viruses and there

8 is very appropriate rationales, we want tropism, we

9 want higher efficiency of integration, et cetera,

10 and I guess I am just asking the questions of what

11 are the sorts of if we now want to go from

12 pioneering molecular studies to clinical trials, I

13 think the job of the Committee is to try and help

14 identify those issues that we should be\--you know,

15 there are certain experiments you might not do as

16 an academician trying to develop a new area, that

17 are now critical to go back and do if we are going

18 to go forward safely in a clinical trial, if we can

19 identify those things, that would be a big

20 advantage I think.

21 DR. DELPH: I just wanted to ask whether

22 there were any different or additional safety

23 concerns between someone who were HIV-positive and

24 given HIV gene vector therapy as against someone

25 who were given HIV gene vector therapy and then

> 101 1 seroconverted, and if so, how should those be

2 addressed.

3 DR. TAKEFMAN: So, you are trying to say

4 what if an HIV\--oh, I see\--so, you gave them the

5 gene transfer vector and then they acquired

6 wild-type. I am not too sure how to answer that

7 one.

8 DR. VERMA: That would be no different

9 than if the vector doesn\'t mobilize, it should be

10 the same reason as the one who has the other way

11 around.

12 DR. TAKEFMAN: Yes, it should make no

13 difference.

14 DR. ALLAN: I don\'t really think that that

15 will ever happen, because I don\'t know that you

16 will be doing gene therapy on infected populations

17 is all I would think, at least with HIV.

18 DR. VERMA: Well, I think that\'s not fair.

19 DR. SAUSVILLE: That depends on the nature

20 of the therapeutic intent. I mean I could

21 certainly imagine issues where\--I mean we saw

22 examples of potentially replacing a defective gene

23 that, you know, might have anything to do with HIV,

24 but if the nuts and bolts of getting it there were

25 HIV derived, and that subject were to become

> 102 1 infected with HIV, then, that is an issue.

2 DR. DELPH: I think one of the reasons I

3 asked this question is that very early in HIV

4 infection, you probably get the highest HIV viral

5 loads that you see at any other time in the

6 disease, and I don\'t know that we really even know

7 the details of exactly how that differs from and

8 the consequences of that.

9 DR. SALZMAN: Rachel Salzman from the Stop

10 ALD Foundation. I just want to comment that we

11 definitely are interested in using lentiviral

12 vectors in patients that don\'t have HIV, and that

13 is why we attend these kind of meetings, to be

14 concerned that it is a safe vector and that it can

15 be useful.

16 I also do happen to be a veterinarian and

17 from my experience with VSV and learning about

18 zoonotic diseases, there is a population of people,

19 maybe not so much in the United States, but maybe

20 more in Third World countries that have antibodies

21 to VSV, have been exposed to it, their cattle get

22 VSV, and they probably also have AIDS in the same

23 population.

24 So, there is sort of like kind of this in

25 vivo real model, and I think that they haven\'t been

> 103 1 optimized and we are just dealing with natural

2 infections, but there are places in the world where

3 animals have VSV, humans have VSV, and humans are

4 HIV-positive, and I don\'t know if that can be used

5 or not.

6 DR. SAUSVILLE: My comment on that is, you

7 know, one recognizes that exists, but to me it

8 seems like that is a different situation than where

9 one consciously restitches the hardwaring, as it

10 were, so that the part of one is now intimately

11 related to the part of the other, so I am not sure

12 that that natural history would necessarily be

13 relevant to a new construct base.

14 DR. SALOMON: That is very interesting. I

15 would comment that from our experience with dealing

16 in xenotransplantation, one of the interesting

17 things was a comment like that in one of our

18 advisory committees led to a worldwide study of

19 patients who had been exposed to pig tissues in

20 this case, looking for porcine endogenous

21 retrovirus, and that came up with the very

22 surprising group of several hundred patients in the

23 former Soviet Union that had gotten pig spleen

24 perfusion.

25 So, it wouldn\'t be crazy for someone to go

> 104 1 to these countries and see whether or not you could

2 track a group of HIV-positive patients who got

3 VSV-G. I mean to the extent I have no idea what

4 the incidence of the zoonosis is in that

5 population.

6 DR. SALZMAN: It\'s fairly high, in some

7 places, it is fairly high. I know as

8 veterinarians, they teach us sort of not to worry

9 about getting VSV no matter what other diseases we

10 may or may not have.

11 DR. ALLAN: That also comes back to the

12 point of cell tropism, and even though the VSV-G

13 has a wide tropism, I don\'t know whether or not

14 regulation of expression is limited to epithelial

15 cells or other cell types, or whether if you took

16 an intact VSV, whether it would replicate in

17 lymphocytes or not. I don\'t know that answer, so

18 the question is whether or not you get both viruses

19 in the same cell. I think that is something that I

20 am sure it is in the literature, so it is just a

21 question of looking.

22 DR. SALOMON: I would say one of the

23 interesting questions that just came up this

24 morning, and I would like to return to this

25 afternoon, is this sort of conflict of are

> 105 1 lentiviral vectors more appropriate to test in HIV

2 patients or in non-HIV patients, and I think there

3 are strong feelings on both sides.

4 I mean some people clearly feel that there

5 is a safety issue and it should be tested in HIV

6 patients, and others, I think who feel equally that

7 there is a safety issue and it shouldn\'t be tested

8 in HIV patients.

9 So, I think that will be an interesting

10 discussion to enter in this afternoon. Certainly

11 if it fits into the thread of any of our

12 conversations this morning, I would encourage you

13 to bring it up.

14 Well, if there is no driving thing, I

15 think this a great time for a break, 15 minutes.

16 \[Break.\]

17 DR. SALOMON: Now, the next part of the

18 session will be two presentations from Cell

19 Genesys. The first will be presented by Dr. Gabor

20 Veres on LentiKat Vectors Overview.

21 LentiKat Vectors Overview

22 Dr. Gabor Veres

23 DR. VERES: Thank you very much for the

24 opportunity to present some of the data that we

25 generated with lentiviral vectors at Cell Genesys.

> 106 1 You will see that obviously, some of my

2 presentation and even some of the slides will be

3 kind of redundant after the two excellent

4 introduction by Dr. Verma and the FDA

5 representative, but I hope that I can provide you

6 some additional data particularly related to

7 testing strategies and even some data which might

8 be helpful to have a discussion how we want to go

9 forward and what assay sensitivities we have to

10 achieve for the different applications.

11 \[Slide.\]

12 Just to go back a little back, as you all

13 know by now, we are using HIV as a basis for the

14 vector system, and the work was all done in Dr.

15 Verma\'s lab by Luigi Naldini originally to generate

16 these vector systems.

17 So, contrary to the fairly broadly used

18 murine retroviruses, HIV is a fairly complex

19 retrovirus, so on top of the basic structure of

20 proteins gag, pol, and envelope, we are dealing

21 with the regulatory proteins rev and tat and quite

22 a few accessory genes which are very important for

23 the in vivo infectivity and the in vivo

24 pathogenesis.

25 \[Slide.\]

> 107 1 So, what is the advantage of the Lenti

2 over the existing retrosystem concerning all the

3 issues and probably safety concerns, why won\'t we

4 use a lentiviral system? It is clearly the

5 transduction efficiency is substantially better

6 than the existing retrovectors. In particular, it

7 transduces non-dividing cells, which is retroviral

8 vectors murine, retroviral vectors are not really

9 capable of.

10 It can provide the same long-term

11 expression than the other retroviral system. It is

12 also highly efficient for in vivo delivery

13 particularly when it is pseudotyped with VSV

14 envelope, and to make a qualifying statement here,

15 as far as we know, the majority of the population

16 doesn\'t have preexisting antibody against VSV

17 envelope or the major HIV proteins.

18 The recent progress which has been

19 reported from several places are the improved

20 biosafety, which means that we try to minimize the

21 HIV sequence in a vector, development of stable

22 producer lines, which is also reported from

23 multiple labs, and a couple of places has now

24 large-scale production capability and also some

25 level of purification.

> 108 1 \[Slide.\]

2 Our lentiviral system originally is from a

3 parental HIV isolate LN4-3, and there was a

4 significant departure from the original

5 organization of the virus. We split the HIV genome

6 into four components which I will show you in the

7 next slide, and we tried to minimize the HIV

8 sequence on the vector, so the transfer vector

9 itself has approximately 10 percent of the HIV

10 sequences, removed all the accessory genes, which I

11 mentioned is important for in vivo infectivity and

12 probably plays a substantial role in pathogenicity,

13 so nef, vif, vpu, or vpr are all removed and also

14 the system doesn\'t require tat for efficient

15 transcription.

16 We split the rev on a separate construct

17 and this provided in trans \[?\] to regulate the

18 gag-pol gene expression, and finally, the LTR is

19 deleted, so this is a so-called self-inactivating

20 vector.

21 \[Slide.\]

22 Dr. Verma showed almost the same slide.

23 Again, third generation, put it this way, this is

24 the current vector, what we are using, so these are

25 the four components which consist of the third

> 109 1 generation vector, the helper plasmid codon for the

2 gag-pol.

3 The rev is on a separate construct and

4 this is absolutely essential to provide high level

5 of gag-pol expression. VSV-G is a heterologous

6 envelope, and then the transfer vector with

7 appropriate promoter and the transgene of interest.

8 \[Slide.\]

9 So, again, this is a summary of the kind

10 of evolution of the vector system. The very first

11 generation had all the HIV sequences except the

12 envelope, and that was pseudotyped with VSV

13 envelope.

14 The second generation has the minimal

15 packaging construct, but all the accessory genes

16 were deleted, and the gag sequence was minimized on

17 the transfer vector to prevent potential homologous

18 recombination.

19 Finally, the last generation vector is

20 tat-less. It has the same phenotype and certain

21 other modification has been made to further reduce

22 the overhead between the helper construct and the

23 vector construct.

24 \[Slide.\]

25 As it also has been shown, one of the

> 110 1 major advantage of the vector system, that it can

2 transduce non-dividing cells. I think one of the

3 primary interest in several laboratories to use it

4 for hematopoietic stem cell transduction.

5 Retroviral vectors would transduce

6 unstimulated CD34 cells very, very poorly. This

7 example shows immobilized peripheral blood CD34

8 cell population, which was transduced overnight

9 without any cytokine stimulation with lentiviral

10 vector construct expressing GFP, and if you look at

11 the total 34 population, there is a substantial

12 high level of the cell population is transfused,

13 and even the subset, which sometimes it is claimed

14 that it represents the more primitive subset of the

15 CD34 cells, even that cell population has a

16 substantially high transduction rate.

17 \[Slide.\]

18 For in vivo application, obviously, the

19 central nervous system is a very obvious target.

20 In this experiment, we used the SIN vector

21 expressing the luciferase gene under the CMV

22 promoter, and the vector was 10

8 infectious unit in

23 a volume of 100 microliter.

24 This vector was injected into the

25 vertebrae, somewhere here, into the mice, so it\'s

> 111 1 interatrial injection, and you see a luciferase

2 expression in the spinal cord and also in the

3 brain.

4 This is our imaging system from xenogene

5 is being used. The substrate is injected IP, and

6 about 30 minutes later, the mice are imaged with

7 the CCB camera, and you can see a very high

8 expression in different parts of the central

9 nervous system. The expression is fairly stable,

10 and these animals are still alive, and we haven\'t

11 seen any particular adverse effect.

12 \[Slide.\]

13 The other major target if you think about

14 in vivo delivery of the vector system, is the

15 liver. Again, luciferase vector was injected in

16 this case directly through the portal vein, and as

17 you see, there is a fairly stable expression and

18 long-term expression in these animals, and the

19 expression is confined pretty much into the liver

20 of these animals.

21 \[Slide.\]

22 Obviously, in this case, one would want to

23 look at the potential toxicity of the vector. We

24 repeated these experiments at different vector

25 doses up to 109 infectious particles per animal,

> 112 1 and we look at one of the most characteristic liver

2 enzymes, and independent of the route of delivery,

3 so either it was injected through the portal vein

4 or into the tail vein, PBS control, we haven\'t seen

5 any substantial elevation of the major liver

6 enzymes.

7 This vector does in this case represent

8 approximately to 2 x 10

8 infectious units.

9 \[Slide.\]

10 The biodistribution is something which is

11 probably related to what we discussed previously in

12 the VSV. Depending on the route of delivery of the

13 vector, if it is delivered directly through the

14 portal vein, as you can see the great majority of

15 the vector ends up in the liver.

16 This is a DNA real time PCR analysis of

17 the animal after approximately 30 days, so the

18 majority is in the liver, but quite substantial

19 part of the vector is actually transfusing the

20 spleen.

21 In case of the tail vein delivery, the

22 vector distributed approximately 50 between liver

23 and the spleen, and one can see a trace level of

24 the vector, integrated vector DNA in a couple of

25 tissues, lymph nodes in particular, and a little

> 113 1 bit in the lung.

2 \[Slide.\]

3 The production of the vector obviously is

4 an important issue. One is clearly a safety

5 concern, the other issue from a more practical

6 point, how much vector one can make transiently or

7 in the stable producer cell line.

8 Currently, for most of the application we

9 use a transient production system. The explanation

10 for this, we are still testing different vector

11 construct, so establishing a producer cell line for

12 each of the vectors is very time-consuming, but

13 obviously, a final vector will be put in the

14 packaging cell and that system will be also tested.

15 But for convenience sake and also for

16 certain application, the transient production might

17 be quite suitable. So, we co-transfect the four

18 plasmids at a certain ratio into 293 cells using

19 so-called cell factory, and after three days, we

20 collect the supernatant treated with benzonase to

21 remove the DNA, and the vector undergoes a

22 purification step, which provides us also

23 concentration, but also removal of the cellular

24 protein, protein from the tissue culture media and

25 also substantial portion of the plasmid DNA.

> 114 1 \[Slide.\]

2 Obviously, one of the major issues is

3 biosafety concerning these vectors. The vector

4 system, what we are using obviously is

5 replication-defective, and since we removed almost

6 everything from the vector, as I said, only 10

7 percent of the HIV sequence is still present, we

8 are not going to transfer any viral genes into the

9 target cells.

10 The vector is pseudotyped with an

11 unrelated envelope, so wild-type virus cannot be

12 generated, but, of course, someone cannot exclude

13 the possibility that a non-homologous recombination

14 happened and this heterologous envelope might be

15 incorporated into the vector.

16 The safety concerns are the generation of

17 replication-competent virus, insertion of

18 mutagenesis into the chromosome, and in the case of

19 population which already has HIV, remobilized the

20 vector.

21 \[Slide.\]

22 With this vector system, we tried to look

23 what would be required to generate the

24 replication-competent virus. Based on the

25 characteristic of the system, we believe that at

> 115 1 least four steps are required to restore a fully

2 functional virus.

3 One is to restore a functional LTR, which

4 would mean that the sequence should be acquired

5 from the chromosome nearby, which has the promoter

6 and enhancer potential to be able to generate the

7 full length transcript from the LTR.

8 Then, a homologous recombination

9 potentially could happen between the vector

10 sequence and the helper plasmid coding for the

11 gag-pol. Fortunately, this wouldn\'t be still

12 sufficient because that construct still would

13 require rev to generate the gag-pol sequence, and

14 the rev has no overlay with this part of the vector

15 construct, so that has to be an non-homologous

16 recombination or a plain insertion, and then this

17 construct has to acquire an envelope from the cell

18 to generate a fully functional

19 replication-competent virus.

20 \[Slide.\]

21 So, this is the schematic. If you look at

22 the vector design, what we are currently using,

23 there is an overlap, which is really a few base

24 pair between the 5-prime and the helper gag

25 construct and the vector. This is needed for the

> 116 1 efficient packaging of the vector.

2 There is a direct overlap in the center of

3 polypurine tract, which again is an important

4 component of the vector system for efficient

5 transduction and also for efficient vector

6 generation.

7 There is no direct overlap between the RRE

8 sequence because we use it from HIV-2, and this is

9 an HIV-1 RRE sequence, so this has only a 60

10 percent homology. The rev, as I mentioned, has no

11 overlap with the vector whatsoever, and the same is

12 true for the envelope construct.

13 \[Slide.\]

14 So, what are the criterias when we think

15 about developing an RCL assay? You have to

16 appreciate that we are trying to develop an assay

17 for a potential vector which actually doesn\'t

18 exist.

19 It has been pointed out that under the

20 best circumstances, the optimal control would be an

21 HIV which carries a fully functional VSV envelope,

22 and that could have been a positive control.

23 Clearly, we have no wish to generate this and use

24 it in an assay what we are developing, so what are

25 the alternatives?

> 117 1 A pseudotyped vector, which would be

2 sufficient to control the initial infection, but

3 then if you do further amplification, that vector

4 wouldn\'t be amplified further on because it has no

5 envelope.

6 We can use attenuated HIV. Attenuated HIV

7 is lacking all the accessory genes, but otherwise,

8 has all the function, envelope, gag-rev and

9 gag-pol. This would be limited to cells which

10 could be infected with HIV.

11 Finally, we can use an HIV pseudotyped

12 with the VSV-G envelope, which is probably the

13 closest one to the real life situation.

14 The amplification system, it should be a

15 cell line which is highly susceptible to HIV

16 infection, so we need very few particles to start

17 the initial infection, and that infection could be

18 amplified with the further passage of the cells.

19 Finally, what is the endpoint? I mean the

20 most obvious one is to follow p24 production

21 because that is a fairly well established assay to

22 detect the progression of HIV infection.

23 \[Slide.\]

24 So, what we are using as a positive

25 control, as I mentioned, HIV without the accessory

> 118 1 genes, it has a full envelope, and generally

2 particles where we add also the VSV envelope in

3 trans, so actually, this generates a chimeric

4 envelope for the first round of infection.

5 We look at this, the wild-type HIV in

6 different cell lines to see which is really

7 susceptible for the infection of either this

8 control construct wild-type HIV or just the plain

9 attenuated HIV which doesn\'t have accessory genes.

10 If you look at the numbers here how many

11 the TCID 50 needed to

establish infection, you should

12 appreciate that this is required on primary human

13 CD4 cells to start HIV replication. Actually, with

14 this construct using the C8166 cells, we could

15 lower the threshold, so we are at the range of one

16 TCID50 to start efficient HIV replication.

17 C8166 is a lymphoblastoid cell line which

18 is available from ATCC commercially.

19 \[Slide.\]

20 So, the test system could look the

21 following. We put the viral stock or the producer

22 cell line on the detector cells, so in this case

23 it\'s infection or co-cultured with the producer

24 cells, detector cells, the C8166, then, passaged

25 five times to further amplify a potential RCL in

> 119 1 the supernatant, so if there is one

2 replication-competent particle in the system at

3 the beginning, that should be greatly amplified at

4 the end, and the endpoint is p24 ELISA.

5 \[Slide.\]

6 We started to test the system to establish

7 the sensitivity, so in this table we summarize one

8 of the experiments that we conducted lately using

9 the attenuated HIV which, as I said, was

10 pseudotyped also with VSV-G for the first round of

11 infection.

12 We know that the physical to infectious

13 particle in this particular preparation was

14 approximately 100. So, if you look, we put a

15 different number of particles in the system, 1,600,

16 160, 16, and, of course, zero, and what you can see

17 here that we could detect approximately 100

18 particles throughout the infection.

19 What this represents here, that obviously,

20 in this case, that is probably one viral particle

21 which started the initial infection, in this

22 scenario, probably more than one particle was in

23 the system. So, that is why we estimated, it is

24 probably one TCID

50, what started the infection.

25 We are going to do further analysis in

> 120 1 this range to completely establish the final

2 sensitivity.

3 \[Slide.\]

4 In summary, the detector cells,

5 T-lymphoblastoid C8166 cells, the score

6 replication-competent recombinants, detects

7 recombinants also with the heterologous envelope,

8 and we can measure 10 fg p24 of attenuated HIV-1.

9 When we spiked this positive control into

10 production lot, currently, we have a detection

11 limit of 1 TCID

50 in 100 ng of p24, which represents

12 approximately 1.2 x 10

9 physical viral particles.

13 \[Slide.\]

14 So, what we would suggest at least for an

15 ex vivo application as a testing strategy is the

16 following. In the vector production, and whether

17 that is transient or a producer cell, we know that

18 we can achieve even currently approximately 8 x 10

12

19 viral particle as a total, and we can test 5

20 percent of that.

21 That corresponds to the requirement that

22 is currently being used for retroviral testing.

23 That means that we would test 5 x 10

11 physical

24 viral particle. On the other hand, for ex vivo

25 cell therapy, which Dr. Ando will give you the

121

1 details of the protocol, we estimated we would use

2 approximately 4 x 10

7 cells as a starting material.

3 If we transduce this with the MOI of 5,

4 that represents 6 x 10

10 viral particles, and we

5 suggest to test 1 percent of these transduced cells

6 as a final product, which is approximately 4 x 10

5\.

7 So, if you look at these numbers, testing

8 5 percent of the final production lot, it would

9 mean that we are testing 8-fold of the clinical

10 dose which one would use in a clinical protocol.

11 So, even if the detection limit is not

12 just a single particle, but let\'s say a particle

13 between 1 to 5, using the multiple of a single

14 clinical dose would allow us to detect the

15 replication-competent viral particles in this

16 scenario.

17 Finally, I would like to acknowledge my

18 co-workers and also people who contributed

19 previously to all of this work, in particular

20 Luigi, Dr. Verma, Didier Trono, Anatoly Bukovsky,

21 and my current co-workers who work on both vector

22 construction, packaging line construction, some of

23 the in vivo studies, large-scale production and

24 purification, and RCL assay in particular.

25 Thank you very much.

> 122 1 \[Applause.\]

2 DR. SALOMON: The second part of this will

3 be by Dr. Dale Ando, Cell Genesys, on Lentiviral

4 Gene Therapy.

5 Lentiviral Gene Therapy

6 Dr. Dale Ando

7 \[Slide.\]

8 DR. ANDO: What I wanted to emphasize was

9 that actually none of us have worked in lentivirus

10 in the clinic, but a lot of us have worked on

11 retrovirus for about the last 10 years, and I think

12 a lot of the clinical systems and regulations In

13 terms of testing and evaluating patients, I think

14 we can benefit a lot from the previous decade with

15 respect to that, in the same way that the

16 construction of the vector has benefited from the

17 previous experience.

18 Again, I think a lot of us who have worked

19 in this area are familiar with some of the common

20 themes of germline transmission, insertional

21 mutagenesis, and the strategies for testing the

22 virus in manufacturing and in the clinical trial

23 subjects.

24 \[Slide.\]

25 With respect to the unique lentiviral

> 123 1 clinical issues, there are several, and what we

2 would like to do in our first approach is to really

3 focus on the issue of replication competent and

4 lentivirus and recombination.

5 This has been approached as we have seen

6 with respect to the design of the vectors to

7 minimize that, and then, which we will get into a

8 little bit more, is the testing strategy, but the

9 idea here is that we would like to test with an

10 assay that we feel can give us limit detection of

11 hopefully at least one particle, completely the

12 vector, and then a portion of the ex vivo product.

13 Obviously, we can\'t test the complete

14 product prior to infusion in the patient, but there

15 may be a way actually in what are called

16 qualification lots or practice lots prior to the

17 study to really evaluate whether or not you have

18 RCL in a total ex vivo product.

19 So, there are strategies of trying to

20 approach this, so that we can get some data to see

21 whether or not our systems are working. So, for

22 this particular application, we are sort of not

23 addressing the issue of mobilization because we are

24 using a SIN vector and going into a situation of

25 patients who do not have HIV.

> 124 1 \[Slide.\]

2 After sort of extensive looking and

3 discussing with several investigators and

4 internally, we feel like we would like to move

5 ahead in adenosine deaminase deficiency, and I

6 would like to go through some of the rationales for

7 that.

8 Again, this is a proposal, we haven\'t

9 finalized this. I know there are a lot of

10 limitations in addition to benefits of this

11 indication.

12 As you know, this is a severe combined

13 immunodeficiency with a fairly marked loss of T, B

14 and NK cell function, high mortality without

15 treatment, and 20 percent of the cases are related

16 to a specific genetic deficiency in adenosine

17 deaminase gene.

18 This has been defined genetically in 1972,

19 the gene has been cloned, and actually, there is

20 enzyme replacement therapy available. There has

21 been a gene therapy trial, and actually one of the

22 first gene therapy trials in genetic disease

23 occurred in 1990.

24 So, again, there has been a lot of

25 clinical experience in gene transfer in this area.

> 125 1 \[Slide.\]

2 Wide ranges of ADA expression levels are

3 tolerated and modest levels are needed for

4 replacement. Again, for the eventual efficacy,

5 there is a selective advantage for ADA expressing

6 cells in patients, and to be able to functionally

7 and clinically benefit the patient, the selective

8 advantage is very important.

9 The other important factor is that enzyme

10 replacement therapy is available, so we are not

11 basically limiting the patient with respect to any

12 maximal clinical benefit, that can come later, so

13 it really allows a stepwise evaluation of this

14 setting with the first step being safety and

15 understanding gene transfer in the periphery,

16 because there a lot of preclinical studies you can

17 do to see whether or not a particular gene

18 transduction and marrow culture procedure works,

19 but you never really know actually until you get

20 into the clinic.

21 So, again, the studies first will be

22 safety in gene transfer and then if we can achieve

23 an adequate level of gene expansion, then, the

24 PEG-ADA can then be actually decreased in the

25 second portion and the efficacy and T cell

> 126 1 immunologic endpoints can be pursued.

2 So, in some cases, to us, it represents a

3 \"best case scenario\" for the general area of gene

4 therapy targeting hematopoietic stem cells.

5 \[Slide.\]

6 Three clinical trials of Moloney

7 retroviral gene transfer to hematopoietic stem

8 cells in bone marrow and cord blood have been done.

9 Actually, that number may be five, and there are

10 some unpublished reports of possibly two patients

11 who have been successfully reconstituted using

12 Moloney vector.

13 Frequency, however, of gene corrected

14 cells in most of these studies was very low and

15 little evidence of gene expression.

16 So, really, the efficacy that may have

17 been seen in those two patients probably depends

18 mostly on the fact that there is a selective

19 advantage of the T cells, so that is key.

20 What would turn this area actually into a

21 fairly uniform or fairly efficacious clinical trial

22 would be to get good levels of gene transfer. That

23 is the real key I think to moving this ahead and

24 then to moving it into other areas of stem cell

25 therapy.

> 127 1 \[Slide.\]

2 Again, the rationale, I think it has been

3 described fairly clearly in what Dr. Verma and Dr.

4 Veres have shown in terms of the hematopoietic

5 progenitor cells, and we still have some work to do

6 with respect to figuring out a minimal gene

7 transfer system between hematopoietic stem cells to

8 preserve function and increase transduction

9 efficiency.

10 Again, the key question is whether there

11 would be greater benefit with increased levels of

12 gene transfer in the study.

13 If we can achieve low levels of gene

14 transfer, even on the level of 1 percent, this most

15 likely will result in fairly significant clinical

16 benefit in the setting of ADA deficiency and help

17 us in the future in development of in vivo

18 methodologies for human stem cell therapy, gene

19 therapy.

20 \[Slide.\]

21 So, the proposed trial\'s evaluation of

22 safety and administration of autologous CD34 cells

23 transduced with a lentiviral vector carrying a

24 normal human ADA cDNA in children with ADA

25 deficiency and SCID.

> 128 1 We are using an investigator who is very

2 experienced in this area, Don Kohn, and his group

3 at Children\'s Hospital in L.A. The objectives are

4 the standard clinical and laboratory safety, and

5 gene delivery to hematopoietic cells and gene

6 expression.

7 The patients will be infants and children

8 with ADA-deficient SCID, less than 1 percent ADA

9 enzyme activity in peripheral blood, laboratory

10 documentation of impaired T and B cell functions,

11 and subjects basically who are not eligible for

12 HLA-matched sibling transplants, and again negative

13 for HIV.

14 \[Slide.\]

15 The basic trial will be screening to

16 determine eligibility, and this is actually a

17 fairly complex process at Children\'s Hospital.

18 Treatment, to remind you, includes taking

19 bone marrow out from the patient, isolation of the

20 cells, and then a manufacturing process at the site

21 with transduction of CD34 cells and infusion.

22 So, really, there are two manufacturing

23 processes, the manufacture of the lentivirus at the

24 company and then the gene transduction at the site.

25 Then, the observation period, looking at

> 129 1 safety, gene expression, immune function, RCL

2 testing, and then long-term follow up.

3 In general, these patients are followed

4 fairly closely by these types of specialists

5 throughout their life.

6 \[Slide.\]

7 The lentiviral manufacturing, as we have

8 previously discussed, will be the transient viral

9 production using DNA transfection in 293 cells, and

10 replication-competent lentiviral testing and

11 release. So, basically, we feel that our current

12 paradigm will allow us to test and have less than

13 one copy per lot in a lot that is probably 8-fold

14 higher than the clinical release.

15 So, we would feel fairly confident that we

16 have the best sensitivity achieved in the viral

17 testing. Then, this virus will be released, then

18 used in the clinical site for transduction of the

19 CD34 cells, at which point we will be testing 1

20 percent of the cells.

21 \[Slide.\]

22 So, in summary, we are planning on using

23 our latest generation ADA SIN lentiviral vector.

24 This is a 4-plasmid system without accessory genes.

25 We will transduce CD34 cells with ADA lentivector

> 130 1 infused patients.

2 RCL testing, as we have noted, in the

3 virus will be extremely complete, and 1 percent of

4 the cell product, and clinical evaluation to follow

5 up the patient according to current guidelines for

6 retrovirus.

7 Thanks.

8 \[Applause.\]

9 DR. SALOMON: That was excellent.

10 Again, I just want to remind everyone that

11 questions to these sponsors are very appropriate,

12 but they are not here today to tell you that they

13 are getting ready tomorrow to do a gene therapy

14 trial, so I think we need to just temper the kinds

15 of questions that we ask.

16 Questions & Answers

17 DR. SALOMON: One of the issues that I

18 think is kind of coming here as a theme that I want

19 to raise just for discussion, clearly from a

20 scientific point of view, a strategy that everyone

21 in the field seems to be using is designing their

22 different plasmid vectors with reduced homology to

23 prevent these potential events of homologous

24 recombination, RT strand transfer, et cetera.

25 That is very molecularly appropriate, yet,

> 131 1 the weakness, it seems to me, is that if as little

2 as 10 to 25 base pair homologies are adequate for

3 homologous recombination, and certainly in the work

4 we are doing with DNA arrays and things very

5 accurate, and then when you say there is 60 percent

6 homology in basically 100 or more base pair

7 crossover, et cetera, it raises a question.

8 So, the fallback position seems to be,

9 well, you know what, if we do it and can\'t

10 demonstrate replication-competent lentivirus, then,

11 what\'s the problem.

12 So, the question I have is can we have

13 some discussion about the concept of how much do

14 you have to prove in terms of all this homology or,

15 in the end, is that really just a good way to start

16 and it\'s all based on proving replication-competent

17 lentivirus doesn\'t exist.

18 DR. ANDO: My comment on that would be

19 that if you look at what happened in the Moloney

20 retrovirus, we went through a number of generations

21 of the Moloney, but really the mainstay, and this

22 took several years and actually Richard was

23 involved with this, was really coming up a very

24 sensitive mouse study co-culture assay, of which we

25 based our release specifications.

132

1 Actually, there are a lot of different

2 cell lines, so the tack that we are taking now is I

3 guess parallel to that. We have designed

4 scientifically a very nice system, minimized

5 homology, but the real key now is to get unit one

6 viral particle sensitivity and be able to test that

7 one viral particle in a signal-to-noise ratio in

8 something that would be a clinical lot.

9 We are producing 40 liter scale or 14

10 liter scale 1 x 10

11 virus. We would like to

11 detect, be able to have a sensitivity to detect one

12 viral particle in that, and that has been a

13 paradigm that has been safe at least in the Moloney

14 area. At least for us, that is a starting point

15 for discussion.

16 DR. KAPPES: I would like to follow up on

17 your comment. I think it is, for me at least, one 18 of the central
issues, but I would like to raise

19 the question, maybe perhaps to a more defined level 20 as it relates
to at least what I understood that

21 you said, and that is whether or not any system, no 22 matter how
sensitive it is, that we use in vitro

23 for detecting RCL is really an adequate predictor 24 of the outcome
of treatment.

25 I could discuss this more, but I think

> 133 1 perhaps I will wait until after my presentation

2 today, because this is the very issue which I will

3 try to address.

4 DR. SALOMON: Dr. High.

5 DR. HIGH: I have a question related to

6 your comment. In your lot release criteria, do you

7 look at contaminating plasmid DNA or mammalian cell

8 DNA in the vector, because, you know, I guess

9 benzonase digestion can\'t really get to plasmid

10 that may be sort of stuck near the capsid, this

11 sort of thing?

12 DR. VERES: No. I mean the purification

13 procedure, we really look at the residual protein

14 residue of DNA, also PCR specific to 293 cells, so

15 actually, it is a quite complex assay event, so it

16 is not just a crude DNA\'s digest, but actually we

17 look at the final product, and I think some

18 specifications are there defining how much DNA is

19 really allowed in a certain product.

20 DR. MULLIGAN: One of the issues that we

21 may get into at some point is whether any

22 packaging-derived sequences are shown to transfer,

23 and obviously, there is assays for

24 replication-competent retroviruses, assays for tat

25 function, other things, but in principle, there is

> 134 1 assays for any HIV-derived sequences being

2 transferred, if you have ever looked or developed

3 an assay where you would simply, for instance, with

4 PCR, move all the way down, gag involved with

5 little primer sets, and asked the question do you

6 detect any transfer of HIV-derived sequence in

7 recipient cell.

8 DR. VERES: So far we haven\'t done any of

9 this, and I think it would be very, very difficult

10 to perform these assays because I mean we would

11 have to assay for multiple components both for rev

12 and also gag-pol, and all of the different\--helper

13 constructs are slightly different, I mean not

14 everybody is using the same helper constructs, so

15 we can detect the conserve \[?\] sequence, for

16 example, in the gag, but that would be only just

17 one part.

18 DR. MULLIGAN: I missed why that would be

19 difficult. If you just take your helper construct

20 and you ask the question whether any of those

21 sequences transfer to recipient cells, why would

22 that be difficult?

23 DR. VERES: I mean we can do that. I mean

24 the question is where are we going to draw the

25 line, what is the minimal sequence we are looking

> 135 1 for, are we looking for the whole gag or just part

2 of the whole gag.

3 DR. MULLIGAN: That is a different issue.

4 I just raise it as a question that may surface in

5 terms of is there any difference between our

6 concerns about MLV versus HIV in terms of the

7 notion of transferring any HIV sequences.

8 Obviously, you can adopt the case that,

9 well, what difference does that make if you don\'t

10 have a coding sequence, but I am not aware that

11 anyone has really done this in the past, certainly

12 not in the MLV case, but I think it might be a very

13 revealing activity.

14 The second question was just my eyesight

15 isn\'t actually so hot, but the biodistributions you

16 showed and the one I looked at, it looked like

17 there is just a tiny, tiny little bar graph bar in

18 the testes, but I couldn\'t tell whether that was

19 the last thing.

20 Was there any detectable, after the I.V.

21 or the I.P. inspection, any detectable signal in

22 testes?

23 DR. VERES: Yes, there is DNA in the

24 testes, but as Dr. Verma showed, I mean it is

25 nothing\--I mean we didn\'t look explicitly which

> 136 1 part of the testes, just took the testes and there

2 is a detectable level by PCR.

3 DR. SALOMON: Dr. Allan.

4 DR. ALLAN: I wanted to follow up on his

5 question, which is you are primarily looking at

6 whether you get replication-competent recombinants

7 and also whether you get like tags, pressures, and

8 these other things.

9 In the case where the patient could be

10 exposed at some point to HIV, which is one of the

11 issues for tomorrow, so even if you transferred a

12 small portion of a gag or pol, or whatever, into

13 the patient, even though it is not

14 replication-competent, and then you come back in

15 with a wild-type HIV, it could rescue partial genes

16 from that patient, and so the issue then is, well,

17 wild-type is worse than\--and we will get into that

18 tomorrow\--but that possibility still exists, is

19 that even though you don\'t get

20 replication-competent virus, you may be

21 transferring pieces of genes to the patient, isn\'t

22 that correct?

23 DR. VERES: As I said, we don\'t have any

24 data showing that we would transfer the gag-pol

25 sequence. Obviously, this is something we can look

> 137 1 in the final product, but I am not aware of any

2 data that would really happen, but I cannot exclude

3 it either.

4 DR. ALLAN: You are not doing PCR for gag

5 and pol in your product, are you?

6 DR. VERES: No, currently, we haven\'t done

7 any experiment addressing this.

8 DR. ALLAN: I have another question that

9 is more general. The SIN vector with the LTR that

10 has basically taken on all promoters and enhancers,

11 do you think you can get recombination in the

12 portion that integrates with wild-type HIV?

13 DR. VERMA: I think it is a general

14 question. You asking the question of

15 recombination. I think you just have to look at

16 the numbers. If you are asking is there a chance

17 that there could be 1 percent, zero percent, I

18 can\'t tell you if there is a zero percent chance,

19 but you have to look at the effect.

20 True, there are 10 nucleotide which

21 overlap, but look at the number of recombination

22 events that must occur in order for it to become a

23 viable particle. It has to have the six genes

24 which are gone, it has to have parts of the LTR, it

25 has to have parts of the gag, it has to have part

> 138 1 of the envelope.

2 It is not that it is impossible to imagine

3 that can happen one day, but a priori, if you look

4 at it, there are many, many levels of recombination

5 to occur before you can get such a molecule.

6 So, I agree with the general comments here

7 that the more you assay for it, so there is no

8 reason why one cannot check gag and pol in the

9 final product, yes, it is a perfectly doable thing

10 to reduce the chances, because the fact of the

11 matter is, it is not a MLV. If it was MLV, we will

12 be less concerned it is HIV, so you want to make

13 sure that you go the extra distance.

14 So, I agree with you, it should be done,

15 more assay, but the probability just by experience

16 of recombination is very low.

17 DR. SALOMON: I think that is a really

18 important point, and I think to kind of focus what

19 I was asking is, at least my impression now, and

20 again, you know, it is up for discussion, is that

21 from the FDA\'s point of view, going on to the first

22 clinical trials and think about regulation, it

23 seems to me from everything I have heard, that the

24 type, the definition, the sensitivity, and the

25 confidence we have in the RCL assays is going to go

> 139 1 way beyond any of these theoretical discussions of,

2 you know, we degenerated that and we took out this,

3 and we self-inactivated the 3-prime LTR, et cetera.

4 If that is true, then, from a safety point

5 of view, a lot of these, you know, very high-level

6 academic discussions of the molecular biology

7 probably ought to be put aside because it is not

8 going to get us to the most important thing. The

9 most important thing is going to be to focus on

10 what is the best RCL assay, what is the attributes

11 of the ideal RCL assay meaning specificity and

12 sensitivity.

13 Now, that is my premise, and that is

14 certainly open for disagreement or discussion.

15 DR. DELPH: I may be way off target here

16 because I really don\'t know enough about molecular

17 biology, but it seems to me that there are two

18 different questions.

19 On the one hand, can you reconstitute the

20 virus from which you deleted all of these various

21 genes, and on the other hand, if you have already

22 replication-competent HIV present, can that

23 integrate some of these genes, and what is the

24 probability of that latter aspect happening?

25 DR. SALOMON: Just to put that in the

> 140 1 context of my comments, I agree, and so that is a

2 separate issue, in other words, but very important

3 one. I am asking just should we be focusing on the

4 design and integrity of the RCL assay, and now you

5 have added the next point, and that is, if there is

6 also wild-type HIV, what is the additional risk,

7 and then can we model that.

8 Dr. Naldini and then Dr. Sausville.

9 DR. NALDINI: I would like to point out

10 that we do have information, part information in

11 terms of those issues that you are raising, that

12 went into the validation of those generation of the

13 system. This information was acquired by tests

14 made at the experimental level. They may not

15 necessarily have been translated into standard

16 tests to be used as release criteria, but we do

17 have information, for instance, that the level of

18 residual packaging, packaging RNA in the producer

19 cell, which is an important risk factor for

20 recombination, because, of course, recombination

21 not only requires some knowledge, but also requires

22 that two different RNA are packaged.

23 We have data showing that early generation

24 system allows a certain level of residual packaging

25 which was lost when we went into the advanced

> 141 1 generation system in which we sort of clean the

2 packaging system.

3 A second type of data is looking for

4 transfer of packaging function like gag and pol,

5 which are expressed, so now are functional to

6 produce particle which would require an envelope to

7 be infectious.

8 We have looked at that and we have been

9 able to find evidence of that again in early

10 generation system, and not detectable one to

11 certain level of sensitivity in later generation

12 system. All of these data are available in terms

13 of validating the safety of the system. Whether

14 they would be required for release criteria, I

15 think is a matter really for discussion.

16 DR. SAUSVILLE: I have a question that

17 again may be off base, but I mean it gets to the

18 philosophy of all this. I have no doubt that we

19 can establish a criteria for a release assay, that

20 we will feel confident will yield a low probability

21 of an adverse event, such as the generation or a

22 recombination of either a new virus or an HIV

23 virus.

24 This is where I turn to our FDA colleagues

25 in terms of guidance. This new type of vector

> 142 1 clearly is different than other vectors that have

2 been conceived in many cases for gene therapy.

3 Does the Agency have a position with

4 respect to what the toleration is, because if there

5 is any possibility of recombination, then, in a

6 certain sense, a lot of this discussion becomes

7 moot if it renders something as a problem.

8 So, speak to us on this matter.

9 DR. NOGUCHI: Specifically, for

10 recombination, I don\'t think that is limited to

11 lentiviral vectors. That obviously could happen at

12 very many levels in production and in vivo with

13 other retroviral vectors, even with some of the

14 \"non-integrating,\" and we could even envision it

15 for adenovirus as an example.

16 DR. SAUSVILLE: But you do agree that the

17 pathogenic risks intrinsic or as a result of that,

18 seem to be somewhat different compared to the

19 current circumstance?

20 DR. NOGUCHI: Well, there is certainly

21 that potential, which is the reason we bring it to

22 discussion, but I would say that we do not have a

23 position set in stone as to something absolutely

24 not.

25 We have taken that position actually only

> 143 1 in the case of cloning a human being, where we said

2 we have jurisdiction and no, you can\'t do it.

3 Short of that, we are really looking for the very

4 best advice that we can get.

5 We are looking for advice on are these

6 safety concerns being addressed in an adequate

7 fashion, if not, what more is needed, are the

8 patient populations the appropriate one for this

9 point of development, or if not, what are the other

10 indications.

11 But in terms of an absolute yes/no on this

12 particular question of recombination, no, we don\'t

13 have a set position.

14 DR. VANIN: Elio \[ph\] Vanin from St Jude.

15 People keep on quoting there is 10 base

16 pairs, and I think that that comes from Adolto\'s

17 \[ph\] paper, and we have to remember what that was.

18 That was a recombination, that was packaging of two

19 different RNA species into a retrovirus, that then

20 got transferred and recombined to make an RCR. So,

21 that is basically two transcripts and one

22 retrovirus, because that came from a producer line,

23 so it wasn\'t DNA recombination.

24 The other thing is, the way the lentiviral

25 vectors are made, you have to package four

> 144 1 different constructs into the same particle and

2 then you have to have the recombination to give you

3 an RCL, and I think we have to remember that.

4 DR. CHAMPLIN: I think you should be

5 commended for the ability to get a positive control

6 for the RCL assay, but I have some questions about

7 that.

8 The data shows that this virus, the HIV-A

9 virus, the SDVG suicide, has a lower PCID 50, maybe

10 100 to 1000 times lower, for certain cell lines.

11 But if you were to then spike your final lentiviral

12 product with this HIV AIDS pseudotype virus, can

13 you still detect it as well or does then it be

14 competed out by the other viruses? Or, more

15 worrisome is that HIV can inhibit other HIVs. Have

16 you looked at that?

17 DR. VERES: Yes. That is a very good

18 question. Actually, we are testing the sensitivity

19 of the system. That is why I had one sentence down

20 there that currently we are able to detect this one

21 PCID50 of the background of 10

9 physical particles.

22 We are doing additional experiments to address what

23 is really the limit of this assay and how

24 reproducible this is. That is the goal of

25 continuing this RCL assay.

> 145 1 DR. CHAMPLIN: I had a question on the in

2 vivo tropism of the virus, your biodistribution

3 studies in the mouse suggested a high level liver

4 uptake, but is that in hematopoietic cells or in

5 liver parenchymal cells, is it the spleen and the

6 liver were the only two organs with substantial

7 uptake?

8 DR. VERES: We didn\'t look, but Dr. Verma

9 looked I think in the liver, it\'s both hepatocytes

10 and sinusoidals as transfused, those are Kupffer

11 cells to a certain extent.

12 DR. SALOMON: Changing the subject just a

13 little bit, in your trial, you know, just rough

14 bones proposal, you do the CD34 purification and

15 the transduction, so I guess one of the things that

16 comes out then is you didn\'t specify, do you freeze

17 the CD34 cells before you do your testing for RCL?

18 I know you have thought about it

19 obviously, so how would you do this in terms of

20 product lot test release?

21 DR. ALDO: That is something we haven\'t

22 defined. I think there is some controversy now as

23 to whether or not you can freeze these cells and

24 maintain the stem cells viable and gene transduce,

25 and that is actually a major issue to be resolved

> 146 1 hopefully in some of these animal models, but if we

2 are going to test, obviously, these tests take four

3 to six weeks, can\'t immediately infuse.

4 You picked up on that, but that will be a

5 major issue with this, with any bone marrow Lenti

6 protocol, and it will be very different from T cell

7 protocols, for example, where you can freeze, and

8 it is fairly well established.

9 DR. ZAIA: I would also like to change the

10 subject. I would like you to justify your choice

11 of disease that you have chosen to \"treat.\" For

12 the first patient who is non-HIV-positive, who gets

13 an HIV vector, you have chosen a very

14 immunosuppressed patient.

15 You could argue that it would be better to

16 choose an immunocompetent patient because if there

17 were a problem, there may be less of a problem in

18 that patient, so that it may be a Fanconi\'s anemia

19 or a hemophiliac, a different patient and a lesser

20 of a problem to confront.

21 Have you thought about that?

22 DR. ALDO: We have actually looked at a

23 lot of different genetic diseases. Every one has

24 some problems or issues. Hemophilia is I don\'t

25 think much simpler. The real positives on this

> 147 1 indication are the fact that there is some effort

2 for the last 10 years on this, and these patients

3 are getting PEG-ADA, so in that sense, their

4 immunodeficiency, although not completely treated,

5 is at least partially treated.

6 We looked at some of the other SCIDs and

7 say, for example, you may have to do neonates, et

8 cetera, and the other SCIDs do not have any

9 alternative therapy other than, say,

10 transplantation.

11 There are other biochemical defects that

12 you can look at, but how clearly stem cell transfer

13 would work in those particular diseases isn\'t as

14 clear, because you have to get something secreted

15 by cells to other tissues and reverse storage

16 problems or specific biochemical defects in certain

17 tissues.

18 So, given the overall balance, I agree

19 this is not perfect, but we thought this would be a

20 good place to start. I am sure there will be a lot

21 of discussions between ourselves and in public

22 concerning this choice.

23 DR. SALOMON: It is an interesting issue,

24 immunosuppression and HIV, and it kind of gets back

25 to a question you brought up, and that is the

> 148 1 differences between de novo infection and

2 established infection.

3 So, in the one piece of one group of data

4 points that has gotten to be quite interesting in

5 organ transplantation has been given successful

6 heart therapy, the HIV community has come to the

7 transplant community and said, you know, we now

8 have long-term lives and we deserve transplanted

9 organs. So, it has become a big issue in the last

10 couple of years. There is an NIH study group now

11 specifically looking at what effects

12 immunosuppression in HIV transplantation has.

13 One of the things that has come out from

14 the history is that if you take patients who have

15 HIV and get an organ transplant and then are fully

16 immunosuppressed, there is really, interestingly

17 enough, little data suggesting that you enhance or

18 increase the progression of the natural HIV disease

19 in that patient group.

20 That would be an argument that

21 immunosuppression might not be such a critical

22 issue. There are, however, just to put it in the

23 other light, evidence from a small group of

24 patients who got HIV from the transplanted organ

25 and then were fully immunosuppressed, and there,

> 149 1 there was a really dramatic proliferation of the

2 virus that exceeded the norm, a very rapid,

3 compressed clinical course that led to death in a

4 couple of patients in a couple months, very small

5 numbers of patients, though.

6 This is by no means I am suggesting, you

7 know, established fact. It is an interesting area,

8 and there will be some more data coming out

9 hopefully from the trials getting set up.

10 Dr. Kappes.

11 DR. KAPPES: Your choice of targets is

12 also interesting and I think poses special concerns

13 for recombination. That is, if you do have

14 recombination, and that recombinant is integrated

15 into the pluripotent stem cell, you certain face a

16 situation where you will amplify the presence of

17 that recombinant.

18 What considerations have you given for

19 this, but with respect perhaps to safety, with

20 respect to monitoring, any comments?

21 DR. ALDO: Really, just what we have seen

22 there in terms of the testing of the 1 percent, but

23 I think prior to this, we are planning to undergo a

24 fairly extensive in vitro and possibly some of

25 these SCID-reconstituted animals to look at these

> 150 1 issues.

2 Hopefully, if there is something that

3 comes up there, we should be able to see it. In

4 the end, reducing the probability of this is

5 difficult. I would say the validation of full

6 clinical lots and showing that we didn\'t see this

7 kind of recombination would be probably the best

8 paradigm that I could think of right now.

9 I don\'t know if that answers your

10 question.

11 DR. MULLIGAN: Could you guys weigh in on

12 the stable packaging cell transient transfection?

13 When the first speaker spoke, I thought the

14 implication was that we are using transient system

15 because it is very easy to use, but\--and I forget

16 what the last part of that sentence was\--but it was

17 something to the effect, I thought, that, you know,

18 of course, we would move to the stable packaging

19 cells and we have that technology in-house, but

20 then the presentation here was that you would go

21 for the transient transfection.

22 So, what is your philosophical point of

23 view about the differences between the two systems?

24 DR. VERES: The philosophy is that the

25 transient transfection system is fairly well

151

1 established and we are capable of producing up to

2 14, 16 liter of material with a particle number of

3 well over 10 10 and

1011\.

4 We do have a third generation packaging

5 cell line which we actually just started to

6 evaluate, for example, one of the issues, what you

7 raised with the SIN vector, can we establish a

8 clone which will have a high enough titer that it

9 is really practical to use, what are the production

10 criterias, how long we can generate viral

11 supernatants.

12 I think we hope that in the next three to

13 six months, we can have answers to some of the

14 questions and we can make enough material both from

15 the transient system and the stable system, and we

16 can put them into this RCL assay in different test

17 systems to really establish the safety, and based

18 on that, we can make decision which one we would

19 move to the clinic.

20 For application like the ADA where the

21 number of patients are fairly limited, the

22 transient production obviously is a possibility

23 because we can easily make enough materials. For

24 other applications, for example, systemic delivery,

25 for example, hemophilia, which probably would

> 152 1 require a much higher particle number, I don\'t

2 think that a transient production system would be

3 suitable.

4 DR. MULLIGAN: So, in your hands, have you

5 found difficulty making very high titer, stable

6 producer cells with SIN constructs?

7 DR. VERES: As I said, we are testing it

8 currently, so I don\'t really have hard data, and I

9 really cannot comment right now. It is a couple of

10 more months before I can answer this.

11 DR. SALOMON: That was great. Again, I

12 want to say thank you from all of us on the

13 Committee for your willingness to step up at a

14 preliminary point in your work and share it with

15 us.

16 The last talk of this morning, certainly

17 not the least and no priority implied, is from Dr.

18 Kordower, on Lentivirally Delivered GDNF for

19 Parkinson\'s Disease.

20 It is nice to welcome Dr. Kordower back.

21 He was a valued colleague in the deliberations on

22 neural stem cells about a year or so ago. It is

23 nice to have you back.

24 Lentivirally Delivered GDNF for Parkinson\'s Disease

25 Dr. Jeffrey Kordower

> 153 1 DR. KORDOWER: Thank you. It is nice to

2 be back. I like gene therapy more than I like stem

3 cells and for my types of applications, closer to

4 the clinic.

5 What I am going to talk to you about today

6 is all preclinical work using gene therapy in

7 animal models of Parkinson\'s disease.

8 \[Slide.\]

9 Parkinson\'s disease, unlike many other

10 neurodegenerative disorders, has a face, a face for

11 America, in fact, multiple faces, and Muhammad Ali

12 and Janet Reno and Michael J. Fox, and the Pope all

13 have Parkinson\'s disease, as you are all well

14 aware, although what I am going to talk to you

15 about today really doesn\'t apply to any of these

16 individuals, because these individuals all have

17 advanced Parkinson\'s disease, and what I want to

18 talk to you about today is using gene therapy which

19 most likely will be most efficacious in patients

20 with early Parkinson\'s disease, because these

21 patients, because they have advanced Parkinson\'s

22 disease, their nigrostriatal degeneration is very

23 advanced and there are few nigra neurons left in

24 the midbrain and little dopamine left in the

25 striatum.

> 154 1 \[Slide.\]

2 So, these patients require different types

3 of strategies, strategies such as neuronal

4 replacement, which can be accomplished with fetal

5 neurons, possibly stem cells, and a variety of

6 different types of stem cells can be used in this

7 type of application.

8 \[Slide.\]

9 The type of gene therapy that I am

10 interested in, the mechanism is both

11 neuroprotection and neuroregeneration, and that

12 requires having some residual nigrostriatal system

13 left for your compound to work on. Now, the way in

14 which I want to try and neuroprotect and regenerate

15 the nigrostriatal system is with the use of trophic

16 factors.

17 Now, what I am going to do is spend the

18 first part of my talk, talking about why gene

19 therapy is needed for the delivery of trophic

20 factors, and then the second and third parts of my

21 talk showing why lentiviral delivery is a very

22 potent and promising way to deliver trophic factors

23 to the parkinsonian brain.

24 The trophic factor I am going to talk

25 about exclusively today is GDNF or glial

> 155 1 cell-derived neurotrophic factor, although it

2 should be noted that there are other trophic

3 factors, such as BDNF and other gene therapy

4 approaches, such as transfecting cells to make

5 certain enzymes that make dopaminergic drugs work

6 better are also in the experimental stage.

7 \[Slide.\]

8 Now, when I mention trophic factors for

9 neurologic diseases, if there are any neurologists

10 in the room, they usually start to roll their eyes

11 at this point, because they say here is another

12 basic scientist with his trophic factor and he has

13 given us NGF for Alzheimer\'s disease, BDNF for ALS,

14 CNTF for ALS, GDNF for Parkinson\'s disease, et

15 cetera, et cetera, and the one thing you can say

16 about all these clinical trials, they have all been

17 failures.

18 Now, have they been failures because the

19 preclinical state doesn\'t predict clinical outcome?

20 Well, if that is true, we have a lot of problems,

21 but I don\'t think that is true.

22 What has happened in all of these clinical

23 trials, that the trophic factor has never been

24 delivered in a way in which the factor ever reached

25 the vulnerable cells that were dying in the

> 156 1 disease, so therefore, there is no reason to

2 suspect or to expect that the trophic factor should

3 have worked in these clinical trials.

4 \[Slide.\]

5 Now, let me give you a little bit of

6 background about GDNF and why we are interested in

7 using this particular trophic factor for

8 Parkinson\'s disease.

9 Lin, et al., initially discovered GDNF by

10 its ability to support the viability of midbrain

11 dopaminergic neurons in vitro, and then it was

12 subsequently found that when you give toxins to

13 these cells, which is MPP+, to dopaminergic cells,

14 GDNF also prevented degeneration caused by these

15 toxins.

16 GDNF has some effects upon normal rats,

17 but the real reason that people got very excited

18 about GDNF in Parkinson\'s disease is that no matter

19 what animal model you use, whether it\'s toxins,

20 methamphetamine, age arrest, no matter how you try

21 and destroy dopaminergic cells, GDNF will prevent

22 that degeneration, and if it is applied

23 appropriately, the animals that receive these

24 lesions will not display functional deficits and

25 will have functional benefits from the trophic

> 157 1 factor.

2 Don Gash and his colleagues then extended

3 these studied to rhesus monkey, and that led Amgen

4 to start a clinical trial which tested the safety

5 and efficacy of GDNF in patients with Parkinson\'s

6 disease.

7 \[Slide.\]

8 Rush Presbyterian Medical Center, where I

9 work, was one of the centers that participated in

10 this trial, and one of the patients came to autopsy

11 that was in this trial, came to autopsy from events

12 totally unrelated to the GDNF, but it gave us a

13 window to determine whether (a) GDNF was

14 functioning in this patient, and whether

15 anatomically, there was any evidence of

16 regeneration or neuroprotection.

17 Let me just give you a little bit of

18 information about this patient. He was a

19 65-year-old male with a long history of PD.

20 Initially, he had a good response to levodopa, and

21 that is critical because you don\'t want to give a

22 dopaminergic trophic factor if a patient doesn\'t

23 respond to dopaminergic pharmacology.

24 He initially entered into a double-blinded

25 trial, and still the blind hasn\'t been broken to

> 158 1 me, but then he entered an open-label phase in

2 which, through an Elmira reservoir, this patient

3 received monthly interventricular injections of

4 GDNF into the ventricular space in an ascending

5 dosing limit. You can see the doses here.

6 Following his final dose of 300

7 micrograms, three weeks later he died at home from

8 a heart attack, and we were able to get the brain

9 from this patient and examine both the behavior and

10 the anatomy in this patient.

11 Clinically, this patient was evaluated

12 using the UPDRS or Unified Parkinson\'s Disease

13 Rating Scale. There are two types of scores here.

14 There is a motor score and then the ADL is an

15 Activity or Daily Living Score. \"On\" means this

16 patient was tested while on levodopa, \"off,\" while

17 off levodopa.

18 The details of the scale really aren\'t

19 important right now, but all you have to realize is

20 high scores is bad, low scores are good, and what

21 you can see here at baseline to last visit is no

22 matter how it was measured, the scores continued to

23 rise, and the patient\'s parkinsonism continued to

24 worsen.

25 Not only did his parkinsonism continue to

> 159 1 worsen, but there were side effects related to the

2 interventricular injections, loss of appetite,

3 nausea, Lhermitte\'s sign, which is like an

4 electrical stimulation down the back of the neck,

5 and these are all temporally related to the

6 injection, so as soon as he got the injection,

7 these symptoms were seen.

8 Then, there were other side effects that

9 were quite serious, that weren\'t necessarily

10 temporally related to the injection, but we think

11 were involved related to GDNF infusions -

12 hallucinations, this person did not hallucinate

13 prior to the GDNF trial, inappropriate sexual

14 conduct, and depression.

15 \[Slide.\]

16 So, clinically, nothing good happened and

17 some bad things happened. When we got the brain of

18 this patient, we basically saw that the GDNF did

19 not enhance nigrostriatal function. Now, you might

20 see here in the top panel, Panel A, this is

21 tyrosine hydroxylase staining through the forebrain

22 of this patient, and there is some staining here in

23 the caudate nucleus.

24 However, down in Panel B, this is a

25 patient, Parkinson patient, that did not receive

> 160 1 GDNF, and so this is a typical finding that can be

2 seen in PD patients.

3 The critical region that must be

4 reinnervated is the putamen, this region here, and

5 you can see in this GDNF-treated patient, and in

6 Panel D at higher magnification, there is virtually

7 no dopamine in the striatum as a result of the

8 interventricular GDNF infusion.

9 \[Slide.\]

10 So, we thought, well, maybe let\'s look

11 down at where the cell bodies of origin are, the

12 substantia nigra, and basically, we found that

13 there was no effect in the substantia nigra. On

14 the left panel, you see TH staining in the normal

15 patient, the loss of cells in the middle panel of a

16 PD patient without GDNF, and the third panel, the

17 patient that did receive GDNF even had fewer

18 dopamine cells within the nigra.

19 \[Slide.\]

20 So, basically, no clinical efficacy, side

21 effects, and no evidence that there was any kind of

22 regeneration or neuroprotection in the brain.

23 Now, I just showed you a slide previously

24 where we had all this preclinical data that

25 suggested that it should work, so why didn\'t it

> 161 1 work in this patient, and, in fact, it didn\'t work

2 in the clinical trial in general?

3 \[Slide.\]

4 The reason it didn\'t work is because the

5 GDNF never got to the cells that were vulnerable in

6 Parkinson\'s disease. On the left here, we have two

7 monkeys that received, not monthly injections of

8 GDNF, but high-dose chronic injections of GDNF into

9 the lateral ventricle, and you can see here in the

10 brains that were stained for GDNF, just trivial

11 amounts of GDNF staining in the caudate nucleus and

12 here in the septum, which is an irrelevant

13 location, and the monkey, too, it all backed up

14 into the singular gyrus.

15 So, basically, the reason it didn\'t work

16 is because the GDNF was not delivered in a way in

17 which it could work, and that is why you need to

18 have gene therapy, a site-directed delivery of GDNF

19 if this is ever going to be an efficacious

20 strategy.

21 Just, if I can step back in general,

22 putting trophic factors in the ventricular system

23 in general is a very bad idea. Lots of things do

24 happen, most of them are bad. So, you want to have

25 site-specific delivery into the parenchyma and gene

> 162 1 therapy allows that.

2 \[Slide.\]

3 Well, the previous speakers did a far

4 better job than I could in describing why we want

5 to use lentivirus for gene therapy, so for time

6 sake, I am not going to go into it, but we are

7 going to use lentivirus GDNF in our animal models.

8 \[Slide.\]

9 Before we go into our monkey models using

10 GDNF, we wanted to do a quick study just to see

11 whether we get any kind of transfection in monkey

12 at all, so we did three consecutive rhesus monkeys

13 and injected with lenti beta-gal or marker gene.

14 The first two monkeys were sacrificed at a

15 month, the third at three months, and each one of

16 these little dots here represents a successfully

17 transfected cell with lenti- beta-gal.

18 \[Slide.\]

19 Look at just how many cells there are,

20 just really\--and I will give you the quantitation

21 of this in just a moment. Just look at Panel C and

22 D here. It is interesting that things change over

23 time. At one month, just the cell bodies seem to

24 be transfected or expressing the marker gene, but

25 at three months, the marker gene is now not only in

> 163 1 the cell body, but expressing both in dendrites and

2 in axons.

3 \[Slide.\]

4 Well, how many cells were transfected?

5 Let\'s just look at the striatum total. In monkey

6 1, we had 930,000 cells; monkey 2, a million 2, and

7 this was actually a transposition; monkey 3 was a

8 million 5.

9 That is a lot of cells, and to just give

10 you some kind of context, back in \'95, we published

11 the first report of a postmortem case of a fetal

12 transplant that came to autopsy, and on one side of

13 the brain we had 125,000 cells, and the other side

14 of the brain we had 85,000 cells, and we couldn\'t

15 be happier.

16 We were so excited to have so many of

17 these cells surviving and doing what we wanted them

18 to do, and here, we have an order of magnitude

19 greater in terms of the number of cells doing what

20 we wanted them to do. These are direct injections

21 into the striatum.

22 So, our first study on three consecutive

23 monkeys demonstrated very successful transfection.

24 \[Slide.\]

25 Now, what cells were transfected? Most of

> 164 1 them on neurons. The top panel on each side is the

2 beta-gal. On the left here we have NeuN, a

3 neuronal marker. On the right we have GFAP, an

4 astrocytic marker, and then the merged image where

5 the yellow shows that between 84 and 88 percent of

6 the cells that were transfected were neurons, the

7 rest were astrocytes.

8 \[Slide.\]

9 Now, we have talked a lot today about

10 safety, and safety involves not only just some of

11 the issues that were discussed, but also in vivo

12 toxicology, and I will talk a little bit more about

13 immune status and toxicology a little later in the

14 presentation, but I just want to use this slide to

15 illustrate a couple of points.

16 Here is a needle track right here, and

17 this is a blow-up of where this needle is right

18 here. This is perivascular cuffing. That was the

19 only vessel I ever saw that had perivascular

20 cuffing in any of these three monkeys, and it was

21 sitting right on top of the needle track, and you

22 can get perivascular cuffing just from putting an

23 injection in the brain.

24 Panel D, the needle track is right here,

25 and this is a NeuN stain, and I want to illustrate

> 165 1 the fact that there are many, many healthy neurons

2 right adjacent to our injection sites. So, from a

3 toxicological point of view, not only do you have

4 lots of cells being transfected, but the striatum

5 appears to be intact, and not expressing any kind

6 of toxic insult from the injections.

7 \[Slide.\]

8 So, now we are ready to go into our animal

9 models. In our initial studies, we chose what is

10 often an unusual animal model for Parkinson\'s

11 disease, and we decided to use aged monkeys. I

12 have a large colony of aged monkeys, and I define

13 an aged monkey as 22 years of age or older. Every

14 monkey year is about 3 human years, so it is about

15 66 to, in this study, 66 to 90 years of human age.

16 \[Slide.\]

17 Now, there are many reasons why it shows

18 aged monkeys as our initial step. One of them is I

19 wanted to make sure that we had somewhat of a

20 present nigrostriatal system there for the GDNF to

21 work on, but there are a number of other advantages

22 to using aged monkeys as a model of PD.

23 One is that the changes that occur in the

24 nigrostriatal system are slow and occur over

25 decades, much like it does in Parkinson\'s disease.

> 166 1 Now, many of us who do lesions write our NIH

2 grants, and routinely we get some reviewer who

3 says, \"Well, your lesions, and they occur over a

4 week or a month period of time, that doesn\'t mimic

5 what occurs in the disease state.\"

6 It is true, it is also very often a

7 trivial comment, but on the plus side, using aged

8 monkeys does allow us to have the temporal changes

9 occur in a manner that is more analogous to

10 Parkinson\'s disease.

11 The reason I chose aged monkeys, though,

12 is point 2. Aged monkeys don\'t lose nigral

13 neurons, they lose their ability to synthesize

14 dopamine in existing nigral neurons, and that is

15 one of the first things that happens in the nigra

16 of a Parkinson\'s patient.

17 A cell doesn\'t just go into an apoptotic

18 cascade and just die or become necrotic and

19 explode. One of the first things that happens, it

20 shuts down its synthesis of dopamine, and that is

21 what we can model here using aged monkeys.

22 There are a number of other interesting

23 aspects of using aged monkeys. They are all

24 progressive motor declines that are associated with

25 nigrostriatal degeneration. For other reasons, you

> 167 1 may want to study the cognitive impairment or

2 concomitant age-related problems, so there are a

3 lot of reasons to use aged monkeys.

4 There is a disadvantage. Aged monkeys do

5 not have Parkinson\'s disease, and they do not

6 respond to levodopa, so the first study I am going

7 to show you is purely anatomical, and then we will

8 switch model systems and I will show you the

9 functional and anatomical studies using a different

10 model.

11 \[Slide.\]

12 Well, we gave injections of our lenti-GDNF

13 into the caudate nucleus and the putamen and the

14 substantia nigra, and the first inkling that we

15 were on the right track came from our PET scan

16 studies. We used fluorodopa uptake, which is a

17 measure of dopaminergic terminals in these aged

18 monkeys.

19 This is one monkey at four different

20 levels preoperatively and three months

21 postoperatively. We put the injections on what is

22 your left side. You can see the caudate nucleus

23 and putamen here, and you can see the dramatic

24 increase in fluorodopa uptake in all of these

25 panels on the side of the lenti-GDNF injections.

> 168 1 \[Slide.\]

2 Right after that we sacrificed the

3 monkeys. I already showed you this slide showing

4 the lack of GDNF expression in the brain when you

5 infuse it into the ventricle. I want you to

6 compare that to what happens when you give

7 lenti-GDNF right into the striatum, and look at the

8 panel on the right.

9 \[Slide.\]

10 Don\'t worry about these holes. These were

11 punches taken for postmortem analysis. But here is

12 the caudate nucleus and here is the putamen, and we

13 can virtually cover the entire striatum with GDNF

14 expression. This is three months postoperatively.

15 Just to show you that it is not due to

16 just putting needles in the brain, when we do

17 lenti-beta-gal, and we stain for GDNF, we don\'t see

18 anything.

19 There is one other point I want to make

20 here. Cliff Saper, who is the editor and chief of

21 JCM, one of the best journals, always says you are

22 supposed to present your representative case, but

23 you have got to show your best case because if you

24 show your representative case, people will think

25 that is your best case.

> 169 1 Well, this is both our best case and our

2 representative case, and one thing I want to

3 emphasize about the data that we have collected

4 that is incredible to me is that every single

5 monkey shows virtually the same thing. We have yet

6 to have any monkey fail in having outstanding gene

7 expression, whether it be lenti-beta-gal or

8 lenti-GDNF.

9 So, all our monkeys look like this three

10 months postinjection.

11 \[Slide.\]

12 I would like to show this slide. Here is

13 the cerebral aqueduct here, and here is the

14 cerebral peduncle down here. This is one,

15 5-microliter injection of lenti-GDNF, and we can

16 cover virtually the entire hemi-midbrain with this

17 one lenti-GDNF injection.

18 \[Slide.\]

19 Not only that, the lenti-GDNF gets

20 transported throughout the basal ganglia system.

21 Here is an injection in the putamen, and this

22 staining is not from an injection, but an

23 anterograde transport of the GDNF from the putamen

24 to the globus pallidus.

25 Look how the staining respects the

> 170 1 boundaries of the globus pallidus, outlining the

2 striatal pallidal pathway, and there is also

3 staining down in the substantia nigra, pars

4 reticulata, outlining the striatal nigral pathway.

5 There is also retrograde transport of the

6 secreted GDNF following the striatal injections.

7 \[Slide.\]

8 Well, how much GDNF is actually being

9 made? This is now a different set of monkeys that

10 were sacrificed 8 months following the injection.

11 Their immunocytic chemistry was identical to what I

12 just showed you from our short-term studies, and

13 the punches we took went through GDNF ELISA.

14 This is a typo here. This should be

15 nanograms per milligram of protein. But from these

16 punches, we got 2,500 and 3,500 nanograms per

17 milligram of protein.

18 Each one of those holes I showed you was

19 about a milligram of protein, and so if you examine

20 the type and number of the staining, what I am

21 telling you here is that for a least eight months

22 postinjection, we are getting chronic microgram

23 doses of GDNF being synthesized and secreted from

24 the lenti-GDNF injections. That is an incredibly

25 high dose.

> 171 1 \[Slide.\]

2 Biologically, what happened in the aged

3 monkeys? Here is a lenti-beta-gal-treated animal.

4 Here is the low intensity of TH staining that is

5 seen in the striatum of an aged monkey, and here is

6 the site of the injection that received lenti-GDNF,

7 and I think you can appreciate the dramatic

8 increase in TH staining on the side of the

9 lenti-GDNF expression relative to the intact site.

10 \[Slide.\]

11 Both dopamine and HVA levels are

12 dramatically up following lenti-GDNF on the side of

13 the injections both in the caudate nucleus and in

14 the putamen.

15 \[Slide.\]

16 Some of our most dramatic effects were

17 actually seen back in the level of the substantia

18 nigra. Here is the nigra of an aged monkey that

19 received lenti-beta-gal treatment, and here is a

20 monkey that received lenti-GDNF treatment.

21 There are three things I am going to show

22 you on the next three slides - more cells, bigger

23 cells, more good stuff in the cells.

24 \[Slide.\]

25 In terms of more cells, let\'s just

> 172 1 concentrate on the right side here, because that is

2 the injection side. There is an 85 percent

3 increase in the number of TH-positive cells on the

4 side of the lenti-GDNF injection. That is an

5 incredible response.

6 What is interesting are the absolute

7 numbers. We have previously published that aged

8 monkeys have about 60,000 nigral neurons, and young

9 monkeys have about 120,000 nigral neurons. So,

10 basically, what we have done is made an old

11 substantia nigra into a young substantia nigra with

12 lenti-GDNF expression, and we believe this is not

13 due to any neurogenesis, but basically, all those

14 cells that downregulated their expression of

15 tyrosine hydroxylase has now been boosted up and

16 the downregulation has been prevented, so now they

17 can be counted, just what we had hoped for when we

18 designed this study in the beginning.

19 \[Slide.\]

20 The volume of each one of these nigral

21 cells is increased by 35.7 percent, and for those

22 of you who aren\'t familiar with quantitative

23 morphology, a volumetric increase of 35 percent is

24 a huge increase in cell size.

25 \[Slide.\]

> 173 1 Finally, I said there is more good stuff

2 within each cell. This is tyrosine hydroxylase

3 mRNA staining, lenti-beta-gal-treated animal on the

4 left, lenti-GDNF-treated animal on the right.

5 Obviously, there are more cells here, but

6 I want you to appreciate that each cell is darker

7 due to the fact that there is more TH mRNA within

8 each cell, and when you do the quantitation of the

9 optical density for TH mRNA, there is a 21.4

10 percent increase in the relative optical density.

11 So, we have no toxicity, we have

12 consistent and robust gene expression that is long

13 term, and we have robust effects at the level of

14 the striatum and the nigra with lenti-GDNF

15 delivery, but still we are missing one thing, we

16 are missing recovery of function, because that

17 model system, as I mentioned, does not respond to

18 dopaminergic drugs.

19 \[Slide.\]

20 So, now we have to switch model systems.

21 I think most of you may be familiar that the best

22 animal model of Parkinson\'s disease is the primate

23 model of MPTP.

24 MPTP was discovered in California as a

25 byproduct of drug abusers making synthetic heroin

> 174 1 in their basement and created this byproduct called

2 MPTP, and they were wheeled into emergency rooms in

3 San Jose with all the symptoms of Parkinson\'s

4 disease, and were for all intents and purposes were

5 end-stage Parkinson\'s disease cases with the

6 exception that they were all 20 years old.

7 Bill Langston and Irwin went on this

8 remarkable detective story, in which they went to

9 their houses and they got the drug, and they found

10 that the offending agent was MPTP. Actually MPTP

11 is a protoxin, the actual toxin is MPP+, and the

12 MPTP is broken down by monoamine oxidase into MPP+.

13 It doesn\'t work very well in rats, it doesn\'t work

14 at all in rats, it works somewhat well in mice,

15 works exquisitely well in monkeys.

16 So, now we are using this model system.

17 What we do is we train these animals on a fine

18 motor task and also we score them on a modified

19 Parkinson\'s disease rating scale. It is analogous

20 to the UPDRS scale that I talked to you about

21 previously.

22 Then, all the monkeys get a single

23 injection of MPTP up the carotid artery. Now,

24 there is one problem with this model system, that

25 monkeys don\'t always get parkinsonian symptoms with

> 175 1 a single injection, so what we have to do is we

2 start out with a large number of animals, in this

3 case it was 20, and we inject them all.

4 Three or four days later, you go into the

5 room, and I can take any one of you into the room

6 and say which one of these animals are

7 parkinsonian, and you would be able to pick out

8 those that are parkinsonian. They have this

9 crooked arm posture, they drag their leg, and many

10 of them will rotate around in their cage. It is a

11 very obvious, obvious clinical phenomenon.

12 Then, what we do is we just take those

13 animals because we know from experience that those

14 animals will always be parkinsonian unless you

15 intervene and will never display spontaneous

16 recovery.

17 So, after we take those animals a week

18 after the MPTP, we then distribute them based upon

19 parkinsonian rating scores into a lenti-beta-gal

20 group and into a lenti-GDNF group. We test them

21 for three months on the same behavioral tasks, we

22 give them a fluorodopa PET scan and then we run

23 them through the same anatomical studies that I

24 just showed you previously.

25 \[Slide.\]

> 176 1 Here is a cartoon of the pick-up task.

2 Basically, it\'s a modified home cage. We put apple

3 in these recessed food wells, and simply just time

4 the animals for how long it takes them to remove

5 the food treats or apple out of the food wells.

6 \[Slide.\]

7 Let\'s not worry about the red bars. This

8 is for a different talk. A normal animal can

9 perform this task in about eight or nine seconds.

10 You give them MPTP, and then if you look at the

11 yellow diamonds, which are the lenti-beta-gal

12 group, these animals get worse and worse and worse,

13 and the longest we let them go is at 30 seconds.

14 There are no error bars here because all

15 controlled-treated animals cannot perform this task

16 within 30 seconds.

17 In contrast, animals receiving the same

18 lesion, same lentivirus injections, but now

19 encoding for GDNF, they initially get a little

20 worse, but then they get better and better, and

21 stay stable significantly better for the duration

22 of the study.

23 You may notice there are pretty big

24 standard error bars here. That is because one

25 animal did not recover, but all the rest recovered

> 177 1 completely, and went back down to normal. I will

2 talk to you in a little bit about why that one

3 animal didn\'t recover.

4 \[Slide.\]

5 In terms of the parkinsonian rating scale,

6 a normal animal will score a zero. Once they are

7 given MPTP, they score about 11 or 12 on this task.

8 Lenti-beta-gal-treated animals stay stable

9 parkinsonian throughout the duration of the study.

10 Lenti-GDNF-treated animals get better and better

11 and better. It is relatively small, so these

12 changes did not get statistically significant

13 through the last four evaluation points, but still

14 a robust anti-parkinsonian effect.

15 \[Slide.\]

16 We had our first indication again that

17 things were going well anatomically. Certainly,

18 that was excellent news behaviorally when we looked

19 at the fluorodopa uptake. This is the side of the

20 MPTP infusion, the side of the lenti-beta-gal

21 injections, and you see basically you lose all

22 fluorodopa uptake on the side of the MPTP

23 injection.

24 In contrast, when you give the lenti-GDNF

25 to parkinsonian monkeys, you are able to prevent

> 178 1 the degeneration of the nigrostriatal system

2 completely. In fact, in this monkey, there is more

3 fluorodopa uptake here than here.

4 \[Slide.\]

5 When we looked at the brains of these

6 animals, these are coronal sections through the

7 anterior commissure. Here is the caudate nucleus,

8 here is the putamen. You can see

9 lenti-beta-gal-treated animals lose virtually all

10 their dopamine within the caudate nucleus and the

11 putamen.

12 In contrast, animals receiving the same

13 lesion, same virus, but now encoding for the

14 trophic factor, we get not only complete

15 preservation of the nigrostriatal system, there is

16 more dopamine here than there is even on the intact

17 side.

18 \[Slide.\]

19 When you do the quantitation,

20 lenti-beta-gal-treated animals lose TH optical

21 density dramatically, and it appears to be a

22 normalization here in lenti-GDNF-treated animals,

23 but if I would have culled out that one animal that

24 didn\'t recover, there is actually an overshoot and

25 there is more dopamine in the striatum as a group

> 179 1 under those conditions than on the intact side.

2 \[Slide.\]

3 Well, what about the nigra? The same type

4 of phenomenon. At the level of the entopeduncular

5 fossa, here is the intact side, this is the

6 MPTP-treated side, and this is an animal that

7 received the controlled vector lenti-beta-gal, and

8 you see the dramatic loss of TH-positive cells on

9 this side. Same vector, same lesion, just now

10 encoding for GDNF, and there is a complete

11 preservation of the nigrostriatal system.

12 You can see the gold staining up here.

13 This is regenerating fibers, of sprouting fibers

14 that have resulted from the intranigral injection

15 of the GDNF.

16 \[Slide.\]

17 When you do the quantitation, lenti-beta

18 gal-treated animals lose almost 90 percent of their

19 cells, and this is completely prevented with the

20 lenti-GDNF, and, in fact, there are more cells

21 here, and we don\'t think that this is due to again

22 any neurogenesis.

23 What we think happens with the MPTP going

24 up the carotid artery, there is a little bit of

25 leakage to the other side, and so basically, we

> 180 1 think we have protected everything on this side of

2 the GDNF, but we didn\'t protect the small loss that

3 is seen on the opposite side.

4 \[Slide.\]

5 Again, if you look at the volume of the

6 changes, the changes in volume of nigral cells, of

7 the remaining cells in the lenti-beta-gal group,

8 these cells shrink by 32 percent, just like they do

9 in Parkinson\'s disease. In contrast, not only is

10 that prevented, but these cells hypertrophy, and if

11 you look at the difference here, there is almost a

12 76 percent difference in the size of these cells.

13 \[Slide.\]

14 Again, if you look at TH mRNA, again, the

15 remaining cells, there is a loss of TH mRNA within

16 the nigral cells, just like it is in Parkinson\'s

17 disease, and again not only is this prevented, but

18 there is an augmentation of TH mRNA within

19 individual nigral neurons.

20 \[Slide.\]

21 I am going to skip all this.

22 \[Slide.\]

23 Again, so we have all this great stuff.

24 We have got functional recovery, we have got

25 anatomical preservation to the max, just what we

> 181 1 would hope, but nothing is worthwhile if have

2 immune responses and toxicity.

3 So, we carried out detailed immune studies

4 using CD45, CD8, and CD3 markers, and what I am

5 showing you here is all CD45, which is the most

6 ubiquitous of those stains. What I am showing you

7 in Panel A and Panel B is the worst response we

8 got\--oh, excuse me\--the most intense staining we

9 got from any of these markers on the worst or most

10 intense section from that animal, and this is all

11 that we have ever seen, just a little bit of

12 staining here, a couple of cells with microglial

13 morphology even in other brains, right through the

14 needle track, and that is an antiimmune response.

15 We got nervous that maybe we were having a

16 problem with our staining protocol, so we threw in

17 an Alzheimer\'s piece of tissue that stained up

18 beautifully to illustrate the specificity of this

19 response.

20 So, there is no immune response following

21 lenti-GDNF injection in these animals.

22 \[Slide.\]

23 Finally, there is one other bit of caution

24 I did want to pass along. Now, we did our

25 injections in the caudate nucleus and the putamen

> 182 1 and the nigra, and why did we choose all three

2 sites? Because we were gutless in the beginning.

3 These are very expensive studies, we wanted to show

4 which sites would be more important, so we figured

5 we are going to inject all of them.

6 Well, it turns out it is interesting that

7 we injected the nigra, and I showed you all those

8 good things that did happen, but bad things can

9 happen also. This is the lateral septum, and look

10 at this very robust sprouting response seen here in

11 the lateral septum.

12 From an anatomous point of view, that is

13 pretty cool, that\'s things we would like to see,

14 but the problem is the cells of origin here are not

15 nigral, they are from the adjacent ventral

16 tegmental area, and when you augment the adjacent

17 ventral tegmental area, that is what in part causes

18 schizophrenia.

19 So, I think it is very important that we

20 do not put dopaminergic trophic factors down in the

21 midbrain, because you are not going to be able to

22 control them sufficiently to ensure yourself that

23 you are not going to augment an adjacent nucleus

24 that can cause very severe side effects in patients

25 that are taking levodopa and are potentially

> 183 1 teetering on hallucinogenic behavior anyway. So,

2 that is one important point I wanted to make.

3 \[Slide.\]

4 So, in closing, where do we go from here?

5 I think it is absolutely essential that no one goes

6 into a clinical trial with gene therapy, at least

7 the types of trials that I am discussing here,

8 without your ability to control gene expression,

9 and it is not just enough to be able to control

10 gene expression, you have to be able to show that

11 you can shut off your gene, and that shutting off

12 your gene reverses whatever you did, because, for

13 example, too much dopamine can cause abnormal

14 involuntary movements called dyskinesias, and many

15 of you may be aware of the recent report about

16 fetal transplants that cause these runaway

17 dyskinesias in these patients, and they have no way

18 of reversing that.

19 What we are doing is we have a study

20 ongoing right now in aged monkeys where we are

21 putting the lentigene in with the tet-Off system,

22 and we will do fluorodopa uptake on PET scan.

23 Then, half the animals will get tetracycline, we

24 will attempt to shut off the GF gene, and we will

25 also measure dyskinesias in these animals and see

> 184 1 whether we can reverse the fluorodopa uptake and

2 reverse any changes in dyskinesias.

3 I think it is absolutely essential that

4 these first two points be met before anyone goes to

5 the clinic with a therapy such as lenti-GDNF.

6 One of the big questions that will be

7 asked by regulatory agencies is what is the

8 appropriate patient population to go into.

9 Typically, trials start with more advanced

10 patients, especially safety trials, especially in a

11 disease state that has other therapeutic strategies

12 available to them, but this type of strategy, GDNF

13 strategy theoretically should work best in, as I

14 mentioned earlier, the less advanced patient.

15 So, we are also doing studies to model,

16 instead of modeling early Parkinson\'s disease,

17 modeling late-stage Parkinson\'s disease to see

18 whether GDNF will be efficacious in that system, if

19 it is not, that would question whether we should be

20 doing trials from the beginning in earlier patients

21 rather than late stage patients.

22 Then, just in closing, I showed you a lot

23 of work, and I tend to go around giving the talks

24 while all the people back in the lab are doing all

25 the work. I am very proud of my group who

> 185 1 collected all this data, as well as Patrick

2 Aebischer and Jocelyne Bloch and Nicole Deglon who

3 provided all the vectors, University of Wisconsin

4 group that did all the PET scanning, as well as

5 Philippe Hantraye and Didier Trono who participated

6 in other aspects of the study.

7 I will stop there. Thank you.

8 \[Applause.\]

9 Questions & Answers

10 DR. SALOMON: So, one of the things you

11 started out by saying is that when any of us went

12 in the room, we would be able to detect the

13 animals, so after you did the gene therapy, would

14 we now have difficulty detecting the animals?

15 DR. KORDOWER: Yes, you very much would

16 have difficulty detecting the animals. In fact,

17 the fact that these animals have some score on the

18 Parkinson rating scale really attests to the

19 experience of the observers and the trained

20 observers who do this all the time.

21 If just someone who didn\'t do this for a

22 living went in there, you would have a hard time

23 detecting which animals were parkinsonian and which

24 were untreated, which were GDNF treated and which

25 were untreated. I am sorry.

> 186 1 DR. SALOMON: Is that what you meant to

2 say?

3 DR. KORDOWER: Excuse me - which are

4 normal and which are GDNF treated.

5 DR. VERMA: Were you not surprised that if

6 you are using it for eight months uncontrolled

7 expression of GDNF, the monkeys, that there was

8 nothing bad that happened to them by and large?

9 DR. KORDOWER: There was nothing bad that

10 happened at all. In fact, all the caveats I

11 brought up are theoretical, there is no empirical

12 data at all to suggest that bad things will happen,

13 but there is one big caveat, and I think this is

14 the caveat that the Freed \[?\] people ran into. No

15 one ever did fetal transplants in monkeys into

16 levodopa-prime Downs, and it is undoubtedly initial

17 clinical trials with gene therapy will go into

18 patients that have been on levodopa, and that could

19 be a major variable.

20 So, monkeys are not parkinsonian patients,

21 and that is a key parameter that needs to be

22 tested, and we are testing that currently.

23 DR. MULLIGAN: This is kind of an

24 irrelevant question vis-a-vis the meeting, but it

25 is interesting one I think. The lac-Z infections,

> 187 1 you showed a time course and you showed that the

2 cell bodies looked like they were making some

3 lac-Z, but then over time you saw protections.

4 Do you have any idea what that is, what

5 accounts for that, and have you looked ever in

6 these to see whether directly there is integrated

7 sequences, on integrated sequences, is there a

8 transition from unintegrated to integrated

9 sequences?

10 DR. KORDOWER: We haven\'t looked at that.

11 What I think is basically happening is that the

12 gene product, both lac-Z and GDNF, is being made

13 and is just being integratedly transported down

14 axons to normal target cells.

15 DR. GROSSBARD: Elliott Grossbard, Amgen.

16 Would I be correct in inferring that some

17 of the preclinical studies with proteins were done

18 in MPTP primates?

19 DR. KORDOWER: That is correct.

20 DR. GROSSBARD: So, you haven\'t really

21 explained the inconsistency because you suggested

22 they were trivial delivery of the neurotrophic

23 factor even in the primates, and yet they had a

24 clinical response.

25 DR. KORDOWER: If you read those papers

> 188 1 carefully, some of those clinical responses are

2 pretty trivial.

3 DR. GROSSBARD: Oh, okay.

4 DR. ALLAN: How long can you wait after

5 you induce Parkinson\'s in the monkeys before you

6 won\'t have an effect?

7 DR. KORDOWER: Well, we are not sure. We

8 think we were right on the bubble. I mentioned

9 there was one animal that didn\'t recover. That

10 animal had great gene expression. We think what we

11 ran into with this particular animal is that there

12 is some variability in the speed at which the

13 fibers regress, and that particular animal may have

14 had quicker fiber degeneration than the others, and

15 the gene that was not able to capture that.

16 It is interesting that that animal had

17 complete protection at the level of the nigra, but

18 did not have protection at the level of the

19 striatum, and that animal did not recover.

20 I don\'t want to appear too flippant about

21 my response to the previous questioner. A lot of

22 those MPTP studies involved interparenchymal

23 injections. The trivial response that I was

24 referring to were studies that used

25 interventricular administration of the protein.

> 189 1 DR. HIGH: You described adverse events in

2 the patient that you took care of who had GDNF

3 protein therapy, and I was wondering if any of

4 those adverse events were accurately modeled in the

5 primates.

6 DR. KORDOWER: No, we didn\'t see any

7 adverse\--what we would call an adverse event in the

8 monkeys, and that is quite typical. You know, with

9 other trophic factor deliveries, when we did

10 studies with NGF, and we put NGF secreting cells in

11 the ventricle of monkeys, they did very badly and

12 they had significant side effects. You do those

13 same studies and put them in parenchyma, and you

14 don\'t see the side effects. It gets back to the

15 point I made earlier, I don\'t think trophic factors

16 should be put in the ventricle.

17 DR. RAO: It seemed implicit in your

18 statement that GDNF is not causing sprouting, if

19 you think that the failure to see response was

20 because you couldn\'t reverse the regression?

21 DR. KORDOWER: No, there is evidence for

22 sprouting, certainly at the level of the nigra, but

23 to get the sprouting, the trophic factor has to get

24 to those fibers that have the receptors on them,

25 and the distance may have been too great for that

> 190 1 to occur.

2 I didn\'t have a chance to go into it,

3 there is other evidence, and we are presenting some

4 of that in the Science paper, I believe, to suggest

5 that there is both protection and sprouting.

6 DR. SALOMON: Was this a VSV-G

7 pseudotyped?

8 DR. KORDOWER: Yes.

9 DR. SALOMON: So, at least we could say

10 that in vivo injections into the brain, VSV-G was

11 an effective delivery system.

12 DR. KORDOWER: Correct.

13 DR. SALOMON: Did you ever take any of

14 these tissue biopsies at, let\'s say, a month after

15 delivery, take them out and put them in co-cultures

16 with cells that would be, you know, H9 or\--

17 DR. KORDOWER: No, that is something we

18 have to do.

19 DR. SALOMON: Do you have any studies at

20 all that would address the issue of

21 replication-competent lentivirus?

22 DR. KORDOWER: None that have been

23 currently finished.

24 DR. SAUSVILLE: You introduced in a

25 prominent way the possibility that having

> 191 1 regulatable expression would be important, and you

2 used the tet system as an example. Although tet

3 promoters are used very avidly in preclinical

4 models, they tend to be at either the On or Off.

5 Is there evidence that you can actually

6 grade the level of expression using that particular

7 promoter system, or do you think this would be

8 relevant, for example, to use in humans?

9 DR. KORDOWER: My answer is totally

10 speculative. This is such a potent trophic factor.

11 My guess would be that you would not be able to

12 dose it with the tet-On system.

13 DR. SAUSVILLE: Although it\'s comforting

14 that you can turn it on and turn it off and

15 regulate it, whether that would be practically have

16 value in terms of grading doses is unclear at this

17 point.

18 DR. KORDOWER: Right. For me, the

19 necessity to have it is totally a safety issue.

20 DR. RAO: I was also curious about the

21 fact that the lentivirus seems to be relatively

22 more specific towards the neurons. I mean would

23 you care to say? I mean the relative ratio at

24 least published would be 10 to 1 for astrocytes and

25 oligodendrocytes.

> 192 1 DR. KORDOWER: In culture.

2 DR. RAO: Even in the brain?

3 DR. KORDOWER: No, I think Dr. Naldini is

4 here, I don\'t know whether he has data, but I know

5 the original rat studies, I believe were also

6 predominantly neuronal, and I think that is quite

7 consistent. That is my understanding.

8 DR. VERMA: That may have to do with the

9 promoter off.

10 DR. KORDOWER: The PGK.

11 DR. VERMA: But some PGK, CMV, many of

12 them have very often, but some of them, like

13 EFN-alpha, does not do as well in neurons as it

14 does in other cells. It is a matter of the

15 promoter, too.

16 DR. RAO: But it seemed better in neurons.

17 DR. VERMA: Depending upon the nature of

18 the promoter you use.

19 DR. RAO: Is there any culture data from

20 this lentivirus suggesting that there is a cell

21 bias?

22 DR. KORDOWER: I am not aware of any.

23 DR. VERES: If anything, I think this is

24 related to the envelope, the VSV envelope. In this

25 regard, I think there is some data published, at

> 193 1 least from meeting reports, they are using either

2 rabies or the other retroviral envelope which claim

3 to have tropism to the glial cells.

4 DR. MULLIGAN: You mentioned on several

5 occasions that you didn\'t think neurogenesis was

6 responsible for the effects. I thought one of the

7 effects of GDNF purported in the past was indeed

8 neurogenesis. Why isn\'t that happening or why

9 wouldn\'t that happen?

10 DR. KORDOWER: We have actually pulsed a

11 couple of animals with BODU and didn\'t see

12 anything, and also, the cells are always in the

13 exact cytoarchitectonic location that they should

14 be, and you never see any streaming.

15 You saw, I guess the best example was the

16 nigral injection where basically, half the midbrain

17 was filled with GDNF, and so you would figure if it

18 is going to cause neurogenesis, it should do it

19 throughout. You don\'t see that. It is only in the

20 nigra.

21 DR. MULLIGAN: What were the original data

22 suggesting that that was a GDNF effect, was there

23 injection made in the past by other people

24 suggesting that this occurred?

25 DR. KORDOWER: Yes, I think in neonates.

> 194 1 DR. ZAIA: Can you repeat one more time,

2 in terms of your rationale, is the GDNF inducing

3 dopasynthesis I presume?

4 DR. KORDOWER: The GDNF is preventing

5 neurodegeneration, and GDNF is increasing tyrosine

6 hydroxylase expression, which is the rate-limiting

7 step of dopamine synthesis, causing regeneration of

8 fiber, so it is doing three things.

9 DR. ZAIA: But then are you suggesting

10 that if you had gone in with the enzyme that you

11 needed to increase dopa, that may have failed? Had

12 you done the control of using whatever the

13 dopasynthetase is, I don\'t remember the enzyme\--if

14 you had gone in with TH after the challenge, would

15 you have protected?

16 DR. KORDOWER: You wouldn\'t have

17 protected.

18 DR. ZAIA: Why not?

19 DR. KORDOWER: Because TH isn\'t a

20 protective enzyme, it\'s a synthesizing enzyme.

21 DR. ZAIA: But it would raise dopa levels,

22 wouldn\'t it?

23 DR. KORDOWER: It would raise dopa levels.

24 DR. ZAIA: And so you are saying that that

25 is not sufficient to protect?

> 195 1 DR. KORDOWER: I am saying because the

2 cells are going to die anyway, so if you are not

3 preventing their death\--you may get a bump in

4 symptomatic benefit, but you are not going to\--

5 DR. ZAIA: I see. So for the rationale,

6 then, it requires the trophic factor.

7 DR. KORDOWER: Correct.

8 DR. ZAIA: Okay.

9 DR. SALOMON: But if we follow that, then,

10 again deferring to my neurology colleague, the way

11 this model was set up is he creates an acute

12 injury, so during that period of time, there is

13 cell injury death and, you know, this quasi-state

14 that maybe some cells can be rescued, and that

15 would be your target, right?

16 You give your GDNF gene therapy then,

17 right, it is not\--you didn\'t show us any data where

18 you caused the injury, waited for two months, at

19 which point the animals have the 30 second or

20 greater fruit-sorting test, and then gave the GDNF

21 therapy.

22 So, when we now make the jump between how

23 one would use that animal model to what is going on

24 in a human patient with Parkinson\'s disease, a lot

25 of it has to do with where in the state of the

> 196 1 disease progression we are at, which you

2 acknowledged right at the beginning, but it also

3 goes to what extent is neural cell loss and

4 destruction occurring.

5 DR. KORDOWER: Versus phenotype.

6 DR. SALOMON: Versus, you know, just

7 changes as I think Dr. Zaia was getting at, where

8 it would be metabolic or enzymatic pathways that

9 are being altered, so what\'s new, you know, animal

10 models are tough to do. I didn\'t mean to go too

11 far beyond it.

12 One thing that I find sort of interesting

13 is you do this injection and then it\'s a little

14 tricky with the slides, because what you are doing

15 a lot of times is you are showing GDNF staining.

16 DR. KORDOWER: Right.

17 DR. SALOMON: And what you don\'t show a

18 lot of is how many cells actually got hit by the

19 vector and how that relates to where you find GDNF.

20 I mean it\'s too wonderful, but you do this

21 injection and you get only the putamen or only the

22 substantia nigra.

23 So, how much spread of the original

24 lentiviral vector occurs outside the needle site

25 and how much spread afterwards occurs of virally

> 197 1 infected cells, and how much is the rest due to

2 just spread of the GDNF?

3 DR. KORDOWER: We are getting a handle on

4 that. Part of the vector system has the

5 woodchuck-enhancing element, and we have an in-situ

6 probe against that. So, you put the injection in

7 and you probably have 3 to 4 millimeters on either

8 side of the injection filled with cells, labeled

9 cells, but the secretion is much farther than that,

10 and we can fill out the entire striatum.

11 In fact, there is even more\--what I showed

12 you immunocytochemically is an underestimation of

13 what is there, because when we do our punches, and

14 we don\'t know where the injection is, I am just

15 doing it on a piece of fresh tissue, sometimes you

16 get a punch that is outside the area of immunocytic

17 chemistry, and although the level of protein there

18 is greater than background, significantly greater

19 than background, although it is not as much as what

20 is in the number of the staining, it is still much

21 greater than background.

22 So, it is even greater than what I showed

23 you, and we can basically fill the entire striatum

24 with GDNF.

25 DR. VERMA: Didn\'t you have a construct

> 198 1 with GDNF, area of GFP?

2 DR. KORDOWER: No.

3 DR. VERMA: Oh, you haven\'t. I thought

4 you had that construct that would tell you.

5 DR. KORDOWER: No.

6 DR. SALOMON: He threw us\--I am kind of

7 disappointed in you guys actually, because Jeff set

8 you up with the statement you cannot go forward

9 with lentiviral gene therapy unless you have a

10 regulatable promoter, and the resounding silence

11 here\--

12 DR. VERMA: Or trophic factors\--

13 DR. SALOMON: I don\'t know. Okay. I mean

14 do you guys want to take it or\--there is consensus

15 here from the Committee that you have to have a

16 regulatable promoter.

17 DR. CHAMPLIN: Here, there is the

18 functional possibilities, as well as dangers from

19 systemic effects. In the brain, obviously, you can

20 make things worse symptomatically, as well as

21 better, and if they get worse, you could turn it

22 off by using the tet-Off system, so it is not so

23 much worrying about the killer virus emerging as

24 much as the functional effect on the patient.

25 DR. KORDOWER: I must say that the reason

> 199 1 I feel that way now, I didn\'t feel that strongly

2 about it six months ago, before the Fried, et al.

3 report, but you put in a fetal transplant, you have

4 got five patients, and I don\'t know if anyone has

5 seen the videotapes, they are horrific, they are

6 horrific, and you don\'t want to be doing, you know,

7 you don\'t want to have put a dopaminergic trophic

8 factor in, have something similar happen, and you

9 can\'t turn it off.

10 DR. SALOMON: You ought to do a suicide

11 gene.

12 DR. KORDOWER: Now you are getting

13 complicated.

14 DR. SALOMON: In a fetal cell transplant

15 you can. I mean I think the principle here is

16 really important, and there is two principles. One

17 is okay, I mean I was partially being facetious. I

18 realized that Dr. Kordower was making the point

19 specifically for intraneural applications, but

20 still that is really a bold point from a regular

21 point of view to say that.

22 The second issue is to what extent do we

23 have confidence in tet-On/tet-Off systems. I mean

24 I thought this was, man, this is a lob for you

25 guys. I mean everybody goes nuts every time you

> 200 1 mention a tet-On/tet-Off system is leaky, it turns

2 off, it gets silenced, and no one said a word.

3 DR. SAUSVILLE: I did protest a little bit

4 about the tet, if you remember, and we established

5 that pharmacologically, it is probably not going to

6 allow regulation, which leads to what I think you

7 stated it was the worst case scenario regulator

8 rather than something that you are going to\--but,

9 also, isn\'t that rather context-dependent?

10 I mean one could imagine replacement

11 therapy is where the consequences of having more or

12 less are not quite the same, but that I guess needs

13 to be judged on a case-by-case scenario.

14 DR. VERMA: Also, I think in the case of

15 the tet, it is not really a question of people have

16 been talking about 100 percent off and on, that is

17 not what they are asking for. If you have a small

18 leaking, it is very different than absolutely zero.

19 So, these systems don\'t have absolutely zero, but

20 small leaking is tolerable in many cases.

21 DR. KORDOWER: As long as your biological

22 effect can be reversed, you are fine.

23 DR. VERMA: I tend to agree with you that

24 it\'s a good idea to have regulation in general, but

25 it is not necessary for every disease candidate,

> 201 1 but I think once you have good regulatory systems

2 available, easy to use, I suspect most people will

3 probably tend to do that.

4 DR. SALOMON: I have no problem with that

5 except carefully looking at what regulatable

6 systems are out there right now and the amount of

7 general concern that these regulatable systems

8 won\'t function as well as one would hope.

9 I think it is a big issue when one says,

10 look, there is a lot of major questions for gene

11 therapy right now, and then if one predicates a

12 gene therapy on top of that, proving that a

13 regulatable system works, that\'s, you know, adding

14 a whole another layer of complexity. I mean should

15 you be doing a lentiviral vector gene therapy in

16 the brain where you need to have the regulatable

17 system work as well as the lentiviral system work.

18 DR. SAUSVILLE: Isn\'t the whole issue

19 related to criteria of proof? I mean, in other

20 words, at one level we have proof, it\'s up, it\'s

21 down. I mean if you ask at the level of proof,

22 does this have functional consequences in the long

23 term, I am not aware of any treatment that the FDA

24 regulates that you have to sort of prove that you

25 have an antidote to what you are giving. You may

> 202 1 want to comment on this.

2 DR. SALOMON: That would be an argument

3 not to require regulatable promoter.

4 DR. SAUSVILLE: The issue of requiring and

5 saying it\'s a good idea, and that is I think the

6 distinction.

7 DR. SIEGEL: I am not sure what the list

8 of precedence is, but I think it would be fair to

9 say for most of the products given, if there isn\'t

10 an antidote, you can just stop giving them, and

11 there are different considerations when you can\'t

12 do that in terms of the implications.

13 That doesn\'t necessarily mean that there

14 is a requirement that if you can\'t do that, you

15 have to have some other mechanism to turn the

16 product off, but it does raise safety concerns that

17 need to be addressed.

18 DR. NOGUCHI: In addition to that, just

19 the ability to turn things on and off is as yet

20 untested in any gene therapies clinically. We

21 don\'t know if the supposed cure for an

22 inappropriate secretion might even be worse, so

23 there is always that caveat with any of these

24 systems.

25 The more complex you make them, the more

> 203 1 opportunity you have for nature to reassert

2 herself.

3 DR. ALLAN: Two things. One is, and I

4 don\'t remember it, does tetracycline cross the

5 blood-brain barrier? It does, okay.

6 The second thing is in the areas where you

7 injected the virus, and you only saw expression in

8 the cells that you wanted to see, the other

9 cells\--it comes actually to another question that

10 someone else had\--but in the other cells, are you

11 getting expression, but it is just those cells you

12 get accelerated turnover of the protein? In other

13 words, are you still getting expression by the

14 proteins turning over, so you are not seeing it?

15 DR. KORDOWER: I think it has to do with

16 insensitivity of the antibody to detect it.

17 DR. ALLAN: So, you think it is just low

18 level expression rather than turnover?

19 DR. KORDOWER: Right.

20 DR. RAO: If I can add to that whole idea

21 of regulatable things, even in trophic factor

22 system, it is important to remember, as Dr.

23 Kordower said, that the effect of GDNF is also to

24 cause anatomical changes. There might be neuron

25 outgrowth, there might be better connections that

> 204 1 have taken place. Even if you dial off the GDNF at

2 the end, it doesn\'t mean that you have reverted

3 back to normal.

4 So, it is not necessarily either an

5 absolute requirement or it is not necessarily a

6 cure either way to necessarily say that we have a

7 regulatable system we have changed the underlying

8 situation.

9 DR. SALOMON: Later this afternoon, we are

10 going to talk about animal models, and I think this

11 is very valuable in that you have done some

12 wonderful work in developing an animal model here,

13 so the question I had was I guess it always makes

14 me a little nervous in thinking about validating an

15 animal model when you say, you know, there is

16 absolutely no toxicity.

17 I would almost be happier if you could

18 say, in terms of validating, that there was

19 toxicity and, you know, we did this and that and

20 avoided it. I guess it seems to me that one

21 element in validating an animal model is to

22 demonstrate that you can under some circumstance in

23 that model develop toxicity.

24 DR. KORDOWER: We are interested in doing

25 dose escalation studies, and those will

> 205 1 presumptively, some high dose will cause some

2 toxicity, so we might be able to address it in that

3 study.

4 DR. RAO: I guess it is not directly

5 related to the virus, but I was curious that

6 despite having milligram quantities of protein\--

7 DR. KORDOWER: Microgram.

8 DR. RAO: \--microgram quantities of

9 protein, that the improvement was not back to

10 baseline in terms of behavior improvement. Do you

11 have any\--

12 DR. KORDOWER: On the objective hand reach

13 task, if you cull out the animal that didn\'t

14 recover, the other animals were all back to normal.

15 On the rating scale, difficult. Other people have

16 asked me the same question, I don\'t really have a

17 good answer for you, but you are right, on the

18 rating scale they weren\'t back to zero, but they

19 were good.

20 DR. MULLIGAN: How about that one animal?

21 You never told us what happened to it.

22 DR. KORDOWER: Well, what happened was he

23 had complete neuroprotection at the level of the

24 nigra, but trivial neuroprotection at the level of

25 the striatum, and I think as I mentioned earlier,

> 206 1 what happened in that animal, given the lesion and

2 the fibers regressed too quickly for the GDNF to

3 stop it.

4 In fact, there was this sprouting response

5 in the globus pallidus of that animal where we

6 couldn\'t encourage the regrowth back into the

7 striatum.

8 DR. SALOMON: Go ahead.

9 DR. VERMA: I just want to know when can

10 we have lunch.

11 \[Laughter.\]

12 DR. SALOMON: You beat me on that one.

13 What I was going to say is I think it is time for

14 lunch. Yesterday, we made it in under 45 minutes,

15 so if we can try and back here in about 35 or 40

16 minutes, we will get started.

17 Thank you.

18 \[Whereupon, at 12:45 p.m., the proceedings

19 were recessed, to be resumed at 1:30 p.m.\]

> 207 1 AFTERNOON PROCEEDINGS

2 \[1:40 p.m.\]

3 DR. SALOMON: I suppose we have still got

4 one or two people who are out to lunch.

5 \[Laughter.\]

6 DR. SALOMON: I couldn\'t resist that,

7 forgive me.

8 There are two announcements. The first is

9 that Marina O\'Reilly will be representing OBA in

10 place of Amy Patterson, who had prewarned us that

11 she had an afternoon meeting, and she will join us

12 at the table.

13 The second announcement that I am

14 personally disappointed at is I guess Dr. Cornetta,

15 Ken Cornetta, from Indiana University and Director

16 of the National Gene Vector Laboratory there, was

17 unable apparently because of weather to get out of

18 Indianapolis. I think that is the loss to the

19 Committee.

20 But using the latest in technology, I

21 understand that he has also joined us by telephone,

22 so perhaps just to test that connection, Ken, are

23 you there and can you introduce yourself to us?

24 DR. CORNETTA: This is Ken Cornetta. I

25 can hear you, Dan. Hopefully, you can hear me.

> 208 1 DR. SALOMON: Actually, we hear you fine.

2 Everyone at the beginning sort of gave a quick

3 two-sentence thing. Can you introduce yourself?

4 DR. CORNETTA: I am Ken Cornetta from

5 Indiana University. I am a Professor of Medicine,

6 trained in hematology/oncology, and also have been

7 interested in retroviral and now lentiviral vectors

8 and their use clinically. For the past about six

9 years now I have been coordinating the National

10 Gene Vector Lab, which is funded through the NIH,

11 and its goal has been to produce clinical grade

12 vectors for academic investigators performing

13 clinical gene therapy protocols.

14 Indiana has been the center for production

15 of retroviral vectors, so I have been keenly

16 interested in the discussion here today in regards

17 to lentiviral vectors.

18 DR. SALOMON: Thanks for joining us, Ken,

19 and like I said, I only regret you are not here

20 personally along with Dr. Emerman.

21 Dr. Emerman, are you still on?

22 DR. EMERMAN: Yes, I am still here.

23 DR. SALOMON: Okay. One of the things,

24 Dr. Emerman, you could reassure me is, are you

25 comfortable in jumping in, because that same issue

> 209 1 is with Dr. Cornetta, it is a little hard, as

2 chair, to stop and ask for the telephone all the

3 time as I forget, but can you jump in, and will I

4 be able to year you?

5 DR. EMERMAN: I don\'t know, we can try.

6 DR. SALOMON: Don\'t be inhibited, either

7 of you.

8 DR. EMERMAN: Okay.

9 DR. SALOMON: It is my pleasure to

10 announce that the first talk of the afternoon is

11 from Dr. Susan Kingsman of Oxford BioMedica,

12 Lentiviral Vectors for the Treatment of Cancer,

13 Neurodegenerative Diseases and AIDS.

14 Lentiviral Vectors for the Treatment of Cancer,

15 Neurodegenerative Diseases and AIDS

16 Dr. Susan Kingsman

17 DR. KINGSMAN: Thanks very much for

18 inviting me to come and talk. Oxford BioMedica is

19 a publicly quoted UK company, and we have got a

20 subsidiary in San Diego called BioMedica, Inc.

21 headed up by Doug Jolly, who is in the audience,

22 and is familiar to many of you.

23 This morning, Dr. Salomon said he was

24 hoping to perhaps see if there was any consensus

25 that might emerge within the field, so I am not at

> 210 1 all embarrassed that many of the slides and the

2 concepts that I am going to present to you have

3 already been presented by other people very well

4 this morning, but I think part of the process is

5 just to go over it, so I will repeat some things

6 that have been said.

7 \[Slide.\]

8 The Retroviridae, a nice, ancient

9 classification, lentiviruses fall within that

10 classification, but they are quite distantly

11 related from the type C viruses, but nonetheless, I

12 think it is legitimate to use the experience that

13 stretches back over at least 10 years from the

14 Mammalian C-type viruses to inform the concepts

15 that we are aiming for in trying to develop this

16 set of vectors for the clinic.

17 BioMedica currently has a clinical trial

18 with a retroviral vector in breast cancer, and Doug

19 Jolly has tremendous experience through his work

20 with Biogene and Chiron, so the collective

21 experience in taking retroviral vectors to the

22 clinic is good, and we hope to use that experience

23 to inform the way we move forward with lentiviral

24 vectors.

25 \[Slide.\]

> 211 1 The lentiviruses are themselves a rather

2 diverse group of viruses. This is a dendogram

3 where the pol gene is related. Now, there are

4 functional constraints on that enzyme obviously,

5 and there is some conservation between the

6 different members of the groups, but the HIV-2 up

7 here, for example, bears virtually no sequence

8 relationship outside the pol gene with something

9 like BIV.

10 They divide into two distinct sets, the

11 primate lentiviruses and the non-primate

12 lentiviruses. We have chosen to study HIV-1 as the

13 archival lentivirus, and we also decided to look at

14 a non-primate lentivirus, and we chose equine

15 infectious anemia virus as our non-primate

16 lentivirus to study. This is because it\'s far

17 apart from HIV, it\'s a non-primate virus, and this

18 particular virus does not cause an

19 immunodeficiency.

20 I think it is far too soon to say whether

21 any one lentivirus will provide a universal vector

22 for all applications. There are people that are

23 working on FIV, SIV, and I think that is very good,

24 we should explore this group of viruses and see

25 what the range of possibilities is, but you can

> 212 1 only do so much, so we have just picked two to work

2 with.

3 \[Slide.\]

4 Now, the one key fact that we have got to

5 be all very sure of is that there is some reason

6 for developing another type of virus for gene

7 therapy because there are lots of viral vectors out

8 there. We don\'t want to just go and invent and use

9 one more unless there is some real benefit.

10 I hope I am just simply going to add to

11 what other speakers have said and just reminding

12 you that they have a constellation of properties

13 which, together, make up something which I believe

14 is unique, relatively simple compared to something

15 like herpes and adenoviruses, they can carry up to

16 11 kb, so you can put a nice cargo in these

17 vectors.

18 You have a defined integration of genes.

19 You sweat in the lab to set up precise gene

20 expression configurations in therapeutic genes.

21 You know that they are going to be docked into the

22 chromosome in the same way that you invented them

23 in the laboratory, and that is a very useful,

24 important feature, especially if we are going to go

25 on to look at some more advanced applications where

> 213 1 we might need regulation.

2 This, they share with the retroviruses,

3 but here we are starting to see some differences.

4 This long-term expression increasingly we are

5 finding that we can see gene expression from

6 lentiviral vectors for much longer periods than

7 retroviruses, and then this key feature,

8 transduction of non-dividing cells, postmitotic

9 cells, and very important, the transduction of

10 slowly dividing cells. The retrovirus is going to

11 hang around in the cytoplasm waiting for the

12 nuclear membrane to break down before it can get in

13 there. Chances are it is going to get trashed, and

14 this is what happens. Lentivirus can just go

15 straight into the nucleus and deliver its cargo

16 even if the cell cycle is 48 hours, 72 hours.

17 So, I believe that these vectors do have

18 some unique advantages for long-term, stable

19 therapy of chronic diseases, and they will be

20 vectors for delivering treatments for unmet medical

21 needs.

22 \[Slide.\]

23 We heard this wonderful talk by Dr.

24 Kordower this morning, and I can\'t hope to emulate

25 it, but just briefly, if we take EIAV vectors, and

> 214 1 that is really what I am going to focus on because

2 we have had a lot on HIV this morning, you can do

3 experiments in the rat where you deliver by precise

4 stereotactic injection to particular sites in the

5 brain, and you can show that you get very effective

6 gene transfer in some important regions of the

7 brain.

8 I am not a neurobiologist, but here we

9 have regions that are important in Alzheimer\'s,

10 regions that are important in Parkinson\'s disease,

11 a region that is important in Huntington\'s disease,

12 a region here that is important in addictions.

13 These are data where we have pseudotyped

14 the EIAV with VSV-G, and what happens there is you

15 get a local gene expression out the site of

16 injection, and you get the gene product will

17 disseminate through the projections.

18 If, on the other hand, you pseudotype with

19 another envelope, which is from the rabies virus,

20 you find that you get gene transfer to sites that

21 are distal from the site of injection, and this is

22 where axons are projecting into the place where you

23 injected, but the cell bodies are out there, but

24 the vector goes in and travels up by what is called

25 retrograde transport and lodges into a distant

> 215 1 part, communicating part of the brain.

2 You can harness that property, for

3 example, here you can inject an EIAV vector

4 pseudotypes with rabies into the muscle where the

5 nerve endings are, and the nerve connects with the

6 spinal cord, and you can access spinal motor

7 neurons by peripheral administration.

8 Here, then, you have got an opportunity of

9 accessing these neurons, and the primary target

10 there are diseases of motor neuron degeneration.

11 So, there is clearly great potential of

12 these vectors to access particular neuronal sites,

13 and by changing the envelope, you can do different

14 things. I think another point I might make is we

15 shouldn\'t get too focused on VSV-G. It is what we

16 have got at the moment, most of our advances, it is

17 terribly useful, but there are other envelopes

18 coming along that may have other issues and other

19 uses.

20 \[Slide.\]

21 Long term gene expression, in our

22 experience also, you can see gene expression after

23 eight days, and you can see gene expression after

24 six months. We have been following animals for

25 eight, nine months now, and this gene expression

> 216 1 persists. So, that is a very good feature because

2 we can have sustained expression, minimizing the

3 need for invasive delivery.

4 \[Slide.\]

5 Now, we try and do some type of toxicology

6 and biodistribution in all our animals that we set

7 up as models, but, of course, ultimately, there

8 will be a proper systematic approach, but you can

9 see that we have looked at a lot of animals

10 particularly in the brain.

11 In the previous report that we heard this

12 morning, I can\'t say there is no inflammation, we

13 do see mild acute inflammation. It has resolved by

14 35 days, so you can\'t tell the difference between a

15 PBS control and the vector-injected control, but we

16 see perivascular cuffing and all the signs of mild

17 acute inflammation that you would expect.

18 We don\'t see histological abnormalities,

19 we don\'t see any overt clinical signs of

20 abnormality. We are looking at lentivectors for

21 congestive heart failure. We have done lots of

22 intramyocardial injections, again, no overt signs

23 of toxicity.

24 We have done worst case where we have put

25 vector into the tail vein, we have looked for liver

> 217 1 toxicity and damage by histology. At the moment,

2 we have not found any significant amounts of vector

3 in the liver or lung.

4 Now, the problem with these studies, as we

5 have heard before, is this is maximum feasible dose

6 at the moment, and as our production systems

7 improve, when we generate more material, we are

8 obviously going to escalate the dose and have a

9 look and see what happens, but so far, so good.

10 \[Slide.\]

11 This is not a theoretical exercise that

12 Oxford BioMedica is going through. We are

13 intending to develop product based on lentivectors.

14 One of them is a treatment for Parkinson\'s disease.

15 We have already heard that the provision of

16 dopamine can have therapeutic benefit, and this is

17 the basis for current treatments where you provide

18 the patients with L-Dopa.

19 What we have done is to configure the

20 dopamine pathway into a single lentiviral vector,

21 so we are making the three key enzymes that are

22 limiting for the production of dopamine. Here, we

23 have been staining for dopamine in the side of the

24 brain that we have lesioned with a chemical lesion

25 to abolish dopamine production, and you can see

> 218 1 that we have established a depot of dopamine.

2 We are clearly very interested to learn if

3 this will translate into behavioral correction, and

4 because the established animal model for

5 Parkinson\'s disease is the monkey, then, we will

6 naturally move on and look at efficacy studies in

7 the monkey.

8 So, where we need to go to a higher

9 primate for efficacy, then, we clearly will, and we

10 can do the toxicity studies, as well.

11 \[Slide.\]

12 The other product that we are developing

13 is a treatment for late-stage prostate cancer.

14 This is a rather conservative approach. We are

15 using a promoter that has been in the clinic

16 before. We are using a therapeutic gene, TK. We

17 have tweaked it a bit to improve its activity, and

18 we are going to go into late-stage prostate cancer

19 patients.

20 This is a very slow growing tumor, so it\'s

21 a good target for lentivector. Prodrug strategy

22 will allow us to terminate the therapy easily. The

23 therapy itself is selective for dividing cells, so

24 we have a differential there against normal cells,

25 it is recurrent intractable malignant disease, and

> 219 1 we are going to do local delivery.

2 We have had preliminary discussions with

3 our Medicine Control Agency about the general field

4 of lentivectors. We intend to go to our gene

5 therapy advisory committee, which is the RAC

6 equivalent, and the MCA with a protocol for using a

7 lentiviral vector to treat late-stage prostate

8 cancer sometime next year, and we are obviously

9 doing the gene transfer and the efficacy studies to

10 underpin that.

11 \[Slide.\]

12 Now, our view is that lentivector

13 specification is an ongoing process as with any

14 other drug development process, and there will be a

15 point where we have achieved a basic design and

16 production that we believe to have a good level of

17 safety. There will be a point that we can get to

18 in the future where we have added the endless bells

19 and whistles, and really honed this to perfection,

20 but we would argue it is not absolutely necessary

21 to get to this point for some scenarios. There may

22 be certain constellations of genes or diseases

23 where it is ethical to test the basic design.

24 So, where you are using prodrug-activating

25 enzymes, which are not toxic per se, where you are

> 220 1 looking at terminal disease in adults, then, it may

2 be ethical to proceed with a basic system.

3 Where you are looking at growth factors,

4 cell death regulators, chronic disease, and

5 children, perhaps you may wish to advance your

6 vector design, but I think if the field is pushed

7 into making this type of vector too early, then, we

8 are never going to get to this point, we are never

9 going to see the full clinical benefits of these

10 vectors if we don\'t begin to get some information

11 early on, because we all have to do animal studies,

12 absolutely critical, but they do have their

13 limitations, and clinical evaluation will give us

14 extra information.

15 So, we see specification as an ongoing

16 process, and we would like to promote the notion of

17 doing clinical evaluation where it is ethical early

18 on, and this is where bodies, such as GTAC and RAC,

19 help us to inform those ethical decisions.

20 \[Slide.\]

21 I have considered two major

22 vector-specific safety issues\--obviously, each

23 transgene will have its own safety

24 issue\--replication-competent lentiviruses and

25 mobilization of the transfer vector in the target

> 221 1 cell. We want to minimize the generation and the

2 impact of RCLs, we wouldn\'t be worrying about RCLs

3 if we thought that they had no impact at all, and

4 \"minimize the inappropriate dissemination of the

5 transgene\" is the way I phrased it.

6 \[Slide.\]

7 We have identified six possible ways of

8 dealing with the issue of RCLs, which I am going to

9 go through each of these in turn, and then top this

10 off by saying that trust me, I am a molecular

11 biologist, doesn\'t actually work. You can design

12 these, but ultimately, we have to have a way of

13 testing them, and I will address those issues.

14 \[Slide.\]

15 So, if possible, use a non-pathogenic

16 virus, and the consequences of any RCL might be

17 minimized.

18 \[Slide.\]

19 If we just look at the features of HIV and

20 EIAV, our two chosen viruses, EIAV, equine

21 infectious anemia virus, there is not a huge body

22 of research that has been done on that although it

23 is catching up, so in that respect, HIV was a great

24 one to start with, so much information out there.

25 EIAV is somewhat more simple. It has

> 222 1 three accessory genes compared with six. It\'s an

2 equine pathogen. It doesn\'t replicate in human

3 cells. It causes a self-limiting anemia in the

4 horses, so there is a chronic carrier viremic

5 state. That is not to say that some horses don\'t

6 die, but it is really a chronic or a self-limiting

7 disease with a carrier status.

8 It is endemic in horses in the Tropics,

9 and there are rare outbreaks in stables worldwide.

10 Compare this with HIV, it is a human

11 pathogen, it does replicate in human cells. We

12 know it causes a fatal immunodeficiency. There is

13 a global pandemic, and there are 30 million AIDS or

14 HIV-positive people worldwide.

15 So, there are different profiles,

16 obviously safety profiles between those two vectors

17 at the start. One thing to say is 30 million

18 primates with HIV, and I phrase it like that, not

19 to be inflammatory, but to say that there is a

20 study there of natural infection by HIV in human

21 beings where there is every opportunity to pick up

22 endogenous retroviruses, there is an opportunity to

23 interact with other infections, and at the moment,

24 the course of the disease does not reflect the

25 emergence of any super pathogenic strain that has

> 223 1 picked up additional retroviral-like sequences or

2 any other properties through replicating in

3 patients.

4 So, we have a different profile. There is

5 no a priori expectation that EIAV would be a human

6 pathogen, but we all know that if put it in by a

7 different route, and modify a virus, we can\'t

8 absolutely say that.

9 There is a very low probability of any

10 patient ever encountering EIAV.

11 So, if you could show that your

12 non-primate lentivirus did all the things you

13 wanted it to do, had good efficacy, then, our view

14 would be we would choose to use EIAV rather than

15 HIV, but I think it is far from clear whether EIAV

16 would be able to fulfill every potential of

17 lentivectors.

18 \[Slide.\]

19 Split the vector production system into at

20 least three components.

21 \[Slide.\]

22 We have heard about this. Really, this

23 was established very well for retroviral vectors in

24 endless studies, that if you do split the vector

25 components up, the chance of generating an RCL is

> 224 1 much reduced and we can\'t really see why we

2 shouldn\'t just follow those concepts in generating

3 lentiviral vectors, because there is a lot of data

4 out there from MLV, so the basic system is the

5 therapeutic gene, gag-pol, and to my knowledge,

6 everybody is using a heterologous env in their

7 system.

8 \[Slide.\]

9 If possible, use a stable producer cell.

10 I really just echo in Dr. Verma\'s comments that if

11 you can, an idea situation is to use a cell line

12 that you can build up years of knowledge about.

13 DNA recombination is unlikely. We have had a

14 debate as to whether we think that is relevant.

15 You can show that it is genetically

16 stable. There are conventional manufacturing

17 parameters established, and there are well

18 characterized starting materials.

19 So, if you can use a stable producer, this

20 would be good, but I think there are arguments for

21 using transient systems, and this is obviously a

22 major point for debate. In my view, the transient

23 system should conform to the split vector paradigm.

24 \[Slide.\]

25 Eliminate all non-essential coding and

> 225 1 cis-acting sequences. This is just to reduce the

2 recombination and pathogenic potential.

3 \[Slide.\]

4 This is just a busy slide, just shows you

5 that the basic structure of the retrovirus is

6 there. With EIAV, we have got tat, rev, and this

7 other coding lesion called S2. These are the

8 accessory genes.

9 With HIV, we have got six accessory genes.

10 We have also got cis-active sites, we have got the

11 packaging sequence, the cPPT, which is involved in

12 reverse, cycle reverse transcription, the rev

13 response element, which is required for

14 orchestrating the transport and/or splicing of

15 messages, and the polypurine tract, which is also

16 important in replication.

17 So, transfer vector construction is

18 complicated. It is an easy thing to say we should

19 reduce the virus, get rid of everything, but there

20 are so many introns, spliced donors and acceptors,

21 accessory proteins, that is it not obvious, which

22 is why people started talking about developing HIV

23 vectors early in the nineties, and we are now

24 nearly 10 years on coming to the clinic.

25 \[Slide.\]

> 226 1 It is so difficult, why bother? I think

2 the reason is that we either don\'t know what these

3 accessory proteins do, and when you are doing a

4 risk assessment for your recombinant DNA, an

5 unknown is about as bad as you can get, or we do

6 have an inkling that they are doing something, and

7 mostly it is something that you are not very happy

8 about, possible growth factor interferes with some

9 cellular function, cell cycle arrest, and

10 obviously, in hearts is pathogenicity.

11 With EIAV, we have tried all sorts of

12 functional genomics analyses on S2 to try and find

13 out what it does in vitro, and we can\'t find it

14 doing anything, but it is absolutely clear that if

15 you delete that protein from the virus, it won\'t

16 cause any disease in horses, so it is a classic

17 pathogenicity factor, and that is one of the

18 problems in analyzing these accessory proteins, the

19 in vitro assays may not define the full range of

20 their properties.

21 So, we would say, if possible, get rid of

22 them.

23 \[Slide.\]

24 I am not immune from this generation thing

25 because it is a useful concept to show that we have

> 227 1 all been working hard to understand our system and

2 to develop it. What you can see is that we have

3 working with John Olsen, who we discovered was also

4 working on EIAV, and it seemed little point to

5 compete, we joined forces, and to try and develop a

6 vector producer cell, a basic cell, a thing we

7 called 8Z-20.

8 Really, I just want to show you the

9 evolution of our thinking. Here is our vector

10 genome, and we stripped out most of gag-pol. We

11 mutated the coding region, but were left to make

12 sure that we couldn\'t get any proteins produced,

13 but there is still an awful lot of material there.

14 We provided gag-pol, took away the

15 upstream sequences in the leader, which are

16 presumed to be important in packaging to make sure

17 this wasn\'t packaged, but we still had a lot of

18 sequence down the end.

19 We provided VSV-G with a tet regulation

20 system because if you overexpress VSV-G, it upsets

21 the production system. The problem then, of

22 course, is there is a region of homology between

23 the packaging site and the gag-pol, and there is a

24 region of homology between the RRE.

25 So, here we have got a potential for

> 228 1 recombination and also we are obviously still

2 expressing S2 and rev in this system.

3 \[Slide.\]

4 Nonetheless, we made a producer, stable

5 producer cell, and this has allowed us to scale in

6 roller bottles and we are producing a reasonable

7 titer, 10 6 transducing

units per mL for five days.

8 This means we have a benchmark vector on which to

9 monitor our improvements.

10 It is no good making a vector system which

11 is perceived to be safer, and you are only making

12 100 particles per mL. That is the point in this

13 exercise, so we figured we would start off with a

14 benchmark and work up from there. So, this has

15 been useful.

16 \[Slide.\]

17 But clearly, we would not be able to go

18 clinical with that, so the next thing we did was to

19 look at the transfer vector and strip out as much

20 as we could. This we have done, so we have just

21 retained the packaging sites, and we can dock in

22 two extra sequences that we want.

23 One is the RRE, because for some

24 unexplained reason if you provide rev and RRE to

25 the vector genome, you get improved titers. So,

> 229 1 that is an option, and then this cCPT for as yet an

2 unknown reason in some cell types, the cCPT can

3 optimize gene transfer and expression, but again

4 this is optional, not obligatory, we have a site

5 there.

6 Then, the polyadenylation of this internal

7 transcript can often do with being improved. There

8 is an element that a lot of people use, woodchuck

9 hepatitis element, and this can be docked in here.

10 Now, if we use this minimal vector, it is

11 not expressing EIAV proteins, it has got greater

12 than 8 kb insert capacity, but we have still got

13 homology in this region, and obviously, if we

14 include the PPT, we have got homology here, and if

15 we include the RRE, we have got homology there, so

16 we still need to do something.

17 \[Slide.\]

18 We have got to minimize the potential for

19 recombination, and this really means removing all

20 homologous sequences from the gal-pol packaging

21 plasmid, but we have to do that without

22 compromising the expression of gag-pol, and at the

23 moment, at this point, the dogma was that the

24 RRE-rev interaction was important for gag-pol

25 expression, so we need to examine the requirements

> 230 1 for that.

2 One way of changing the sequence is to

3 alter the nucleotide sequence, but retain the

4 precise meosic \[ph\] sequence because of the

5 redundancy of the genetic code, but it will be

6 pointless to do that if you would compromise the

7 expression efficiency.

8 \[Slide.\]

9 We have biology on our side, however,

10 because lentiviruses are very peculiar in

11 maintaining a most abnormal codon usage. If you

12 were to look at the codons that were commonly used

13 in mammalian cells, for example, for alanine, 53

14 percent of the time, this one is used, whereas, in

15 a wild-type lentivirus, it is only 19 percent of

16 the time, and we obviously don\'t have time to go

17 through this chart, but you can find numerous

18 occasions where a lentivirus will choose to use the

19 rarest TRNA in a cell.

20 What we decided to do is to codon-optimize

21 the gag-pol, so that we changed all the codons.

22 Not only would this alter the nucleotide sequence,

23 but it should give us preferred codons to ensure

24 good expression.

25 \[Slide.\]

> 231 1 So, we have made an entirely synthetic

2 gag-pol where the entire sequence of the gag-pol

3 has been changed with the exception of a small

4 region at the gag-pol overlap because there is a

5 frame-shifting event occurs to fuse gag and pol,

6 and that has a requirement for a particular

7 sequence.

8 There are no sequences flanking this

9 gag-pol cassette, that have been anywhere near a

10 lentivirus, totally unrelated. So, the codons have

11 been changed across gag-pol. It removes all blocks

12 of sequence homology, and a really added bonus is

13 this thing is now rev-independent, so that we could

14 get rid of the rev response, so that we have been

15 able to get all that junk down the end, and we have

16 done that for both EIAV and HIV.

17 \[Slide.\]

18 This allows us to create what we call\--we

19 have given up with the generations now\--what we

20 call a minimal EIAV vector system, where we have

21 the stripped-out transfer vector, completely

22 synthetic gag-pol, and we have our envelope. That

23 is no obligate requirement for any accessory gene,

24 but rev-RRE can improve the yield.

25 There are no functional viral proteins or

> 232 1 significant coding regions in the transfer vector.

2 The transfer vector contains only 1,100 nucleotides

3 of the original EIAV. There is no significant

4 homology between components. What I mean by that,

5 if there is no stretch of longer than 6 nucleotides

6 that is shared between any of these components.

7 It has been approved in the UK by our

8 health and safety executive for containment level 1

9 use depending on the transgene, so if we are using

10 something like feta-gal, this is basic laboratory,

11 good laboratory practice, the lowest containment,

12 and we have a similar system for HIV.

13 \[Slide.\]

14 We have now gone on to create a second

15 generation packaging cell, and this is work in

16 progress. We don\'t know if we are going to be able

17 to achieve this. In the transient systems, yields

18 are fine, but with these three plasmids, we have

19 every expectation that we will be able to generate

20 a production system.

21 The packaging system will have the

22 synthetic gag-pol, it will have VSV-G, and then we

23 will make a version that will optionally have rev,

24 and we have code and optimized that, so again,

25 there is no sequence homology with the original

> 233 1 vector, and we can optimize expression.

2 We aim to meet the 8Z-20 characteristics,

3 so with this improved system, we can\'t hit the

4 level that we got with 8Z-20, then, we will have to

5 have a decision as to whether these improvements

6 really were worth the tradeoff in manufacturing

7 efficiency.

8 \[Slide.\]

9 Reduce the packaging of vector helper

10 components. Well, we are going to learn our lessons

11 from retroviral vectors and we are going to

12 eliminate packaging and dimerization signals.

13 \[Slide.\]

14 We have done a lot of studies which I can

15 only briefly go into. Clearly, when a construct

16 expresses gag-pol, it can package the RNA that went

17 on to express it, that is the problem. Some

18 lentiviruses do package co-translationally, things

19 like HIV-2, so maybe you would steer clear of those

20 for developing vector systems.

21 We have made all sorts of mutations and

22 changes in the leader sequence, various deletions

23 in the presumptive packaging site. We have a

24 completely synthetic gene which should have no

25 packaging site, and we have made a version that has

> 234 1 a two-stop code, so we are only making RNA, RNA

2 control.

3 \[Slide.\]

4 These experiments are all quantified by

5 quantifying the particles using an assay called

6 PERT, which I will talk about, by quantifying the

7 RNA using real-time PCR, by internally controlling

8 with actin RNA, so that you are always trying to

9 compare a packaging situation where you have got

10 similar amounts of RNA, similar yields of protein,

11 so that you can make some comparisons.

12 The type of data that we have, there is a

13 baseline. You will never get rid of background

14 noise in biology. I really believe that that is

15 not possible. So, we have set our baseline as the

16 highest amount of actin that we found in any one

17 sample.

18 Then, we asked how much RNA did we find in

19 the particles compared to how much of that RNA did

20 we find in the cell. The better a particle is at

21 packaging, the more of the RNA it will have picked

22 up from the cell. That is our feeling.

23 So the wild-type gag-pol with the

24 wild-type packaging site packages itself. The

25 deletion really doesn\'t package itself, and the

> 235 1 synthetic gag-pol doesn\'t package itself. So, we

2 have knocked down the packaging to background level

3 by altering the packaging site.

4 It is important to do these studies in the

5 context of a transfer vector because another way of

6 getting your RNA into the cell is if you have got

7 any region of homology, you may get dimerization

8 and piggy backing. In fact, we didn\'t see this in

9 this particular EIAV vector. We have seen it in

10 some mutations that we have made in HIV.

11 The bottom line is the wild-type gag

12 packages itself and the vector. The packaging site

13 mutant gag does not package itself, but it does

14 package the vector, and the synthetic gag-pol does

15 not package itself, but it does package the vector.

16 So, there we have packaging constructs

17 which package the vector, which is good, that is

18 what we need, but which don\'t package themselves

19 above background. So, partial or complete removal

20 reduces packaging to background levels, and we have

21 had no evidence of piggybacking by dimerization

22 with the transfer vector.

23 \[Slide.\]

24 So, we have addressed a number of issues

25 for minimizing RCLs, now we need to do some assays,

> 236 1 and we want to use sensitive, calibrated assays,

2 and we want the assays to relate to the properties

3 of any RCL, and this actually is the conundrum

4 which we have touched on in the morning as to what

5 is the nature of the RCL.

6 \[Slide.\]

7 Well, what I have done is suggested that

8 the only way that RCLs could arise in the EIAV

9 vector system is now by non-homologous

10 recombination because we have removed the potential

11 homologous recombination and by background

12 packaging in the particle.

13 By definition, we can\'t predict the

14 arrangement of genes and the recombination events

15 that would arise from non-homologous recombination.

16 There is no way of doing that.

17 So, we have tried to take a generic view

18 and we have said that all RCLs must have gag-pol.

19 By definition, this is the transferring entity.

20 The most likely gag-pol is vector derived, the one

21 that we put in. If there is any other gag-pol that

22 we have somehow inadvertently empowered from the

23 cell, it is only rendered transmissible with the

24 vector-derived env.

25 So, all our constructs are going to have

> 237 1 the vector-derived gag-pol and the vector-derived

2 env, vector-derived gag-pol and some other env that

3 we can\'t predict, but we can\'t say that it could

4 never be there, a gag-pol that is endogenous that

5 we can\'t predict what it is, but we don\'t want to

6 ignore the fact that it might be there, and the

7 vector env.

8 The universal feature is a gag-pol, so you

9 can screen for transmissible reverse transcriptase,

10 all of these will have that. You can then qualify

11 any ambiguous results by a second screen for

12 transmissible gag and pol, and by screening for

13 transmissible env.

14 So, we have taken a theoretical RCL

15 structure, developed a generic view, and we don\'t

16 want to restrict our view by adjusting the amount

17 of VSV-G. We don\'t want to design VSV-G-specific

18 assays because we may not use VSV-G for all our

19 applications.

20 \[Slide.\]

21 We propose to test vector preparations and

22 propose production cells following the current CBER

23 guidelines for MLV-derived vectors. We are going

24 to use two assays, F-PERT, which is

25 fluorescence-based product enhanced reverse

> 238 1 transcriptase assay, as our primary assay tool, and

2 we are going to use a PCR assay to resolve

3 ambiguous PERT assay results. This is a specific

4 assay for hypothetical recombinant molecular

5 structures.

6 We have developed and strategies in

7 collaboration with relevant UK Government agencies,

8 and these are the Laboratory of the Government

9 Chemist, the National Institute for Biological

10 Standards and Control, and also we have had

11 discussions with a contract manufacturer, Q-One

12 Biotech, because it is important that anything we

13 develop can be transferred to a manufacturer for

14 small companies or groups who are not going to

15 develop their own in-house manufacturing

16 capability.

17 \[Slide.\]

18 F-PERT assay has an advantage. There are

19 many groups working on this. It is being refined

20 all the time. It was originally developed for

21 looking for HIV in plasma. It can detect a single

22 particle. Obviously, one has to qualify that

23 mixing it up with whatever brew you are trying to

24 find the particle in.

25 Basically, you collect particles, you

> 239 1 disrupt them, liberating the pol. You then provide

2 an RNA. This is the MS2 phage RNA and a specific

3 primer. Any pol that is there will then make a

4 cDNA.

5 You then amplify up the cDNA using

6 specific primer pairs and you detect the amplified

7 product using the standard TaqMan technology.

8 This assay is independent of the nature of

9 events that lead to the RCLs. It is broadly

10 applicable and high sensitivity. We have put 10 to

11 100 particles, because I didn\'t want to be held to

12 a figure. We are obviously going to refine that

13 and come up with a standard window of sensitivity

14 that we deem acceptable for our particular

15 application.

16 This assay has been modified with a series

17 of controls to protect against false positives.

18 \[Slide.\]

19 We are looking at the sensitivity. It

20 will detect manganese and magnesium-dependent

21 reverse transcriptases. You can find these over a

22 range of dilutions, and we are obviously spiking

23 mixtures and looking at the sensitivity in the

24 context of the soup, the end of production soup.

25 \[Slide.\]

> 240 1 A qualifying assay is a PCR assay, and

2 here we are looking for gag, we are looking for

3 pol, we are looking for env. We are looking for

4 them individually and we are looking for them

5 linked with each other. We are doing that by just

6 making a set of nested primer pairs.

7 These have just been identified by the

8 clever biomathematicians doing blast analyses, and

9 our primers are specific for our vector, and they

10 can, by working in particular pairs, they will

11 amplify a small region or a large region, and we

12 can look to see what sort of things are coming out.

13 \[Slide.\]

14 Obviously, looking at the sensitivity, and

15 we have defined the sensitivity in the context of

16 the assay cell genomic DNA, and we have set a level

17 that the assay must detect 1 to 10 copies in

18 background of 10

5 genomes.

19 Here, you can see our cutoff points is

20 between 1 and 0.1 for gag, and similarly with pol.

21 In this particular experiment, VSV-G was slightly

22 less sensitive. We obviously can develop these for

23 any envelope that we choose to use.

24 \[Slide.\]

25 So, our procedure that we are putting to

> 241 1 you for discussion, we transduced cells with the

2 test article and we amplified, so we split the

3 cells at an appropriate ratio over an appropriate

4 time, and these are parameters that will be

5 defined.

6 Then, on the supernatant, we do a PERT

7 assay, and then we can qualify that if necessary

8 with a PCR assay.

9 Our amplification is going to be in two

10 types of cell lines - the 293 cells, which support

11 the transduction by a wide variety of vectors and

12 pseudotypes, and they are the production cell line,

13 and also lymphoid cells, and we are currently

14 screening a range of lymphoid cells. This is

15 important because the potential in-patient target

16 for RCLs are hematopoietic cells, and by using two

17 different cell lines, we are sampling a range of

18 viral replication characteristics.

19 As a positive standard, we are using FeLV,

20 because that gives efficient amplification in both

21 these cells. It is a regulatory standard, and we

22 don\'t get any interference with lentivector.

23 We believe the amplification process is

24 essential, and this is readily adapted for

25 screening producer cells also.

> 242 1 \[Slide.\]

2 So, we would propose that we consider the

3 following points when we are talking about RCLs.

4 If possible, use a non-pathogenic virus, split the

5 vector production system, preferably use a stable

6 cell line, eliminate all non-essential coding and

7 cis-active sequences, minimize the potential for

8 homologous recombination, and we don\'t want to set

9 an absolute figure on that; reduce the packaging of

10 vector helper components, and use sensitive,

11 calibrated assays for RCLs after amplification in

12 human cells.

13 \[Slide.\]

14 So, we have gone some way to addressing, I

15 believe, this issue. The next one is mobilization

16 of the transfer vector in target cells.

17 \[Slide.\]

18 We can see two ways of doing this. First

19 of all, use a transfer vector that isn\'t very well

20 mobilized, or reduce the level of the mobilizable

21 RNA in the target cell.

22 \[Slide.\]

23 Well, what do we mean by use a transfer

24 vector that is poorly mobilized, by what? What are

25 we concerned about here? There may be an infinite

> 243 1 number of possibilities for concern, but the one

2 that we thought might be of the most concern is

3 HIV. That is the agent of mobilization that is

4 probably of most concern. It is a virus that is

5 around in the human population, and we should

6 probably ask the question is our transfer vector

7 mobilized by HIV.

8 HIV is not the only virus that we might

9 consider, so we decided to look at MLV, as well, as

10 sort of a generic retrovirus, so, yes, they may be

11 endogenous retroviruses, yes, they may be other

12 things out there that we haven\'t thought of, but if

13 we study mobilization by HIV, as a known human

14 pathogen, and MLV, as a retrovirus that we know a

15 lot about, we thought that might be useful.

16 So, we set up some cross-packaging assays

17 where we take a cell that has a reporter transfer

18 vector, and we put gag-pol and env in this cell.

19 The env is VSV-G, and then we mix the EIAV transfer

20 vector with its own gag-pol.

21 We look by FACS after five days, but also

22 after two serial pathologies, because there is this

23 phenomenon of so-called pseudotransduction where

24 you might score a positive, but you are not sure if

25 that is a genuine integrated event, so you passage

> 244 1 the cells to make sure you really are looking for

2 an integration.

3 \[Slide.\]

4 I am only going to show you some data

5 because I am going to run out of time. These

6 results are quite interesting. What you see is, of

7 course, EIAV mobilizes itself very well, a titer of

8 10 6. It mobilizes HIV at 1,000-fold

less.

9 It mobilizes MLV at a little bit lower

10 than that, a couple of hundred. HIV mobilizes

11 itself very effectively, as you would expect, it

12 mobilizes EIAV at 1,000-fold lower level, and MLV,

13 virtually at 1,000-fold level, so the difference

14 between these two is really not significant.

15 So, the ability of EIAV to be mobilized by

16 HIV is just the same as MLV. MLV mobilizes HIV,

17 and it barely mobilizes EIAV. So, there is a

18 little bit of background cross-mobilization, as you

19 would expect, consistent with these particles

20 picking up RNA.

21 The main thing I want to emphasize here is

22 that HIV does not interact with EIAV any

23 differently from MLV. We already have MLV vectors

24 in the clinic in HIV-positive patients. So, I

25 would argue that there is no reason why one

> 245 1 shouldn\'t put EIAV vectors in HIV-positive patients

2 either, but there is the issue, with HIV, that if

3 there is HIV around, it will be mobilized by HIV.

4 \[Slide.\]

5 So, if you have a situation where you

6 can\'t address the question by using a poorly

7 mobilized vector, or there is some reason you are

8 expressing a very toxic gene, and even dropping the

9 mobilization by 3 or 4 logs is not adequate, you

10 may want to do something else to the vector.

11 There are a number of ways of reducing the

12 level of the mobilizable RNA in the target cell,

13 and one is by the SIN vectors that we have heard

14 about. I don\'t think this will be necessarily

15 required for all lentivectors. I wouldn\'t want to

16 stand up and say I don\'t think it is important,

17 that is why we haven\'t done it.

18 We have actually looked at this in EIAV,

19 made a classical SIN vector. It drops the viral

20 genome down to 900 nucleotides. It deletes the

21 LTR. We had a comment that maybe this was useful

22 in reducing the oncogenic potential. There is no

23 evidence for lentiviruses of any oncogenic

24 potential despite massive viremic states. There is

25 no evidence that having a promoter here is a

> 246 1 problem.

2 We don\'t particularly like the SIN vectors

3 because we think there are advantages to

4 introducing the transfer vector into the genome by

5 transduction rather than by transfection, and this

6 is a jolly good site to put tissue-specific or a

7 regulated promoter, so if you decide you are not

8 going to use that site, it restricts your options

9 for making advance vectors.

10 \[Slide.\]

11 Nonetheless, we have made this.

12 Interestingly, we have compared our SIN version

13 with the basic EIAV LTR, and that is naturally a

14 SIN because it requires tat, EIAV tat doesn\'t

15 interact with human cycline, so it doesn\'t work, so

16 it is a sort of a natural SIN, and we have compared

17 it with R8Z-20 line.

18 This is a pure cell line, this is a

19 population, so we have obviously refined the study,

20 and we predicted we would have very, very low

21 amounts of RNA. Of course, this RNA could be

22 read-through, it could be anything, it could be

23 short bits, long bits, we are not quite\--you know,

24 one doesn\'t know what the nature of packageable RNA

25 is, and the CMV RNA likewise could have some

> 247 1 upstream transcript.

2 So, the first thing we do is measure

3 packageable RNA using our very sensitive assays

4 that I have described, and we can see that both the

5 SIN vector and the LTR vector give low levels of

6 packageable RNA, which is 3 orders of magnitude

7 lower than the CMV, so we have dropped the

8 packageable RNA by 3 logs.

9 When we look at the titer as to whether

10 that RNA is actually going on to do anything, then,

11 there is a differential of, in this particular

12 experiment, 5 logs. So, you can make an RNA

13 completely, or virtually completely I should say in

14 this audience, unmobilizable because you can drop

15 its levels. We are not convinced that that is

16 important.

17 \[Slide.\]

18 We have had before us this question of

19 mobilization of HIV vectors by HIV, is this a

20 special case for the treatment of AIDS, so you turn

21 virus escape into an attribute.

22 \[Slide.\]

23 Yes, there are various versions. You have

24 one before you. We have also been developing one,

25 and there are a number around. The aim here is to

> 248 1 put in an inhibitor of HIV replication, preferably

2 an RNA, because I don\'t think you want to engineer

3 stem cells with any proteins that could be

4 immunogenic. So, a therapeutic RNA, and here you

5 can configure the therapeutic RNA as an internal

6 constitutive transcript or you can configure it as

7 a full-length inducible transcript where when the

8 incoming virus comes in, it switches on gene

9 expression.

10 Now, really, you want your therapeutic, I

11 think, to stop the virus. You don\'t want to close

12 the stable door. You would like to stop the virus

13 actually getting going. So, you want to knock down

14 chemokine receptors, really stop it getting going,

15 but if it does manage to integrate and start to

16 make more virus, then, by having the vector there,

17 you can not only knock down the level of that

18 virus, but the virus can pick up the vector, and it

19 can pick it up two copies, or in a hybrid, and

20 propagate the seeds of its own destruction.

21 So, mobilization amplifies the therapy if

22 virus escapes the first wave of ribozyme. I think

23 there is a certain amount to be said for that

24 strategy, and I think it is one that really does

25 deserve some debate where you are actually

> 249 1 disseminating the therapy through the patient.

2 \[Slide.\]

3 Whenever you do this in the lab, and you

4 have heard this in VIRxSYS\'s proposal, you get

5 escape variants, and we all say, oh, in vitro

6 concentration, too many cells, too close, and it

7 won\'t happen in vivo, but to my knowledge, nobody

8 has actually asked what is the genetic nature of

9 those escapes, and that might be a useful thing.

10 \[Slide.\]

11 After that brief digression into HIV, and

12 I think we will have an opportunity to talk about

13 that more tomorrow, I hope I have convinced you

14 that we have made some progress in addressing the

15 issues of replication-competent lentiviruses, and

16 some progress in addressing the issues of

17 mobilization of the transfer vector.

18 \[Slide.\]

19 I have described a specification for a

20 lentivector with a good safety profile, no

21 pathogenic proteins, very poorly mobilized by HIV

22 to the same extent as MLV. No potential for

23 homologous recombination by definition, less than

24 10 nucleotides is regarded as not to be a site for

25 recombination where RCLs are extremely unlikely.

> 250 1 \[Slide.\]

2 I would like to suggest that we can begin

3 to look at a generic set of guidelines that will

4 cover all lentivectors, but I think that we will

5 have to embellish them on a case-by-case basis for

6 each particular lentivector for each particular

7 indication.

8 I think we must show efficacy in a

9 relevant animal model, must be able to produce this

10 vector at GMP or in the spirit of GMP, good

11 manufacturing practice, and it should be configured

12 with the following points in mind.

13 Eliminate non-essential proteins and

14 sequences. Ensure extremely low, preferably zero,

15 homologous recombination potential between the

16 components. Show a significant differential

17 between self-mobilization and mobilization with

18 HIV, or use a vector that after integration

19 generates a significant reduction in mobilizable

20 RNA, and I suggest we use MLV as a benchmark for

21 mobilization.

22 Use a minimum of three split components in

23 a stable packaging cell line or in a very low

24 homologous-recombination transient system.

25 Use sensitive, calibrated assays for RCLs,

> 251 1 follow MLV as a guide. There is all that

2 experience out there. Put in an amplification step

3 in an appropriate human cell line.

4 Test for no significant acute toxicity in

5 the relevant animal efficacy model.

6 \[Slide.\]

7 There is a series of references that you

8 can follow up what I have said if the spirit moves

9 you, and there is a lot of people in the company

10 that have been developing this work, but

11 particularly mentioned are collaborators John Olsen

12 for the production systems, Karen O\'Malley for some

13 of the neurobiology, James Uney, where we have been

14 looking at long-term correction of various animal

15 models of disease, and our colleagues in the

16 regulatory agencies and at Q-One Biotech who have

17 been helping us design these systems.

18 Thank you.

19 \[Applause.\]

20 DR. SALOMON: Thank you very much for that

21 very nice presentation.

22 Questions & Answers

23 DR. SALOMON: One of the things I was

24 thinking about when we were going through this is

25 can we begin to articulate what would be the

> 252 1 definition of an ideal replication-competent

2 lentiviral assay.

3 Everybody is going to do it a little

4 different, right? I mean we have already heard

5 several different possible cell lines, we know that

6 there are going to be different strategies to

7 engineer the vector. That could have implications

8 into what then would be the target cell line.

9 Can we maybe figure out some elements that

10 if a sponsor brings it forward, you fulfill these

11 elements, it\'s a good RCL assay?

12 DR. KINGSMAN: Well, my view is the way

13 not to go is to try and design artificial viruses

14 to create some positive control, because you may or

15 may not be right.

16 I think the assay for transmissible

17 reverse transcriptase is a very useful one. It is

18 quantitative, it\'s reproducible, it is looking for

19 the entity that you are interested in, in terms of

20 an RCL\--so this is all, I am restricting my

21 comments to a replication-competent entity\--so if

22 there is a reverse transcriptase which is

23 transmissible, then, you need to detect that.

24 That, I think is the important thing, is

25 there a transmissible reverse transcriptase. The

> 253 1 PERT assay is a sensitive, reproducible biochemical

2 assay. Now, 10 years ago, these assays weren\'t

3 available, we used surrogate assays.

4 The mobilization of other vectors, you

5 know, these were indirect assays, and there is a

6 feeling that what is old is good, and we should

7 stick with that, but in actual fact, using these

8 indirect assays, when you have got highly sensitive

9 biochemical assays, may not be the thing to do.

10 With the retroviral field, people were

11 making mouse retroviral vectors in mouse cells, and

12 therefore, there was a whole ethos of looking for

13 these viruses and using mouse assays.

14 We have a horse virus that doesn\'t

15 replicate in human cells, and doesn\'t replicate

16 very well in horse cells either. You would have to

17 get primary dermal fibroblasts. So, there is no

18 meaningful assay that you can do.

19 So, the biochemical assay seems to me to

20 have a lot going for it. It is looking for what

21 you are interested in. If you find a transmissible

22 reverse transcriptase, you have got to ask what it

23 is.

24 Then, you go and you can do your PCR

25 analysis to find out what it is, but there is no

> 254 1 way of second-guessing from these series of

2 illegitimate recombinations and hypotheticals what

3 you should actually do.

4 DR. SALOMON: If we take that principle

5 then, just to kind of make sure that I understand

6 what you are saying, so you are going to base your

7 assay for RCL on the assumption that an RCL has to

8 be carrying an intact gag-pol.

9 DR. KINGSMAN: Yes.

10 DR. SALOMON: I mean it also has to be

11 carrying a number of other things, but at least it

12 has to have a gag-pol.

13 DR. KINGSMAN: That is the bare minimum,

14 yes.

15 DR. SALOMON: And the pol should encode an

16 RT.

17 DR. KINGSMAN: Yes.

18 DR. SALOMON: So, what would be your assay

19 limit, how do you define the lower limit, any RT?

20 I mean it has got to be zero? We both know that

21 quantitative PCR never gives you a zero result,

22 right, it is all based on a threshold.

23 So, how would one validate a number

24 achieved in this wonderful new quantitative assay?

25 DR. KINGSMAN: You would do it with

> 255 1 reference to a standard, a standard that we are

2 going to use is FeLV, and we would do a spike

3 mixture, and we would detect a single RCL, a single

4 virus over our amplification process, so our assay

5 would detect that, and the limits of sensitivity of

6 our assays would allow us to do a plus/minus. I

7 think that is what we would go for, a plus/minus.

8 DR. SALOMON: Okay, so keep going. Now

9 you are just defining the sensitivity of the assay.

10 What I am pushing you to say then is what would be

11 the definition, then, of an appropriate limit for

12 this quantitative RCL assay then, one particle in a

13 background of 100,000 or a million or\--I am not

14 talking about detection now, I am talking about now

15 it is safe, you could use this in a clinical trial.

16 DR. KINGSMAN: In the 300 mL\'s or 1

17 percent of the\--so following the guidelines for

18 MLV, so it would be in 100 mL\'s at 95 percent

19 confidence, and in 1 percent of the post-production

20 cells, if you scored a plus, then, you would sling

21 it out. It is just following the MLV protocol.

22 DR. SALOMON: One last question. Are we

23 saying, then, that we are comfortable with the idea

24 that an RCR limit set out of some empiric

25 experience with MLV is appropriate for lentivirus?

> 256 1 DR. KINGSMAN: Yes.

2 DR. MULLIGAN: Just a quick question about

3 the PERT assay. If the principle is you have to

4 have an intact pol, why not go simple and just do a

5 PCR or an RT/PCR for pol sequences? What is the

6 relative sensitivity of those? It has got to be

7 the RT/PCR, doesn\'t it?

8 DR. KINGSMAN: Yes, I think the RT/PCR

9 relies on having primer pairs, and it relies on

10 knowing something about the sequence. That is

11 really I think why one would go for the biochemical

12 assay of reverse transcriptase, so you are not

13 making any judgments about the sequence in your

14 front line assay. Your front line assay is for any

15 transmissible pol.

16 DR. MULLIGAN: The question is in the

17 normal case where you don\'t have an unusual pol,

18 what is the relative sensitivity. You would hate

19 to miss in your primary screen something. What are

20 the chances you wouldn\'t pick up a normal pol

21 sequence via the PERT assay, but you would by the

22 RT assay?

23 DR. KINGSMAN: We need to do repeated

24 studies, but at the moment, the sensitivity, we are

25 saying is that the PERT can detect 10 to 100

257

1 particles, and the PCR assay can detect 1 to 10

2 genomes in a background of 10

5 genomes.

3 DR. MULLIGAN: So, you would agree that if

4 you moved closer to the PCR, then, well, I guess I

5 would agree that it would make sense if you got

6 closer to the RT/PCR sensitivity, but you may not,

7 right, you may not get to that point?

8 DR. KINGSMAN: Well, the reason I am being

9 conservative in the estimate of the sensitivity of

10 the PERT, other people will say you can detect a

11 single particle, a single virus-like particle, and

12 I think it is perfectly possible to do that, but we

13 don\'t have a full set of data where we have done

14 mixing experiments and said that in the context of

15 the culture supernatant we can detect a single

16 particle.

17 So, what we are relying on is doing an

18 amplification process where we can\--we are not

19 looking for a single particle in the primary

20 harvest, we are looking for the consequence of that

21 single particle amplifying out, so that we can then

22 detect.

23 I would not advocate using either of these

24 assays on the post-production supernatant, that

25 there has to be an amplification step, and then the

> 258 1 sensitivity of your assay is kind of balanced with

2 the amplification. If you have a massive

3 amplification, then, your detection, you may have a

4 tradeoff on the sensitivity of your detection.

5 DR. MULLIGAN: I guess the reason why you

6 have to do it that way is obviously you can\'t do

7 the PERT in the presence of particles that are the

8 pseudotype particles.

9 DR. KINGSMAN: No, no.

10 DR. MULLIGAN: But that is a fundamental

11 difference, so you are not looking, right, you are

12 assaying, you can\'t look for a rare species in your

13 production of virus by this test, you can only look

14 for a transferrable amplified.

15 DR. KINGSMAN: Yes, and that is what we

16 are defining as the issue. Now, if that is not the

17 issue, then, we obviously need to address the other

18 issues, but what we have said is that what we are

19 concerned about is a replicating entity that could

20 turn a therapeutic vector into something that is

21 detrimental to the patient.

22 So, the thing that we can identify is a

23 transmissible gag-pol that may have some

24 unexplained pathogenic potential. We obviously

25 can\'t look at our EIAV vector and think of a

> 259 1 pathogen just looking at the components that we

2 have got left. We wouldn\'t say that means it is

3 safe obviously, because nobody has tested a

4 replicating virus with those components injected

5 into a brain under the particular set of

6 circumstances.

7 So, what we are saying is that we are

8 concerned about any replicating entity, but with

9 EIAV, we have not become concerned about whether

10 there is gag-pol there or VSV-G or some other bit

11 of a vector genome. That, we have not regarded as

12 a major issue. The issue for us is whether there

13 is a replication-competent entity, and that is what

14 we focused on.

15 DR. KAPPES: Susan, I thought I had my

16 question together until you made that last

17 statement, but let me say what I was going to

18 anyway.

19 I think I favor the notion or the

20 principle of looking for recombinants that contain

21 reverse transcriptase, and as you pointed out, what

22 it measures is the potential of that recombinant to

23 produce RCR even though it might not itself

24 represent a replication-competent form of vector or

25 virus.

> 260 1 Moreover, it is a way of measuring prior

2 to administering that vector or those recombinants

3 if they exist, that is, recombinants which contain

4 functional machinery, such as reverse

5 transcriptase, against the possibility that

6 additional recombination will occur in vivo which

7 can ultimately, although probably unlikely, lead to

8 RCR.

9 DR. KINGSMAN: Yes. I think that is

10 right. We are not making any value judgment as to

11 how these things arose or what their complete

12 nature is. We just think it is not a good idea to

13 have them there, and we are proposing to use

14 biochemical assays to measure these. That is our

15 proposal.

16 DR. BORELLINI: Flavia Borellini from Cell

17 Genesys. In my experience with the PERT assay, you

18 have a problem of a background due to cellular

19 polymerases that can actually score in a PERT

20 assay. So, I would anticipate that you would find

21 yourself in a situation where you have a lot of

22 positives, that then you have to go and verify by

23 your cell PCRs.

24 Then, why not do the PCR right away and

25 skip the PERT?

> 261 1 DR. KINGSMAN: Well, we are open to that

2 suggestion of doing both of them. The background

3 with telomerase is well known in PERT, and it can

4 be solved by variously spiking the mixture with

5 calf thymus DNA, and there is a whole series of

6 modifications to PERT that have produced those

7 backgrounds.

8 Obviously, when we do a number of scale

9 runs, if we keep find that we get a level of

10 background positives that require a qualifying

11 test, then, that would inform us to bring the

12 qualifying test up, level with the PERT test, but

13 at the moment, we are going to try and run with the

14 notion of doing a single defining assay and having

15 a secondary backup assay.

16 But clearly, if we find false positives

17 occurring at an irritatingly high rate, then, we

18 will take steps to deal with it, but this issue of

19 telomerase is well known and can be solved by

20 varying the protocol, you can knock it down.

21 DR. BORELLINI: Actually, it is not only

22 telomerase, but it is just DNA polymerases that can

23 cause background.

24 DR. KINGSMAN: Yes, or whatever, I was

25 wrong to imply a mechanism there.

> 262 1 DR. NALDINI: Luigi Naldini. I was

2 questioning again the assay for the RCL. I mean in

3 principle, in your amplification step, you would

4 like to use the most permissive system to allow

5 amplification of a recombinant.

6 Wouldn\'t that be made of a horse cell for

7 an EIAV-derived vector?

8 DR. KINGSMAN: We don\'t really want to

9 study horse cells when we are interested in human

10 cells, and we know that those cells that we have

11 chosen are incredibly permissive for a whole range

12 of vector configurations with different

13 pseudotypes.

14 DR. NALDINI: But they are not permissive

15 for the EIAV virus.

16 DR. KINGSMAN: Yes, but we are not working

17 with the EIAV, we are working with two components

18 from that virus which there is no envelope.

19 DR. NALDINI: You have an LTR from the

20 EIAV, you have gag-pol from the EIAV. So, if you

21 have a recombinant, to have higher chances of

22 detecting that, you should have a system which is

23 permissive to that.

24 DR. KINGSMAN: I really don\'t see that one

25 needs to go down studying a horse virus in primary

> 263 1 pony dermal fibroblasts in order to find a

2 qualification for a vector system which is so far

3 removed from EIAV you can possibly get it, and you

4 are interested in human cells and what goes on in

5 human cells, and what you want to know is does this

6 thing amplify in human cells, not do we amplify an

7 RCL in h horse cells. It is do we amplify one in

8 human cells.

9 DR. NALDINI: You want a biological

10 barrier when you are using the vector in your

11 application. I don\'t think you want to have any

12 biological barrier when you are testing your

13 system. You want to have the most amplification

14 possible. I think it is a matter of debate.

15 DR. MULLIGAN: I would say you are both

16 right. I think, if I could reinterpret his point,

17 to make it sound more interesting, it would be that

18 if a horse cell allowed you to amplify something

19 that then was able to be shown to infect human

20 cells, maybe there would be a reason.

21 He is saying there could be a reason that

22 this would be a uniquely sensitive way to amplify

23 something, not to make it into something different,

24 but just so it is detectable, but ultimately, you

25 would have to show that that virus that you

> 264 1 amplified on horse cells was capable of having

2 human infectivity.

3 DR. KINGSMAN: And I can\'t see the point

4 of doing that.

5 DR. ALLAN: Can I follow that same line of

6 questioning? Just in the sense that your vector

7 now, is it a SIN vector now, you are generating a

8 SIN vector?

9 DR. KINGSMAN: You can, but I don\'t think

10 you need to use a SIN vector.

11 DR. ALLAN: So, you have an intact LTR,

12 EIAV LTR.

13 DR. KINGSMAN: Yes.

14 DR. ALLAN: So, then, the issue then is

15 relevant because you could use a cell line that has

16 tat expressed in it, and then you can look to see

17 whether that LTR is going to function, if you

18 contain the tat in the cell line, so you could

19 actually have a reporter assay.

20 DR. KINGSMAN: Yes, I think you could

21 infinitely manufacture cells with various

22 reporters, you could provide tat, you could

23 provide, you know, EIAV envelope to make sure it

24 maximally picks up the gag-pol, to make sure that

25 you are maximally testing that, but I don\'t see the

> 265 1 advantage of doing that over and above the strategy

2 that we have proposed, particularly as I have shown

3 that the vector is completely stripped out of all

4 EIAV pathogenic entities.

5 There is no tat, there need not be any

6 rev. There is gag-pol and yes, you are right, we

7 don\'t know if that has a fundamental pathogenic

8 potential, but that may be less likely.

9 So, I am a little bit averse to recreating

10 a horse virus out of this, when we are really

11 trying to look for entities that replicate in human

12 cells, and we have stripped this vector system down

13 to such a point that although we are not going to

14 say there is no possibility because that would be

15 wrong, it is going to be very, very unlikely, and

16 it is very difficult to draw out any entity that

17 you could say would be problematic.

18 So, I think I would try to resist

19 artificially creating viruses to validate these

20 lentiviral vectors, which are different from MLV

21 and that they have heterologous proteins, they are

22 much more stripped down than any MLV vector that

23 has ever gone into the clinic, and it may not be

24 sensible to try and artificially create a positive

25 standard.

> 266 1 DR. MULLIGAN: Can I change the topic?

2 There is a mobilization question. We will talk

3 much more about the virtues of mobilization, I

4 guess, but since we have you here and you did give

5 a sales pitch for your approach, one of my concerns

6 would be unless you have a model that would reveal

7 to us convincingly that having mobilization

8 potential would be helpful, I weigh that against

9 the risk of a bad thing happening in vivo, and the

10 question is I can\'t think of how you would possibly

11 test this.

12 How would you possibly test and make the

13 case that having the capacity for mobilization

14 would be useful?

15 DR. KINGSMAN: I agree with you that the

16 sole advantage in my mind of using an HIV vector to

17 treat AIDS is the potential for mobilization,

18 because if you are just asking for tat regulation,

19 then, there are other vectors that can switch on

20 things with tat. If you are asking for

21 CD34-positive gene transfer, you can use retroviral

22 vector.

23 If you are asking for stem cell gene

24 transfer, maybe you could use something that wasn\'t

25 HIV, although I have to say HIV is superb at

> 267 1 putting genes into stem cells, that is superb. But

2 what only HIV will do is mobilize the therapy.

3 So, I think you would make an intellectual

4 statement, first of all, that there is the

5 potential for mobilization. Then, I think in your

6 lab studies, you would study that and make sure

7 that in a coinfection with HIV, sort of studies

8 that Irving Chen did, you can see your vector being

9 distributed.

10 So, you will know that it would be picked

11 up, but how you validate that your therapy was

12 successful in the patient as a result of having had

13 mobilization, I think that would be difficult.

14 DR. MULLIGAN: I meant whether you think

15 there is any animal model system where you could

16 assess the therapeutic virtues of a mobilizable

17 vector, and I can\'t see how that would be tested,

18 and I don\'t buy the intellectual argument that this

19 may be better, and since it may be better because

20 more is better, you know, reinfection is better,

21 doing something where there might be a risk factor

22 associated with it.

23 So, you can\'t think of\--

24 DR. KINGSMAN: No, I mean nobody wants to

25 switch to using higher primates, that would be

> 268 1 outrageous, really in my view. The only thing you

2 could do is some sort of SCID mouse, but I don\'t

3 think that sounds very sensible.

4 I think the standpoint that we have really

5 looked at this from is in terms of long-term

6 reconstitution in pediatric AIDS where you are

7 really trying to give a very limited number of

8 treatments to patients who are going to have this

9 disease for a long, long time, and therefore, the

10 minimum number of manipulations that you can do

11 would be useful. But it is a theoretical argument.

12 DR. MULLIGAN: To pin you down further,

13 sink you deeper in this, mechanistically, even

14 intellectually, what do you think would actually

15 happen, who much mobilization would you possibly

16 need to have happen out of the cells you infect to

17 have it be a therapeutic virtue?

18 That is, you are talking about essentially

19 making a virus-producing cell after you have done

20 your gene transfer, and that the titer coming out

21 of that cell, the mobilized virus, is going to have

22 a therapeutic effect.

23 You must have done the calculations. How

24 could that possibly be a good thing? I mean the

25 amount of virus you would need, the viremia you

> 269 1 would need out of those cells would be fantastic,

2 wouldn\'t it?

3 DR. KINGSMAN: I am going to back down on

4 that really, because I think it is an important

5 question, but it is very difficult to answer. I

6 think you could do studies in vitro where you are

7 looking at the progeny virus that came out of an

8 infectious cycle, and you ask what was the ratio of

9 genomes to transfer vector, and if the ratio of

10 genomes to transfer vector was 100 to 1, you might

11 think the risk wasn\'t worth it.

12 If, however, the ratio of genomes to

13 transfer vector was 50-50, then, you might think

14 that in half the reinfections, those cells then

15 might be protected and maybe that would be a good

16 thing to do.

17 DR. MULLIGAN: I would argue even worse,

18 that if it was that, I would be more worried. That

19 is, once you get up to a point where you are

20 getting very significant mobilization, and you have

21 significant gene transfer, think of how many

22 absolute virus particles you will then have in the

23 person. I mean do you really think that you want

24 to make a producer cell out of a person.

25 DR. KINGSMAN: This is assuming that the

> 270 1 patient has not had their virus load knocked down

2 by anything else. So, yes, you are not\--I see

3 where you are coming from\--you are not demanding

4 that the patient has a rampant HIV infection in

5 order to make your therapy work. That would be an

6 absolute mistake.

7 DR. MULLIGAN: I will stop after one last

8 point, which is that but then if you go down to the

9 point where you have very little infected cells,

10 therefore, you have very little mobilization, how

11 possibly could that be helpful?

12 DR. KINGSMAN: Well, because you have got

13 to that stage.

14 DR. MULLIGAN: I mean how could it be

15 helpful producing out of the small number of cells

16 that are coinfected\--

17 DR. KINGSMAN: At that point, it wouldn\'t

18 be, because you would have achieved your goal, you

19 would have dropped your viral load. I don\'t think

20 anybody is talking about a cure here. You are

21 talking about another way of dropping viral load to

22 manageable levels without having to stay on drug

23 regimens for 30, 40 years.

24 DR. SALOMON: You could argue this is a

25 safety factor. What I think you are bringing up

> 271 1 here is, is mobilization in a gene therapy protocol

2 now in HIV, is that something we should be saying

3 no, there should be no mobilization, and that is a

4 safety issue, or should we say that if there is a

5 therapeutic argument for it, then, that it might be

6 still something to leave on the table, that

7 mobilization under the right circumstances would be

8 a positive thing.

9 That is what I think is the important

10 issue here.

11 DR. MULLIGAN: But I am questioning how

12 even the theoretical of the mobilization, and I

13 think it sounds very reasonable that your

14 mobilization, more virus particles, but I can\'t

15 find any context as we are walking through either a

16 large number of infected cells, infected by the

17 vector, or a tiny amount of vector, virus-infected

18 cells, infected by vector, that it would be

19 helpful.

20 In the one case when you have a large

21 number of cells, then, if it works effectively, you

22 are making just a ton, you are mobilizing a ton of

23 lentivirus vectors into the circulation of the

24 patient.

25 If you have a tiny number, then, the

> 272 1 amount of mobilization is insignificant.

2 DR. SALOMON: What I see from what has

3 been published in this field is, for example, if

4 you take a whole leukophoresis unit, which is a

5 good target, it is readily obtainable, but it is

6 still a whole lot of cells, 10

10 or more

7 lymphocytes, you make your transduction on that,

8 infuse it into the patient, people are excited

9 about seeing somewhere between 3 and 10, maybe 15

10 percent maximum of circulating lymphocytes X months

11 later, right?

12 Now, if there was a survival advantage for

13 that set of lymphocytes, then, the first question

14 would be, would having 15 percent of our T cell

15 repertoire, or T cell numbers, I shouldn\'t use the

16 word repertoire, that that would be adequate, but

17 if you argue that the protection was mobilizable

18 and therefore would spread, so you would have 50,

19 60, 80, 100 percent of your T cells circulating,

20 then, that would be a theoretical argument for

21 arguing for mobilization. I am not, but I am just

22 trying to construct the mobilization argument.

23 DR. MULLIGAN: But just on that point,

24 then, if you said that you had 50 or 60 percent,

25 then, mobilizing your vector, I would think people

> 273 1 would have grave concerns. I mean think of the

2 amount of virus production then you are having or

3 vector production you are having, do people really

4 think they want to do that.

5 DR. SALOMON: I don\'t know. I am

6 perfectly willing to discuss that. I was just

7 trying to follow the argument for mobilization.

8 But I would follow it by saying that if it was

9 true, that mobilization, to the extent that you

10 were effective in reducing HIV replication with

11 your therapy, then, your mobilization would be

12 self-limiting, right?

13 You would stop mobilizing when you were

14 treated, and you could even have fun and argue that

15 if it came back, you know, then, you would actually

16 mobilize again, bring the titer down, and I mean

17 that might be a wonderful way to treat HIV.

18 DR. MULLIGAN: The proof of the pudding

19 would be observing toxicity. If it\'s a non-toxic

20 event to mobilize virus, so what. On the other

21 hand, if people get immune complications or just

22 complications of the vector load, then, obviously,

23 that would have limitations. So, I mean one would

24 ideally like to model it in some animal system

25 before going forward with humans.

> 274 1 DR. DELPH: I guess my other question on

2 that is would the mobilized virus be transmissible?

3 DR. KINGSMAN: Well, you are talking about

4 a transfer vector genome, and then it could be

5 transmissible, so I think that the safety testing

6 of the transgene is absolutely paramount in these

7 type of studies.

8 Actually, we have no intention of putting

9 forward a clinical protocol of this AIDS therapy

10 until there is an awful lot more studies being

11 done, but I think I would only do an RNA

12 therapeutic because the data suggest that there are

13 no significant immune responses against the

14 therapeutic RNA, so the studies that have been done

15 to date suggest that RNA molecules will not create

16 an immune response because the difficulty would be

17 is if in engineering the patient\'s stem cells you

18 suddenly made them targets for an immune response,

19 you would then create an immune deficiency, which

20 is obviously completely bad news.

21 So, I think these type of mobilization

22 strategies are really useful for RNA therapeutics,

23 but I would myself not campaign a protein

24 therapeutic down this route, but that\'s just my

25 view.

> 275 1 DR. MULLIGAN: One last time on the

2 mobilization. The point is that you are

3 essentially in an in vivo context trying to make

4 producer cells that then transfer this vector.

5 DR. KINGSMAN: Yes, that would be the

6 concept.

7 DR. MULLIGAN: Presumably, for that to be

8 therapeutically useful, that is, the mobilization

9 virus, you are going to have to have efficient

10 infection of the cell population by that mobilized

11 virus.

12 So, that mobilized virus is made in the

13 circulation essentially, so what is the

14 concentration that you would have to have of

15 mobilized virus to have a therapeutic effect in an

16 of itself?

17 DR. KINGSMAN: I wish I hadn\'t put those

18 two slides in. I thought, well, it probably is

19 helpful for the discussion. I think the only

20 answer I can make is if you can dilute the potency

21 of HIV genomes with therapeutic genomes, that

22 should be a useful thing to do, but your

23 therapeutic strategy cannot be mobilization per se.

24 I don\'t think anybody is\--well, I hope

25 nobody is saying that. Your therapeutic strategy

> 276 1 is the ribozyme that you put into the target cell

2 to prevent infection in the first place. All I was

3 suggesting is that sometimes that won\'t be

4 successful, you will get virus escape, and that if

5 you do get virus escape, it might be a jolly good

6 idea to try and arm some of that virus with

7 therapeutic gene, so you get an added benefit.

8 I think the concept of the therapy being

9 dependent on mobilization is completely erroneous.

10 That is not what people\--well, it is certainly not

11 what we are saying\--we are just saying it\'s an

12 adjunct, it\'s an added benefit.

13 DR. MULLIGAN: What would be the

14 difference between mobilization, just shooting in

15 the virus I.V.?

16 DR. KINGSMAN: The vector.

17 DR. MULLIGAN: The vector I.V.

18 DR. KINGSMAN: I think if you could come

19 up with a protocol for I.V. administration with

20 sufficient amount of vector, I think that\'s a jolly

21 good approach. It is just that we would have

22 thought that is something that would happen much

23 later on in the development of these vectors, the

24 intravenous administration of vectors, I would have

25 thought would follow on from protocols that did ex

> 277 1 vivo administration.

2 But if you are allowed to go straight I.V.

3 or straight into the bone marrow with an injection

4 of a lot high titer of an HIV vector, then, I think

5 that would also achieve protecting a large number

6 of target cells, which is all that we are talking

7 about here, so I can\'t disagree with you.

8 DR. CHAMPLIN: In vivo production of virus

9 would presumably occur in lymph nodes more so than

10 the blood itself, so you would be adjacent to

11 uninfected lymphocytes, so you might very well have

12 a local concentration that would be adequate even

13 though the systemic concentration would be low.

14 DR. ZAIA: I would like to talk some more

15 about mobilization, but in a different context, and

16 that is the context of whether or not\--I can accept

17 an HIV mobilizing in HIV, and thinking the

18 resultant virus is going to be at least on the same

19 genetic part of the ladder that you showed us.

20 But when I see that HIV could mobilize

21 EIAV, then, I worry that maybe that virus is going

22 to be different. So, can we agree that there may

23 be a greater safety issue involved if we are using

24 EIAV that is being mobilized in the context of an

25 HIV infection, or would you disagree with that?

> 278 1 DR. KINGSMAN: Well, I will turn it around

2 to how do you feel about MLV? I mean MLV is

3 mobilized to the same extent as EIAV.

4 DR. ZAIA: Not quite as much.

5 DR. KINGSMAN: Well, 500 compared to 800

6 in a series of experiments that when they are done

7 to completion, may actually\--you know, it is the

8 same order. I think there is no real difference

9 between them. So, MLV is currently in trial in

10 HIV-positive patients for hemophilia.

11 DR. ZAIA: But still there was a 3 log

12 difference, but it wasn\'t zero.

13 DR. KINGSMAN: It won\'t be zero. I mean

14 you have sticky proteins and sticky nucleic acids.

15 You will get nucleic acids picked up and

16 transferred, and there have been studies with HIV,

17 and really, there is always a background level of

18 packaging of things.

19 So, if you are going to try and shoot for

20 absolute zero, then, I really think that we should

21 shut up shop and come back in maybe 50, 60 years

22 time, because I don\'t think biology gets down to

23 absolute zero.

24 So, there is a level of mobilization, and

25 I think you have to balance the\--

> 279 1 DR. SALOMON: The implication here, and I

2 thought it was interesting, and I thought of it,

3 too, you know, in a sense this would be an

4 iatrogenically induced zoonosis, but the idea here

5 would be if MLV is mobilized and you have done MLV

6 in an HIV-positive patient\--by the way, that is not

7 exactly the best argument in the world, maybe we

8 should stop those trials\--

9 DR. KINGSMAN: It is not, but it is an

10 argument, it\'s not my trial.

11 DR. SALOMON: I just was going to point

12 out that there is a logical flaw there, but let\'s

13 assume that there is. Is there any difference

14 specifically in mobilizing a class of virus, i.e.,

15 in this case, these are both lentiviral vectors,

16 although they are quite different\--

17 DR. KINGSMAN: But what is left there?

18 You know, what are we mobilizing? Are we

19 mobilizing a lac-Z?

20 DR. SALOMON: How do you know you are not

21 mobilizing this incredibly engineered gag-pol from

22 your packaging line?

23 DR. KINGSMAN: Because you have screened

24 for that.

25 DR. SALOMON: And you have screened for

> 280 1 that in your third assay.

2 DR. KINGSMAN: You have screened for your

3 mobilized RT, you can screen for mobilizable RT.

4 DR. SALOMON: But there was one

5 replication-competent retrovirus in X number of

6 particles, so those were the couple that carried

7 the\--

8 DR. KINGSMAN: Yes, that happened to get

9 into a cell that happened to be coinfected with

10 HIV, that happened to then recombine.

11 DR. SALOMON: But it happened to get into

12 the cell with the HIV is your whole purpose, right?

13 DR. KINGSMAN: Well, I don\'t want to

14 muddle up the HIV therapy for AIDS, because HIV

15 therapy for AIDS, I think is a special case, and

16 what the bulk of my talk was, and somebody did say

17 to me you should leave the HIV slides out, and I

18 wish I had done\--but the bulk of my talk was about

19 EIAV vectors and whether these are safe and whether

20 these can now be used in the clinic.

21 DR. SALOMON: My comments were

22 specifically in the HIV case, it was not the case

23 of the EIAV.

24 DR. NOGUCHI: Dan, I would just remind

25 you, and, Dr. Kingsman, we do appreciate your

> 281 1 willingness to come and present, but we were going

2 to be more cordial and not really discuss specific

3 products for our guests who are presenting their

4 future plans.

5 But I do have to say that in terms of just

6 biologics production, going back to the little

7 debate before on sensitivities of assays, really,

8 we are talking about not so much whether you are

9 regenerating a horse virus which has no relevance,

10 we are talking about can you detect something that

11 we want to know about.

12 It may be that a horse cell might be more

13 sensitive for amplification that you are talking

14 about, or it might be something like Moose Dooney,

15 we simply don\'t know, but the point is if there is

16 an actual recombinant that we want to see, however

17 you amplify it to get to be detectable is

18 appropriate even if it happens to be in horse

19 cells.

20 But in the general presentation, I think

21 if we could focus on the science that you

22 presented, that would probably make everybody a

23 little more comfortable.

24 DR. KINGSMAN: I have to say I am not

25 uncomfortable. I think the debate is really very,

> 282 1 very interesting and very useful.

2 DR. SALOMON: I think the principles that

3 I am still trying to struggle with here would be I

4 think we did a pretty good job trying to get a

5 little bit at the definition of an RCL assay, and I

6 think that your approach to it is very reasonable

7 actually.

8 The part that we are talking about now, I

9 guess there is a couple like themes here that I am

10 not sure we have really got a discussion going and

11 completed yet, and that would be, number one, in

12 generating trials for HIV with lentiviral class

13 vectors, one could say you should not have

14 mobilization, it is too early in the field, one

15 should start off with a non-mobilizing strategy,

16 and a principle should be no mobilization, not that

17 you couldn\'t construct a theoretical argument for

18 mobilization, and I was doing that as a

19 point/counterpoint with Dr. Mulligan.

20 That didn\'t mean that I was trying to

21 argue that that was appropriate for the first

22 trials. So, I think one issue that I would like to

23 see the Committee discuss, I am not going to try

24 and even achieve a consensus, but just should one

25 in these first trials say just say no to

> 283 1 mobilization, not saying the next generation of

2 trials might require mobilization.

3 So, that was one point. The second point,

4 which I was kind of getting at, and I think that

5 was the point Dr. Zaia was getting at, is in these

6 HIV trials, is there an argument, a specific

7 argument to be made for using an HIV-based vector

8 or using an EIAV or an FIV or an SIV vector, which

9 are all possibilities, right?

10 In other words, is there a rationale from

11 a safety point of view\--science is not the

12 point\--for using a certain class of vectors or not

13 using a certain class of vectors in these trials?

14 So, if we could kind of deal with those

15 two things, mobilization now or never, and class of

16 vector as it relates to the use in a trial.

17 DR. CHAMPLIN: In the absence of any data

18 that mobilization is really toxic, I mean I

19 wouldn\'t categorically exclude it. I mean it would

20 be the burden of the group bringing forward a

21 project to demonstrate that in the preclinical

22 system that it seemed to be beneficial, at least

23 the overall approach was beneficial, and there

24 wasn\'t undue toxicity, and that as one went into

25 human trials, when that day would come, that one

> 284 1 would select the proper patient group where the

2 risk-benefit relationship would be rational, but I

3 would exclude it completely.

4 I could envision cell-cell interactions in

5 a lymph node where you would actually want to have

6 the vector distributed through the cells and the

7 node to more completely administer the therapeutic

8 effect.

9 So, it very well could be a positive thing

10 if it didn\'t sort of overshoot and produce toxic

11 effects.

12 DR. EMERMAN: This is Mike Emerman. I

13 just want to talk about the mobilization with HIV

14 patients a little bit. First of all, the principle

15 is if the HIV vector gets mobilized, it means it is

16 not working very well, so presumably the vector is

17 there to inhibit HIV production, and if it is

18 officially mobilized, it means it is not

19 efficacious at all.

20 The major problem with mobilization is

21 that the mechanism of recombination in retroviruses

22 is co-packaging. So, if we are worried about

23 possible recombinants between endogenous or the HIV

24 in the patient and the vector, that is how it is

25 going to be occur, is by co-packaging by the

> 285 1 mobilization.

2 So, in both cases, mobilization is a bad

3 thing.

4 DR. SALOMON: That came across.

5 DR. ALLAN: I am not a vectorologist, but

6 can you completely design something that is not

7 going to be mobilized if you are treating a patient

8 that has HIV, and you are using HIV as a vector,

9 can you prevent that? I don\'t think you can unless

10 you have something that is 100 percent effective

11 like Michael was saying.

12 DR. MULLIGAN: Just to come back to your

13 crystallized issues, on the first point, I would

14 argue very simply that no, not now, because I think

15 that there is no one who has ever made the case yet

16 that mobilization for this therapy would be

17 anything more than a bell and whistle or chrome to

18 the concept.

19 No one has said that this mobilization

20 approach was likely to make it work when it

21 wouldn\'t work without the mobilization, so I agree

22 with exactly the way you put it, that the issue is

23 not to say this is never a good idea, but I think

24 we are very far from having any compelling, even

25 intellectual arguments, much less really

> 286 1 experimental data, which I think would be very hard

2 to get.

3 Then, Michael\'s point I think is actually

4 very good, that I hadn\'t even gotten to, which is

5 just as he says, if it allows the virus, if it

6 isn\'t protecting things, and mobilization

7 increases, things aren\'t working so hot. I think

8 it\'s a good point.

9 DR. BORELLINI: When I came up here, the

10 topic was RCL, so I am going to ask my RCL

11 question, and I am picking up again from Dr.

12 Noguchi\'s comment. I think that it is very

13 important to not have barriers to amplification in

14 the design of your assay because as it is designed

15 now, you could have something amplifying really

16 well, and growing out really well if your

17 amplification system was an equine cell line, and

18 with your system now, you would release this, and

19 based on the fact that it does now replicate in one

20 human cell line, but you don\'t really know the

21 tropism, what this thing is going to be, and you

22 are putting a lot of weight on that one human cell

23 line to tell you that it is now going to replicate

24 into any other human cell lines.

25 DR. KINGSMAN: Well, we are going to use

> 287 1 two, we are going to use lymphoid lines and 293, so

2 we will use two cell lines, and we will put in

3 amplification on those two cell lines, and we have

4 designed the system where the probability of any

5 RCL emerging is lower than for any other retroviral

6 or lentiviral vector system that has been designed,

7 and I think that by using the appropriate controls

8 and making sure that we can detect a single control

9 RCL, and we have chosen to use FeLV, that this will

10 be adequate.

11 By taking horse cells and designing a

12 positive control that is some theoretical virus

13 that might emerge and studying this in horse cells,

14 I cannot see how that is going to be\--it might give

15 you a false sense of security, but the type of

16 entities that may come out of this are

17 hypothetical, diverse, random recombinants that

18 will be generated in a human cell, and that we are

19 wanting to know do they amplify in a human cell.

20 That is what we are talking about.

21 DR. KAPPES: I am not sure if I completely

22 agree, and let me explain my vantage point. In a

23 moment, I think, I will show a system that I

24 devised where I have enhanced, and I will show the

25 details, enhanced every opportunity for that

> 288 1 recombinant to be detected.

2 So, what did we learn by doing that? We

3 learned, well, first, we derived sufficient

4 recombinant to analyze, and then by analyzing it,

5 we understood its nature in a way that I believe we

6 are able to address the very questions we are

7 trying to get a handle on, and that is how to

8 design a vector or an assay for QA/QC to minimize

9 the risk of the emergence of RCR in vivo.

10 I know your comments were slightly

11 different, but in a general sense, I point that

12 out.

13 DR. KINGSMAN: Well, yes, I mean I know

14 what you are going to talk about, and I am looking

15 forward to hearing that, and I hope you can move on

16 it fairly swiftly, but this is the nub of the

17 matter, is to how far we recreate what we feel is a

18 suitable positive control based on our best guess

19 as to the likely entity that might emerge, and if

20 we use as the positive control, are we all going to

21 go away feeling super comfortable that we have

22 second-guessed this thing and we have designed

23 something, where it could take quite a long time to

24 recreate this hypothetical virus in studies in cell

25 lines that we are not really interested in knowing

> 289 1 about the pathogenic principle.

2 Really, this is why I go back to one of my

3 early slides, which says that the development of

4 these vectors should be matched with the target

5 population and the disease, and that we can begin

6 to learn about the performance of these vectors in

7 the clinic.

8 Our proposal is to take a vector for

9 prostate cancer into the clinic in late-stage

10 patients who have no other treatment option, a

11 local delivery where we can monitor gene transfer,

12 we can monitor the blood for the presence of

13 sequences, we can monitor the delivery site, we

14 re-biopsy, and we ask about what\'s actually going

15 on, and we learn in the relevant host with relevant

16 assays what these vectors are doing.

17 I certainly wouldn\'t advocate taking these

18 vectors at the current stage of development and

19 going intravenous into young children or who can be

20 treated with an enzyme or some other therapy right

21 now. It is clearly an ongoing process.

22 Now, I think we need to be very careful

23 not to inhibit the developments of the field by

24 making too many scenarios for hypothetical events,

25 you know, we could delay this now for a long time,

> 290 1 and I think there are vectors. We have heard this

2 morning, we have seen some very carefully crafted

3 vectors. I am biased, but I believe that we have

4 carefully crafted some vectors that have a good

5 safety profile.

6 I think one can assay for the major safety

7 components and by trialing them with a transgene

8 that is per se non-toxic. I would suggest that we

9 are nearly ready to make progress with these

10 systems for certain diseases, for certain

11 endpoints.

12 I think the HIV in HIV-positives is a very

13 special peculiar case, and we should not get

14 sidetracked by the special features of that

15 combination when we have much more global features,

16 and we have diseases that can be treated with these

17 vectors, which there are no other vectors out there

18 that can tackle some of these diseases.

19 So, I think we need to bear that very

20 strongly in mind.

21 DR. DELPH: I have two questions, but I

22 would just like to make a comment on a statement

23 that you just made, and that is, I do

24 disagree\--well, I agree that HIV gene vector

25 transfer may be somewhat special in people who are

> 291 1 already HIV-positive. I think we cannot ignore the

2 fact that there is a possibility that people who

3 are HIV-negative can become HIV-positive, so I

4 don\'t think we can behave as if they are mutually

5 exclusive.

6 DR. KINGSMAN: I absolutely agree with

7 you, and I was not intending to imply that, and, in

8 fact, I think any decision that would disadvantage

9 a particular group of patients and exclude them

10 from a therapy is to be guarded against at all

11 cost.

12 This is why I made the comparison between

13 EIAV and MLV, and suggested that the performance

14 characteristics of a vector, such as EIAV, are

15 compatible with using them in any type of patient

16 irrespective of their HIV status, because we don\'t

17 currently prescreen patients for entry into MLV

18 trials for their HIV status. We don\'t exclude

19 people on that basis, and I would argue that there

20 is no reason to exclude a group of patients with

21 EIAV vectors.

22 What I mean is the special case is an HIV

23 vector in a patient who has overt levels of virus

24 where I think there must be special consideration

25 of the biology.

> 292 1 DR. DELPH: I have two questions, and the

2 first one is are there external factors that

3 increase the probability of the emergence of

4 replication-competent retroviruses or of

5 mobilization?

6 The second one is what are the effects of

7 irradiation on cells that have undergone retroviral

8 gene transfer therapy?

9 DR. KINGSMAN: The first question, are

10 there any special circumstances that impact on RCLs

11 and mobilization, I mean we heard the original MLV

12 study in the immunocompromised primate, so there

13 was the suggestion that if the immune system is

14 compromised and an RCL emerges, then, the

15 consequences would be possibly more drastic.

16 I just don\'t think I have an answer for

17 the question are there things that you can do to a

18 cell to make it more likely to make RCLs or to make

19 the RNA more likely to be mobilized. I can\'t quite

20 get a handle on thinking\--somebody might want to

21 help me out on that one\--I can\'t think of something

22 we could do to a patient to add to any issues, but

23 that may be a deficiency in my\--

24 DR. MULLIGAN: I think there are things,

25 but they are probably not relevant here. There is

> 293 1 like demethylation, if you demethylate sequences,

2 viral sequences that are not transcriptionally

3 active, which probably isn\'t going to be the case

4 here, but that would then lead to mobilization

5 increases.

6 DR. KINGSMAN: Yes, I mean these are

7 things you would do in the laboratory, but I can\'t

8 think about things in the patient.

9 The other question was irradiation, what

10 would irradiation do to patient cells that contain

11 a gene therapy transfer vector. I have to pass on

12 that one. I haven\'t got any expectation of any

13 definite.

14 DR. SALOMON: I am pretty sure there is no

15 answer to that one.

16 DR. KINGSMAN: Yes, good, I can\'t think of

17 one.

18 DR. SALOMON: I would like to move on to

19 Dr. Kappes.

20 If it is brief?

21 DR. CORDOVA: Just very brief. We have

22 heard about the biological barriers, however, this

23 is being produced within the human cell line, and

24 so clearly, you are providing whatever barriers

25 there are, you are overcoming them.

> 294 1 What other barriers would there be,

2 biological barriers, would it be the envelope

3 perhaps that lets it only replicate as a wild-type

4 in equine cells?

5 DR. KINGSMAN: Well, the LTR doesn\'t

6 function in human cells, so that is obviously a

7 barrier, and the envelope, we believe you

8 can\'t\--well, we don\'t believe, we know\--even if you

9 get a vector genome into a human cell, if you are

10 relying on the LTR, tat activity to drive it, it

11 won\'t work, and I showed you those data. So, there

12 is an entry barrier, there is a gene expression

13 barrier.

14 I don\'t know if there are any other

15 barriers. Presumably, I mean there are situations

16 with murine retroviruses where the gag-pol is a

17 determinant, but clearly, the gag-pol functions in

18 these vector systems, so one would have to assume

19 that the gag-pol is not a barrier.

20 So, it is envelope and gene expression.

21 DR. CORDOVA: So, within the context of

22 what you created, your vectors, you really have

23 overcome those barriers because you are producing

24 them in a human cell line, correct?

25 DR. KINGSMAN: Yes, be definition, you

> 295 1 would have to have done that.

2 DR. SALOMON: Thank you very much. You

3 became, unfortunately, the point person for a

4 couple key parts of the debate, I really don\'t

5 think they were directed specifically at you,

6 Susan.

7 The last speaker of today is Dr. John

8 Kappes from the University of Alabama at Birmingham

9 on Predicting Lentiviral Vector Safety in Vivo.

10 Predicting Lentiviral Vector Safety in Vivo

11 Dr. John Kappes

12 DR. KAPPES: I would like to thank the

13 organizing committee for the opportunity to discuss

14 my ideas and also, maybe preemptively, point out

15 that there is nothing absolute about my choice of

16 words here for predicting lentiviral vector safety

17 although I think there is merit, and that is the

18 focus of this talk.

19 \[Slide.\]

20 This slide, knowing that I was presenting

21 later in the day, was meant to summarize probably

22 what was presented, and in the case truly is what

23 was presented and discussed in great detail, but

24 let me walk through it.

25 The status of the field obviously, I think

> 296 1 everyone recognized tremendous progress has been

2 made in vector design without removing the

3 potential of these vectors to effectively transfer

4 genes, not just in vitro, but in vivo.

5 The primary challenges that we face

6 include an approach or approaches for safe

7 administration of the vector, and in particular,

8 for issues related to recombination and then

9 generation of RCL. That, of course, relates to in

10 vitro QA and QC.

11 I would like to pause here for a second to

12 try to take what I have heard this morning and

13 place into context the data that I am going to

14 present and the ideas I am going to suggest.

15 There is no doubt that tremendous progress

16 has been made in the design of these vectors, and

17 there are many ideas discussed particularly that

18 related to the use of RCL assays for quality

19 assuring against the generation of RCL in vivo, but

20 we keep coming back to academic theoretical

21 possibilities of is this true or isn\'t this true.

22 For example, let me walk through the

23 vector design. The envelope was removed to prevent

24 specific transfer of any progeny by

25 envelope-receptor interactions. The accessory

> 297 1 proteins were removed because they play a role in

2 pathogenesis of disease, but does the removal of

3 those genes mean a recombinant won\'t be pathogenic?

4 I don\'t think anyone knows, but certainly it

5 wouldn\'t have the pathogenic properties of the

6 parental virus.

7 Tat is completely gone. Rev, in the case

8 of HIV, is provided in trans, although keep in mind

9 in the absence of rev, again, you don\'t have an

10 absolute block in the transfer of RNA from the

11 nucleus to the cytoplasm. So, again, you can get

12 into these theoretical arguments.

13 Similarly, with the SIN vectors, while

14 there is a huge decrease in the transcription from

15 the LTR with the deletion in U3, there still is

16 what I will call basal transcription, and in part,

17 this is affected by the position at which the

18 vector integrates.

19 So, again, you come back to arguments what

20 if, and what if, and how about, or whatever, and my

21 point is how about if\--maybe I shouldn\'t ask\--I

22 will suggest that if we move away from using or

23 focusing on RCL as an endpoint for what constitutes

24 a safe virus, this is our determination of RCL in

25 vitro for safe vector, and think\--I go back to what

> 298 1 Susan was talking about\-- think about a marker that

2 is absolutely required for the generation of RCL.

3 I will give an example. If you remove U3,

4 you have a SIN vector, yes, you can still get

5 background transcription. That transcripts can be

6 mobilized. We just had a half-hour discussion

7 about it. If you use an assay which ensures your

8 vector prior to administration lacks critical

9 reverse transcriptase function, then, perhaps the

10 assay, although it is not for RCR, maybe we will

11 call it pre-RCR, to me has more definition and

12 perhaps more value in advancing these vectors

13 toward the clinic.

14 So, that is what I hope to do, present

15 some data that might help advance the vectors

16 toward the clinic.

17 \[Slide.\]

18 Simplistically, we have the packaging

19 construct, the gene transfer vector, and the

20 envelope, and the concern is that through genetic

21 recombination, now, this is in the primary

22 transduced cell, we would generate a structure here

23 depicted as LTR-gag-pol-env-LTR/RCL.

24 I think that is highly unlikely especially

25 now if we separate rev and other components and

> 299 1 minimize U3 regulated expression, and, in fact,

2 neither the second nor the third generation vectors

3 have been shown to generate RCL, I don\'t think it

4 is going to happen, I don\'t think we are going to

5 find RCL. That is in part why I am stressing the

6 potentially, at least the limited value of its use

7 as a marker for safety.

8 On the other hand, a single recombination

9 event between the packaging construct and the gene

10 transfer vector can generate this kind of

11 structure. That will be the focus of the rest of

12 my talk, but first, hopefully, it is not too

13 laborious, I am going to show five or six slides to

14 try to underpin where I am going with this.

15 \[Slide.\]

16 First point. Genetic recombination, I

17 would be as bold to say is likely. These

18 retroviruses use genetic recombination as part of

19 their life cycle, part of the reverse transcription

20 process.

21 Our past experience with MLV might also

22 suggest that reverse transcription or through

23 reverse transcription, genetic recombination is

24 likely, not just with components that comprise the

25 vector, but also in certain cases with endogenous

> 300 1 genes.

2 This issue of genetic recombination, as we

3 are all acutely aware, underpins our concerns with

4 respect to safety and RCL.

5 \[Slide.\]

6 My point that I am trying to get at is in

7 vitro measurements of RCL are not predictive of the

8 emergence of RCL in vivo in the long term. In

9 fact, the way we could think about the in vitro

10 assays for the generation of RCL would be for the

11 emergence of a structure which had replication

12 potential, while in vivo, the detection of RCL,

13 whether it\'s months to years after administration,

14 would be recognized as failure even though at this

15 step, perhaps the vector didn\'t generate RCL.

16 \[Slide.\]

17 So, what are the requisite safety

18 measures? I think we have discussed each of these

19 today. There is PCR-based assays, there are

20 RCL-based assays, and what I will describe as a

21 gag-pol recombination assay.

22 \[Slide.\]

23 I will quickly try to move through the

24 advantages and disadvantages of each of these,

25 again trying to emphasize why I think a gag-pol

> 301 1 recombinant assay might have certain merits.

2 The advantages of an RCL assay is, of

3 course, it is going to detect\--again, this is in

4 vitro, and I really want to stress that\-- most of

5 the time when I think about concerns for RCL, it is

6 the ultimate RCL, it is the RCL in vivo that is

7 going to potentially cause disease, but the

8 advantages for the RCL assay in vitro is to guard

9 against RCL.

10 I am not saying we shouldn\'t do that, we

11 should, but I do think, under the quality

12 controlled environments, it is ever going to be

13 detected.

14 What are the disadvantages? As I have

15 said, it is not predictive against the emergence of

16 RCL in vivo, and it is not informative. It is not

17 informative in many important respects in my

18 opinion, as it relates to recombinants that will

19 likely be formed.

20 It doesn\'t give you any information about

21 the composition of recombinants, if they are

22 formed. It doesn\'t tell you anything about the

23 functionality or the replication potential, and I

24 think that is a key word which I will come back to,

25 potential of the recombinants.

> 302 1 It doesn\'t give you information about how

2 the host will interact with the recombinants, nor

3 how the recombinant will interact with the host.

4 Finally, I guess bottom line, it doesn\'t,

5 in my view, tell you much about the risk that the

6 vector will pose to the treated person.

7 \[Slide.\]

8 PCR assays. The advantage is they can

9 detect vector and/or packaging-specific DNA, as Dr.

10 Kingsman just pointed out. The disadvantages

11 relate to specificity and the similar points I made

12 about biological specificity for RCL assays.

13 \[Slide.\]

14 The advantages for a gag-pol recombination

15 assay would include the enablement of the

16 monitoring of vector stocks for what I called

17 pre-RCR. Specifically, these recombinants

18 represent a coding region with a functional

19 gag-pol, and would likely contain or be flanked by

20 LTRs.

21 So, what is the significance of this

22 pre-RCR?

23 \[Slide.\]

24 The significance is several fold. It

25 shows gag-pol-vector recombinants are produced, so

> 303 1 it tells you that you are generating a recombinant

2 with functional gag-pol.

3 Without functional gag-pol, and I depict

4 that as this structure, RCL cannot be generated in

5 primary transduced cells, and importantly, nor can

6 that recombinant, if it lacking this structure, or

7 the vector if it is lacking a recombinant which

8 contains this structure, lead in vivo over the

9 course of time to the emergence of RCL.

10 I think this is a different wording of

11 what I have just said. Functional gag-pol is

12 required for the recombinant to generate RCL in

13 vivo.

14 Thus, my point, in vitro monitoring for

15 functional gag-pol-containing recombinants provides

16 a tangible way to analyze lentiviral stocks in

17 vitro for their potential to generate RCL in vivo.

18 \[Slide.\]

19 I have summarized or I have attempted to

20 summarize all of what I have just said here. My

21 hypothesis is that recombination will be formed and

22 that instead of going this pathway, the blue box is

23 supposed to represent the in vivo situation, where

24 you take this vector which contained recombinants

25 that would not be RCL, you place it in the

> 304 1 individual. You don\'t know what is going to happen

2 with respect to recombination in vivo and you don\'t

3 know whether it is going to lead to RCL.

4 Alternatively, if you use the gag-pol

5 recombination assay, my hypothesis is that it could

6 serve as a surrogate to predict the risk of this

7 vector stock for generating RCL in vivo. Thus, you

8 avoid the unknown pathway.

9 \[Slide.\]

10 So, this is where I was going to start.

11 Believing that genetic recombination would occur,

12 we wanted to understand how recombination could

13 compromise or could generate forms that would pose

14 a risk in vivo.

15 At this time, tat transfer assays, gag

16 transfer assays, RCR assays were negative, so we

17 devised an approach to detect, but more

18 importantly, to enrich for the presence of the

19 recombinants, and our idea was that if we could

20 detect recombinants and enrich them, then, they

21 could be characterized both genetically and

22 biologically.

23 \[Slide.\]

24 Here is the approach. This represents a

25 HeLa cell into which is integrated this genetic

> 305 1 structure. It contains an HIV LTR and importantly,

2 puromycin. The point is that we transduce using a

3 lentivector, HeLa cells to introduce this structure

4 and screen these HeLa cells for sensitive induction

5 of puromycin resistance by tat expression, so that

6 with a single copy of virus in this cell,

7 sufficient puro could be expressed, that the cells

8 would grow and confer resistance in the presence of

9 5 micrograms or more per mL of puromycin.

10 \[Slide.\]

11 The notion was that if we could use this

12 cell line where LTR was turned by tat, it might

13 provide a more sensitive means to both detect and

14 then select and enrich for recombinants. So, we

15 generated these vector stocks, and the hypothesis

16 was that if the vector, shown on the ends, and the

17 packaging constructs, shown in the middle, in this

18 case tat is highlighted because the key element

19 that would need to be present in any recombinant is

20 tat.

21 Then, tat would be expressed if this

22 recombinant was formed and integrated, up-regulate

23 the LTR and puro, conferring resistance, allowing

24 us to grow this to large numbers and analyze the

25 outcome.

> 306 1 \[Slide.\]

2 Before showing the data, I would first

3 like to point out that I realize that there are

4 what I am calling state-of-the-art vector

5 components, third generation packaging construct,

6 SIN vectors, and what I am calling the trans-lenti

7 vector, which I will talk more about later, but as

8 I just described, the system we first started using

9 requires tat.

10 It requires tat to up-regulate puromycin

11 selection. That was the design of our approach.

12 So, those initial experiments were done using a

13 packaging construct which contained tat and rev.

14 It is a second generation-like packaging construct.

15 The vector contained GFP as a reporter,

16 and three components were transfected into 293 T

17 cells, vector stocks were produced and titered, and

18 10 7 infectious units of these virions

were used to

19 infect this LTR puro cell line to screen for

20 resistant colonies.

21 What we found is in the absence of

22 nevirapine, which is an HIV-1 specific RT

23 inhibitor, approximately 1,000 colony-forming units

24 were formed, and what does that suggest? It

25 suggests that indeed recombination occurred between

> 307 1 the vector, again flanking the ends, and the

2 packaging construct. It doesn\'t necessarily

3 indicate that a recombinant, as I have depicted,

4 was formed with a gag-pol reading frame, only that

5 tat was present.

6 Importantly, with nevirapine, we find no

7 resistant colonies, indicating that this is

8 specific or dependent upon HIV-1 reverse

9 transcription.

10 \[Slide.\]

11 To try to address more specifically the

12 question as to whether recombinants are formed that

13 contain functional gag-pol, we used the approach

14 depicted here. This is the first step I showed on

15 the last slide. If this recombinant is present,

16 and if the gag-pol open reading frame is

17 functional, what we would expect is for that

18 infected cell\--now, remember what we have done is

19 grown this out, there could have been very few of

20 these originally, but through selection we have

21 grown them out, selected them, and we have a

22 purified culture of recombinant-containing cells.

23 If this recombinant-containing cell

24 contains a recombinant, it is functional in

25 gag-pol, it should produce virions, that if

> 308 1 pseudotyped as I show here, we transfected VSV-G

2 into this cell culture, could be mobilized and

3 mobilize its own RNA.

4 So, what we did in following through on

5 that experiment was transfer the supernatants

6 produced from this cell line, and again transfected

7 not just with VSV-G, but as I was alluding to when

8 I made a comment to Dr. Kingsman earlier, tat and

9 rev, to maximally stimulate and thereby detect the

10 presence of these recombinants.

11 We took the supernatant, applied them to

12 naive LTR puro cells, and detected 540

13 colony-forming units, indicating that indeed in the

14 original transduced cells there were recombinants

15 produced, suggested through the process of reverse

16 transcription by this control, that contained not

17 just LTRs, but open reading frames for gag and pol

18 and tat.

19 \[Slide.\]

20 So, from the expanded culture\--and this

21 isn\'t the expanded culture, of course, but it is

22 what I have used for illustration\--we extracted the

23 high molecular weight DNA and analyzed the five

24 prime end by PCR amplifying this fragment, and that

25 is shown here. This is a proviral control.

> 309 1 \[Slide.\]

2 So, we have a fragment that looks like the

3 appropriate molecular weight, and by sequence

4 analysis, indeed, what we found is that the vector

5 sequence was joined to the packaging construct

6 sequence, and of colonies we picked and analyzed,

7 all 10 contained this sequence as you might surmise

8 based on the requirement for an open gag-pol

9 reading frame.

10 \[Slide.\]

11 If we use the same approach, that is, PCR

12 amplification of a DNA fragment to amplify

13 recombination on the 3-prime end of the vector, we

14 also found joined in between the vector and

15 plasmid, and I depict that differently because I

16 think it is not just an interesting, but an

17 important point with respect to DNA mobilization

18 and recombination.

19 This is the 3-prime end of the vector, so

20 up here somewhere, reverse transcription initiates

21 and generate the strong stuff DNA, the RU5, which

22 jumps or translocates to the 3-prime end of the

23 vector through the sequence homology with R.

24 So, now you have a stranded DNA and the

25 vector RNA. The polymerase continues reverse

> 310 1 transcription and what we have found by our

2 sequence analysis actually switches templates into

3 the packaging construct RNA, and we found of 10

4 analyzed, four different forms. They are depicted

5 as a recombinant that crossed over into the 63rd A

6 of the poly(A) tract, the 53rd and 47th, and this

7 position I believe was at 108 nucleotides upstream

8 of U3. Yet, again that happened at this position,

9 which was 58 or some other number of nucleotides

10 upstream of U3.

11 \[Slide.\]

12 The details of that data which I just

13 showed are depicted here, but I won\'t bother to

14 review it unless somebody has questions.

15 \[Slide.\]

16 So, the question then was if we

17 remove\--let me back up a little bit to try to get

18 to the point I was trying to make early, so I

19 should take advantage of that.

20 All the splits we have made, which are

21 fundamentally accepted as state-of-the-art in

22 vector technology, have these theoretical

23 possibilities associated with them because they

24 don\'t absolutely eliminate the potential for

25 something, whether it\'s RNA expression or

> 311 1 pathogenesis or what have you.

2 With that in mind, our approach was to

3 eliminate the reverse transcriptase and the

4 integrase gene from the packaging construct. The

5 point is without reverse transcriptase and/or

6 integrase, but especially both, even if you had a

7 recombinant that was formed between the packaging

> 8 construct and the vector, that recombinant itself 9 would have no
> potential to potentiate the

10 development of RCL in vivo.

11 Importantly, as Dr. Kingsman pointed out,

12 we can analyze the vector for the absence or the

13 presence of these sequences, either genetically, as 14 she described,
or functionally, as I have begun to 15 describe.

16 \[Slide.\]

17 So, now we take a new stock of vector that

18 I call trans-lenti, trans-lenti virus, TLV,

19 generated where reverse transcriptase and integrase 20 are provided
in trans. They are separated from the 21 packaging construct, provided
in trans via fusion

22 with vpr, and we measure whether that vector can

23 produce resistance colonies, as I have shown

24 earlier, again using the same titer of vector 10

7

25 particles, we infect this cell line.

> 312 1 Then, we collect the supernatant after

2 introducing VSV-G and tat and rev to test whether

3 this vector contains this genome that can be

4 mobilized, and the answer is no. However, I should

5 point out that if in addition to VSV-G and tat-rev,

6 we also transfect back into this culture, vpr, RT

7 integrase. I don\'t have that data to show.

8 Then, we produce positive colonies, and

9 what is the significance of that? It directly

10 tells me direct data, that the block in DNA

11 mobilization that I show here is due to the removal

12 of RT and integrase, where it is provided in trans

13 in the original stock.

14 \[Slide.\]

15 So, to summarize these data, recombination

16 occurs between the lentiviral packaging construct

17 and gene transfer vector. Integrated recombinants

18 express viral proteins. They express, in this

19 case, tat, gag, gag-pol. These recombinants

20 produce progeny virions that are envelope

21 deficient, however, if they are pseudotyped, they

22 can mobilize RNA to naive target cells.

23 \[Slide.\]

24 In particular, I wanted to stress the

25 point I made about recombination within the poly(A)

> 313 1 tract. What does this tell us? It confirms,

2 number one, that genetic recombination has occurred

3 during reverse transcription, and it helps

4 substantiate that this is not an artifact. There

5 are many types of questions and experiments we

6 conducted to try to minimize false positives, if

7 you will, but this is one of the strongest data

8 that we have to indicate that this is actually

9 occurring during reverse transcription as a result

10 of inadvertent incorporation of other messenger

11 RNAs into the very vector you are generating for

12 treatment.

13 It also suggests that removal homologous

14 sequences from the vector and the packaging

15 construct may not itself be sufficient to prevent

16 mobilization.

17 In addition, this might represent a

18 mechanism by which genes without homologous

19 sequence can be mobilized, including endogenous

20 genes, and there is actually data published in 1988

21 using an avian retrovirus to show that oncogenes

22 could be mobilized by a recombination event that

23 occurred in the poly(A) tract of the oncogene

24 message.

25 \[Slide.\]

> 314 1 So, that is really old data. The big

2 question for my lab in the more recent future was

3 how about the state-of-the-art lentiviral vectors,

4 so what I compared are those listed here - third

5 generation packaging construct, a SIN vector, and a

6 trans-lenti, and I should point out these don\'t

7 contain tat or this doesn\'t contain tat, so we had

8 to devise a new approach, but it is also worth

9 pointing out, not because of the approach, but just

10 because it\'s different, that the trans-lenti system

11 still contains tat.

12 \[Slide.\]

13 Vector stocks were generated and I want to

14 point out that for third generation vector, we had

15 a titer of 10 8. We

also generated a stock with

16 third generation packaging constructs/SIN vector

17 also 10 8 and a transvector at

109, not because we

18 can produce more, in fact, just the opposite is

19 true, and we can talk about that later, but because

20 I wanted to understand the differences with respect

21 to the endpoint I will show.

22 Go back mentally at least to the HeLa puro

23 line. The idea is that since now we don\'t have

24 tat, we need to rely on mobilization of something

25 else, so that HeLa puro line contained a lentiviral

> 315 1 vector introduced fragment, which contains a

2 packaging signal.

3 The idea here is when we infect this

4 virus, if a recombinant such as is depicted here

5 was to be produced, when it expressed gag-pol,

6 formed particles, perhaps this message would be

7 encapsidated, and if it was, it could be mobilized

8 and detected in a cell line which constitutively

9 expresses tat under control of CMV, because now we

10 are mobilizing this, remember we need tat still, so

11 once it is in here, once this message is mobilized,

12 reverse transcribed integated, we still need tat

13 for upregulation to confer puro resistance.

14 \[Slide.\]

15 The data for the results are here. For

16 third generation at a titer of 10

8, we detect

17 whatever this is, 50 resistant colonies. For

18 envelope minus virus, it detects zero, and these

19 are really important controls, too, because I think

20 we had to be extremely critical or of ourselves in

21 understanding how these data might not actually

22 represent what I depicted earlier.

23 The vector minus control is a very

24 important control. The concern was the possibility

25 that even without integration, the trans, the

> 316 1 packaging construct, the third generation packaging

2 construct could be expressed and encapsidate the

3 message which transfer puro resistance, but without

4 vector, it, too, is negative.

5 The third generation SIN vector is

6 positive, and this is an interesting result perhaps

7 worthy of discussing later, because if you look at

8 the published data, you would expect there to

9 perhaps be a difference of 1,000 or 10,000-fold as

10 compared to a non-SIN vector, but that is not the

11 case.

12 I have had some ideas as to how that might

13 be explained, and the transvector, even at 10

9 is

14 zero.

15 \[Slide.\]

16 So, in conclusion, at least for this part,

17 the third generation packaging construct and SIN

18 vector generate recombinants with functional

19 gag-pol capable of mobilizing DNA, and when I say

20 \"capable,\" these are envelope minus recombinants,

21 only capable if they have pseudotype properties.

22 Separating RT and integrase from the

23 packaging construct decreases the frequency by

24 approximately 2 orders of magnitude.

25 Again something I said earlier, but I

> 317 1 think it is important, since a functional gag-pol

2 genetic structure is absolutely required for the

3 generation of RCL, and this includes not just in

4 vitro, but over the long term in vivo, monitoring

5 vector stocks for the production of envelope minus

6 gag-pol-containing recombinants may serve as an in

7 vitro surrogate marker to control against the

8 generation of RCL in vivo.

9 The trans-lenti vector design is

10 particularly amenable, but certainly not required

11 for this type of testing.

12 \[Slide.\]

13 I would like to shift gears and show some

14 other data because although I argued against this

15 earlier, most of the data or maybe all of the data

16 is really potential or theoretical based, that is,

17 what value do these envelope minus recombinants

18 really have.

19 Well, I think with respect to utility,

20 they can serve as a pre-RCR measurement that might

21 have some benefits compared with more conventional

22 RCL assays, but the point I am getting at, are

23 there other issues with respect to these types of

24 recombinants that might be generated when it comes

25 to biological safety, that is, are they

> 318 1 biologically significant.

2 \[Slide.\]

3 There is a lot of data that suggests that

4 envelope is not required for virus transmission,

5 and I didn\'t say that well, because it is much too

6 strong, but let me walk through this.

7 It is known for quite some time now that

8 cellular membrane proteins are incorporated into

9 virions during virus binding. It is also known

10 that this initial binding of HIV to its target cell

11 does not require an interaction between the

12 receptor and the envelope glycoprotein.

13 There are other factors, probably those

14 related to the point here, cell-derived factors

15 that are actually necessary for mediating at least

16 predominantly that initial interaction between the

17 virus particle itself and the surface of the cell.

18 Interaction between cell-derived membrane

19 proteins and receptors on the cell surface

20 facilitate initial binding. Interaction between

21 cell-derived membrane proteins and cellular

22 receptors can support HIV infection. There are

23 three or four paper published in 1997 and 1998

24 about this. Let me explain because it is not quite

25 fair I don\'t think without explaining.

> 319 1 In this case, what was demonstrated is

2 that you could express CD4 in a cell that was

3 producing HIV that did not contain an envelope, and

4 that envelope now budding from the cell and

5 acquiring the cell membrane with it as it buds, is

6 expressing CD4, can interact with the

7 GP120-expressing cell, fuse, and infect.

8 But the point is that this relationship

9 between whether a specific envelope is required for

10 infection, and simply by removing it, might also

11 not be enough, and, in fact, the most compelling

12 data was published actually this is 2000, in

13 December, by Irving Chen\'s group, that HIV envelope

14 minus virus can infect CD4-negative cells.

15 It is on that premise that I set up the

16 experiments which I will show now, which actually

17 shows that is not just true for HIV, but also for

18 HIV-based vectors.

19 \[Slide.\]

20 Also, to reiterate, or maybe to emphasize

21 my point, as it pertains to these experiments, why

22 are we concerned about this structure, this

23 recombinant if it exists?

24 If it is an envelope minus recombinant

25 that produces envelope minus virions, and it\'s

> 320 1 mobilized, even independent of specific receptor

2 envelope glycoprotein interactions to another cell,

3 the point is every time it replicates, it

4 potentiates the opportunity for additional

5 recombination especially, if you think about it in

6 vivo over the long term for the possible emergence

7 of RCL.

8 \[Slide.\]

9 So, how do we design these experiments?

10 We took envelope minus HIV-1 vector, real simple,

11 CD4 minus cells, and asked four questions. Do the

12 virions bind, do they synthesize DNA? If they

13 enter, what is the route of entry, and do they

14 actually infect these CD4 minus cells?

15 \[Slide.\]

16 The first question, do they bind? These

17 virions are green fluorescent virions. I won\'t

18 give you all the background, but using vpr, similar

19 to that which I showed for RT integrase, we could

20 incorporate enough green fluorescence protein into

21 the virus particle by expressing vpr-GFP in trans

22 to HIV-1, or in this case, to the vector. So, GFP

23 is incorporated into the virion and under confocal

24 microscopy analysis, we can actually detect single

25 virions. Those are depicted by the green spots.

> 321 1 So, using this method, what we have

2 demonstrated\--I will move directly to the point\--is

3 that even envelope minus virus can bind with equal

4 efficiency to both CD4-positive and CD4-minus

5 cells.

6 \[Slide.\]

7 So, how about DNA synthesis, what is the

8 consequence after binding to CD4-minus cells? Two

9 types are shown, 293T and HeLa. GC53 is a HeLa

10 cell that contains CD4 and CCR5 that is used as a

11 positive control.

12 The pluses indicate infection with the

13 vector, and here the pluses indicate 3TC, which is

14 anti-RT inhibitor as a control. We are detecting

15 early. That is strong-stop and full length or

16 nearly full length, what I call R-gag DNA products

17 of reverse transcription in these cells.

18 What is shown is that the envelope minus

19 vector can not just bind as I show on the previous

20 slide, but synthesize strong-stop and full-length

21 viral DNA in these HeLa, and 293T CD4-minus cells.

22 In the presence of 3TC, that synthesis is

23 blocked meaning that this is not virus-associated

24 DNA. It requires entry into the cell.

25 \[Slide.\]

> 322 1 We were interested in the route of entry,

2 as I mentioned, so we did an experiment, and this

3 certainly isn\'t a comprehensive analysis, but the

4 experiment involved the treatment of the cells with

5 Bafilomycin A. Bafilomycin A inhibits a proton

6 pump that acidifies endosomes.

7 The idea is that if the virus, which

8 contains no envelope enters the endosome, which has

9 been shown by others, and synthesizes DNA, we

10 should be able to inhibit that using Bafilomycin.

11 Indeed, that is the case.

12 With envelope-minus virus in the absence

13 of Bafilomycin, we have DNA synthesis, but in the

14 presence of I think it\'s 100 nanomolar Bafilomycin,

15 we inhibit by about 5-fold the levels of DNA

16 synthesis.

17 Importantly, this control isn\'t affected.

18 This is envelope-positive virus, isn\'t affected by

19 Bafilomycin because it takes a different route for

20 entry, membrane fusion.

21 \[Slide.\]

22 I don\'t think I am going to try to

23 describe all this data in detail, but the important

24 take-home message is that in HT1080 cells, which is

25 a tumor line derived from connective tissue, and in

> 323 1 a TU139 cell line, which was reported by Irving

2 Chen, it\'s a gingival cell line, we find that the

3 envelope-minus virus indeed is infectious, that is,

4 it forms provirus. It\'s a bona-fide infection.

5 This infection, very interesting, in the

6 case of HT1080, is not substantially or is

7 inhibited by Bafilomycin as might be suspected from

8 the slide I showed previously where we inhibit DNA

9 synthesis by Bafilomycin, but in the TU139 cells,

10 it is not inhibited, suggestive of two receptor

11 glycoprotein independent pathways for entry, this,

12 through an acidified endosome, and here,

13 independent of the endosome, perhaps through some

14 other membrane interaction.

15 \[Slide.\]

16 I am going to move through these slides

17 very quickly. I wanted to point out that the

18 trans-lenti virus vector, which I have been

19 discussing, has properties very similar to those of

20 lentiviral vectors with respect to gene

21 transduction in targets that I think are of

22 relevance.

23 Those include hematopoietic stem cells,

24 central nervous system, and the eye.

25 \[Slide.\]

> 324 1 In a study done in collaboration with Tim

2 Townes at UAB, we took purified bone marrow cells

3 from mice, we transduced them with both

4 lentiviral\--and this data is published\--and

5 trans-lenti viral vectors, and grafted them into

6 lethally irradiated mice, and after 16 weeks,

7 analyzed the percentage of those cells which were

8 expressing, in this case, GFP. What we find is the

9 lenti and trans-lenti were very comparable, and

10 importantly, that the bone marrow and this

11 phenotype could be transferred by secondary

12 transplantation, suggesting that we indeed

13 transduced the stem cell.

14 \[Slide.\]

15 That is my depiction of our in vivo data

16 for transduction of neurons.

17 \[Slide.\]

18 Here, the eye. Even after I believe it\'s

19 180 days, injection of trans-lentiviral vector into

20 the subretinal space leads to the RPE cell layer

21 being GFP-positive.

22 \[Slide.\]

23 So, in conclusion, the formation of

24 proviral DNA recombinants with a functional gag-pol

25 coding region may increase the risk of RCL. In

> 325 1 vitro monitoring for functional gag-pol-containing

2 recombinants, pre-RCL if you will, may serve as a

3 surrogate marker to control against the emergence

4 of RCL in vivo.

5 The trans-lentiviral vector design splits

6 the gag-pol function and is therefore particularly

7 well suited for this type of quality assurance.

8 \[Slide.\]

9 I have described this, but I thought I

10 would show it one more time because the transvector

11 design might be better suited for this approach if

12 we find that the lenti design is generating

13 recombinants that contain gag-pol.

14 \[Slide.\]

15 My acknowledgments are to my lab at UAB,

16 the work I mentioned with Tim Townes, Lori McMahon,

17 she was involved in the slide which wasn\'t shown

18 very well, where we transduced neurons, and Jean

19 Bennett for the work in the eye, and John

20 Wakefield, who is an employee of Tranzyme, who did

21 most of the recombination work.

22 Thank you.

23 \[Applause.\]

24 DR. SALOMON: I was trying to think of the

25 best way to do this, and I would accept some

> 326 1 feedback here. I thought possibly one way to do it

2 is as the next thing we need to do is get on to the

3 questions, and the first questions are the safety

4 data which makes a nice segue into what Dr. Kappes

5 just presented, would be to take a break now and

6 then 10 minutes, and come back and have Dr. Kappes

7 still here and begin the questions.

8 I need a break. Okay.

9 \[Break.\]

10 Open Public Hearing

11 DR. SALOMON: One of my duties is to again

12 reiterate a welcome to anyone from the public to

13 address the Committee at this kind of juncture,

14 which at one of several points where we can hear

15 semiofficial or unofficial open public comment.

16 I think, as you all know, I have not made

17 any effort to restrict the public from getting

18 involved at any point here, but I just want to

19 invite anyone who would like to. We haven\'t heard

20 any official requests, and I don\'t see anyone going

21 to the podium.

22 \[No response.\]

23 Committee Discussion of Questions

24 DR. SALOMON: It\'s 4:30, and I am going to

25 do my best in an hour and a half to begin some

> 327 1 discussion on Dr. Kappes\' talk and sort of the

2 implications and weave that into Question No. 1,

3 and then if you will forgive me, at some point I

4 will make an artificial distinction because in an

5 hour and a half I want to get through these three

6 questions.

7 What safety data should be available prior

8 to initial use of HIV-based lentivirus vectors in

9 Phase 1 clinical trials?

10 They are asking us to consider really the

11 elements that I think have been set up very well up

12 until now by the discussions, and I think

13 highlighted very nicely by Dr. Kappes\' talk in

14 which he kind of took a different attitude about,

15 well, maybe replication-competent lentivirus assays

16 isn\'t exactly the best way to go, and I think we

17 have kind of gone back and forth on that.

18 So, why don\'t we start there. I know we

19 had one question waiting at the end. Susan.

20 DR. KINGSMAN: In my past days as a

21 trustworthy academic, I did a lot of work on the

22 stoichiometry of gag and gag-pol in particles

23 because, of course, the way the virus is set up, is

24 to make 20 gags to every one gag-pol. It is quite

25 a precise stoichiometry, and if you get that wrong,

> 328 1 you tend to get a lot of aberrant particles, tubes,

2 and strange shaped things.

3 So, I wondered how your system got the

4 stoichiometry right and whether you have done any

5 EM studies to look at the homogeneity of the

6 preparations.

7 DR. SALOMON: Can I clarify one thing?

8 You are talking about his system?

9 DR. KINGSMAN: Yes, his very special

10 vpr-gag, vpr-pol system.

11 DR. KAPPES: It\'s an important question

12 and fortunately, we began addressing that question

13 far before we had\--\"we\" meaning my lab I

14 suppose\--any interest in using HIV as a vector. I

15 have studied virus assemblies since I went to

16 Birmingham 15 years ago, and we were interested in

17 understanding, using vpr, how questions could be

18 asked relating to the fundamentals of virus

19 assembly, that is, what role does gag-pol really

20 play in virus assembly.

21 So, that from perspective, we were working

22 with HIV and optimizing the system in many regards,

23 which aren\'t obvious from the data that I showed.

24 But your point is well taken and it is not so

25 simple as depicted where you transfect in whatever

> 329 1 it is, 4 micrograms of this and 3 micrograms of

2 that, and wham, you get a vector which is of

3 sufficient titer.

4 We can generate titers similar to Lenti,

5 but there is always a difference. We have never

6 been able to optimize the system to generate a

7 titer that is as high as Lenti. If we had done the

8 experiment 100 times, and you took an average, it

9 is probably 3- to 5-fold less because of the point

10 that you make.

11 There is a stoichiometry. There is even

12 other factors perhaps, and, for example, not to go

13 into\--well, to deviate too far from your question,

14 our data would suggest that RT integrase,

15 intermediate, which is produced during maturation

16 of the virus particle, plays an important role in

17 the formation of an infectious nuclear protein

18 complex.

19 So, there are a lot of factors, and a

20 titer that I showed is the best we can get which

21 compares with Lenti by a 3- to 5-fold reduction.

22 DR. SALOMON: What I want to do is stay

23 focused. I don\'t think that the point here, albeit

24 quite interesting, would be the idea that there is

25 maybe yet another delivery vector in development

> 330 1 after what we are doing with Lenti. I don\'t think

2 that is what we are supposed to be talking about

3 right at this second.

4 I think that what I would like to do is

5 stay on track with what the implications are of

6 what Dr. Kappes presented, what Dr. Kingsman

7 presented, and the others, in the context of this

8 first question.

9 Earlier on, I tried to articulate the idea

10 that, well, one of the things that was in front of

11 us was what would be the definition of a

12 replication-competent lentiviral assay, and we made

13 some discussion of that. I am not certain we

14 nailed it, but we made some discussion of it.

15 But now I guess the question would be,

16 thanks to Dr. Kappes and the discussion that

17 ensued, is a replication-competent lentiviral assay

18 the best sort of assay to hold out, and if it is

19 not, we need to have some discussion about what

20 would be reasonable alternatives to an RCL assay.

21 Have I got that right? I think that was

22 the strategy here.

23 Do you want to start with a comment on

24 that specifically now?

25 DR. KAPPES: I think I will just reiterate

> 331 1 what I presented. I think recombination is likely,

2 I think we will, if you analyzed titers that are

3 likely to be used therapeutically, generate

4 recombinants that contain LTR, albeit perhaps SIN

5 LTR, gag, pol, LTR.

6 If that is true, you essentially have the

7 essential retrovirus architecture minus envelope.

8 So, I would suggest that measuring for recombinants

9 which contain functions that are absolutely

10 required to perpetuate the risk of generating RCL

11 in vivo would be a feasible and perhaps even

12 reasonable means of trying to get out at safety.

13 DR. SALOMON: So, following that along,

14 Dr. Kingsman suggested a similar premise, that the

15 gag-pol was absolutely required for a

16 replication-competent lentivirus, and I think that

17 is the same premise you are making.

18 So, the assay that she and her colleagues

19 are proposing is one where you amplify it in a

20 target cell line. For right now I don\'t want to go

21 into the morass of whether the target cell line

22 should be horse or whatever. I think that is an

23 interesting discussion, but maybe not right this

24 second.

25 You amplify it in a target cell line, an

> 332 1 appropriate target cell line, and then you use a

2 PERT, product amplified reverse transcriptase

3 assay, versus your strategy, which is I guess you

4 would put it into this tat, puro, HeLa cell

5 transfectant, right? And then look for puromycin

6 resistance.

7 Now, there, your amplification, instead of

8 being PERT, is the selection on puromycin, correct?

9 DR. KAPPES: Right, and also you are

10 analyzing for a functional gag-pol, wherein Dr.

11 Kingsman\'s approach, just the presence of reverse

12 transcriptase or whatever was being monitored, that

13 would indicate a recombinant, would be enough to

14 reject the stock perhaps.

15 DR. SALOMON: Right. So, the RT activity

16 in the viral particle itself could give a false

17 positive in the PERT assay. She gets around that

18 by amplifying it in the target cell line.

19 DR. KAPPES: Another difference is that I

20 think you can imagine all kinds of recombinants,

21 and I could be completely wrong, especially from

22 packaging cell line won\'t be recombinants, but if

23 there are, the recombinant itself doesn\'t pose a

24 problem, so detecting the presence of the

25 recombinant, if it\'s not functional, perhaps isn\'t

> 333 1 a biological concern.

2 DR. SALOMON: So, if I have stated it

3 right, we have got RCL assays, we have a PERT assay

4 after amplification in a target cell line, and we

5 have a gag-pol assay that is based on an

6 amplification on the puro resistance in this

7 tat-HeLa model.

8 Any comments about relative values? Dr.

9 Rao.

10 DR. RAO: I actually just wanted to check,

11 and maybe I have missed something here, but let\'s

12 take the cell genesis system where they are looking

13 at an RCL assay, and they tried to do one

14 additional assay where they used a VSV-G envelope.

15 I am trying to see whether they were

16 actually now trying to assay the gag-pol, the

17 combination events that have occurred, and whether

18 they could just use that as a modification or an

19 additional part of the RCL assay to pick up those

20 recombination events, and whether that would be a

21 simple test in their mind rather than having to do

22 a separate test at all, and whether there would be

23 any relevance.

24 DR. VERES: Yes, I had those backup

25 slides. We have a 293G cell, which expresses the

> 334 1 VSV envelope, but under tetracycline control. So,

2 theoretically, if we generate sort of partial

3 recombinant, which is basically a function of the

4 core without an envelope, that could be transfected

5 into the cells and further passage and basically

6 amplified.

7 So, if it\'s a fully functional core with

8 full gag-pol, rev, and everything in it, that could

9 be passaged and basically using p24 assay, it will

10 be amplified and detected after that.

11 DR. RAO: Do you think that that would be

12 a reasonable assay in terms of detecting intact

13 gag-pol?

14 DR. KAPPES: I think it could be, however,

15 an important difference, as Gabor just pointed out,

16 it would require additional recombinations, that

17 is, by adding envelope alone, you wouldn\'t detect

18 the recombinant unless the other genetic components

19 of the original vector were also present, that is,

20 that they also recombined.

21 In my approach, remember\--maybe I walked

22 through it too quickly\--I provided additional

23 elements in trans, such as envelope, such as tat,

24 such as rev.

25 DR. VERES: Can I just reply to that? I

> 335 1 mean I do understand because you detect every sort

2 of recombination whether that is recombinant is

3 being functional or not. I mean the question is

4 whether this is really important if we consider

5 this is a product, do we want to detect which

6 potentially will be dangerous, or just do we want

7 to detect a recombinant which is there, but it is

8 actually not going to go any further, and there is

9 no potential that it is going to be dangerous.

10 DR. SALOMON: That is exactly what we are

11 supposed to be talking about, exactly, and that is,

12 is detecting that a safety factor.

13 DR. KAPPES: Well, let\'s walk through it

14 carefully and see. Again, we get into the

15 theoretical how, but we have a recombinant that is

16 LTR, even SIN LTR, packaging signal, gag, pol, LTR.

17 So, you have no rev, so you could make the argument

18 that that transcript will never be expressed, but

19 you can look at the literature and you find that

20 that is not true.

21 There is a huge difference in rev minus

22 and rev positive expression, but it is not

23 absolute. You look at the effect of the deletion

24 in U3 on expression of the recombinants message.

25 Again, you have a huge difference in the total

> 336 1 amount, but it is not absolute. You still have

2 expression.

3 So, if you are looking at a measure\--and I

4 know I am going to extreme ends\--but if you are

5 looking for a measure where I think you can

6 tangibly say that it is or is isn\'t, I think I

7 would look at an LTR, gag-pol LTR recombinant even

8 if it contains a SIN deletion, even if there is no

9 rev, as a potential risk to safety in vivo over the

10 long term.

11 DR. RAO: I just want to continue with

12 this thought just for one more second. If we

13 assume that you can do a test in 293G line, then,

14 one difference between the assay as Dr. Kingsman

15 suggested, and what you guys have suggested, is

16 really a matter of which method of amplification

17 you are using and how much time the assay takes.

18 In your mind, maybe I missed something, is

19 there a difference other than those two things that

20 I mentioned as being, you think one is more

21 sensitive than the other necessarily, or one is

22 intrinsically better than the other in your

23 opinion?

24 DR. KAPPES: I think one other important

25 difference is the functionality of the gag-pol. In

> 337 1 a biological assay, of course, you are going to

2 require that reading frame to be fully functional.

3 DR. RAO: Which the RCL in the 293G would

4 require.

5 DR. KAPPES: Either that or the assay I

6 described, yes.

7 DR. RAO: But not the assay that Dr.

8 Kingsman described.

9 DR. KAPPES: Not per se.

10 DR. SALOMON: But the assays are

11 dramatically different because, in the one assay,

12 you have to get infection of the target line. That

13 is what got into the discussion of you should use a

14 horse cell line or should you use the 293, or

15 should you use some other line.

16 Once you get infection to that line, the

17 efficiency of amplification of that line is an

18 issue. In Dr. Kappes\' assay, you basically are

19 going into the HeLa, and you are amplifying on the

20 basis of the presence of just having that gag-pol

21 with a packaging sequence, because the rest he is

22 providing, and then you are selecting for puro, so

23 it is not based on the infection of the HeLa line,

24 right?

25 DR. RAO: I understand that. I was just

> 338 1 trying to say that if you have two assays and you

2 have to choose between them, do you have to say

3 that one is necessarily a shorter time period, but

4 equally effective, or is it both are equally

5 sensitive, one takes much longer to do because you

6 have to do an amplification and selection for five

7 weeks because that would be a concentration in any

8 kind of test that you select.

9 I understand that there are differences,

10 but the goal of those tests remains the same.

11 DR. KAPPES: In response to that, I will

12 point out the assay that I described needs to be

13 highly controlled and is difficult. It is very

14 labor intensive.

15 DR. MULLIGAN: I think to try to get done

16 by 6:00, I would very simply separate a real garden

17 grade variety replication-competent virus assay

18 from these other things and see if we can move

19 ahead from the first one.

20 I would say that from what we heard, Cell

21 Genesys, or Sue\'s, they are perfectly okay. They

22 are just like MLV. I wanted waste a lot of time

23 with details of which is a better assay. That is,

24 we want real garden grade variety

25 replication-competent virus tested, and it appears

> 339 1 that there are several different ways to skin the

2 cat.

3 Then, I think I wouldn\'t get too

4 complicated about which one of these other assays,

5 I think that is a significant issue relative to the

6 MLV field, which is do we care that we are

7 transferring HIV DNA sequences to recipient cells,

8 and I wouldn\'t make the distinction whether they

9 are gag coding or whatever. They are not

10 replication-competent. I would lump them basically

11 as they are HIV sequences and do we care.

12 Here is where this dovetails with the

13 different packaging systems, the transient systems,

14 the 2-plasmid, 3-plasmid, 4-plasmid, and I don\'t

15 think we are going to resolve anything more than

16 there are theoretical different values to these

17 different systems.

18 I would argue that I would want to know

19 what is transferred in terms of HIV sequences to

20 recipient cells. I don\'t know whether I would make

21 that a release criteria for the testing assay, but

22 I think it is very important to have that kind of

23 info, but I would not support institutionalizing

24 the need for a gag-pol assay, you know, that

25 transfers gag-pol.

> 340 1 So, I would be very conservative and say

2 we are in good shape with the good old-fashioned

3 assays, and I would leave it to people that want to

4 look for these other things and maybe encourage

5 people to look for these other things, but not to

6 go past that point.

7 DR. SALOMON: So, that is an important

8 answer. That is exactly the kind of answer that I

9 was looking for, and that was do we abandon an RCL

10 assay, which was the premise of some, and you are

11 saying no, you don\'t abandon an RCL assay, in fact,

12 if anything, you allow these other assays to be

13 developed and validated, but you stick with good,

14 old basic RCL assays.

15 Frankly, I am comfortable with that.

16 DR. BORELLINI: So am I. Based on what we

17 know, the point where the highest likelihood of an

18 RCL to be generated is probably not in vivo, it is

19 in the fermenter where you have 40 liters of cells

20 that are spewing out, the viral components.

21 So, this is where I think we need to test.

22 At that point, you need to test what represent the

23 biologically active threat, which is the RCL, not

24 the partial. The partial, I think would be very

25 important to test if we had a lot of data

> 341 1 indicating that once in vivo, there is partial, may

2 go and do all sorts of things, but I don\'t know,

3 from the HIV field, I am not sure the data has been

4 seen in patients that have HIV, picking up of

5 envelope sequences here and there, or endogenous

6 sequences here and there, I am not quite sure the

7 hazard has been seen.

8 I am looking at the HIV person.

9 DR. SALOMON: Well, there is certainly

10 data that HIV, had different strains of HIV move

11 around elements, envelope proteins, and LTRs in

12 patients with more than one species of HIV, unless

13 I am totally getting that wrong.

14 DR. BORELLINI: As has been seen, did they

15 pick up envelopes from, for example, the endogenous

16 retrovirus sequences, or other sequences that share

17 poly(A) or something? I am not aware that that has

18 been shown so far.

19 DR. SALOMON: Any other comments on this

20 question of RCL? Dr. Torbett.

21 DR. TORBETT: I guess I would agree with

22 Dr. Mulligan. I don\'t think we have to go to the

23 extra point of going way beyond what is already

24 standard. I think that the current assays are

25 appropriate. Again, I would agree that going

> 342 1 beyond in experimental systems to validate would be

2 useful, but it seems putting onerous and very

3 serious consideration to how to move forward will

4 slow down the field. The question is do we need

5 those. My answer I personally believe is no.

6 DR. KAPPES: I would like to follow up

7 with a point. If we ignore the recombinants which

8 contain functional gag-pol, we are assuming that

9 those recombinants have no possible harmful effects

10 in the patient who is being treated with the

11 vector.

12 Now, I would also like to compare that

13 scenario with what we have learned or haven\'t

14 learned for MLV, which highlights my concern for

15 these recombinants.

16 I showed data which suggests that even

17 though they are envelope-minus, they might

18 mobilize. In the case of MLV, in our 10 years or

19 whatever of experience, it would suggest to us that

20 if these recombinants existed in that system, that

21 they never grew into some monster, but keep in

22 mind, MLV doesn\'t infect non-dividing cells, so I

23 think we are comparing apples and oranges, at least

24 with respect to the point I am trying to make.

25 These HIV recombinants will have a

> 343 1 property unlike MLV in that they will be able, if

2 expressed, to mobilize their recombinant genome to

3 other cells, again perpetuating the opportunity at

4 least for additional recombination.

5 So, I am not quite so quick to give up the

6 notion at least that detecting these recombinants

7 don\'t have value for predicting the safety of your

8 vector.

9 DR. ALLAN: It just seems to me that the

10 issue of recombinant gag-pol, to me would have more

11 interest in (b) rather than (a) because of

12 mobilization with the wild-type HIV. I just have

13 one question.

14 In your assay system, where you are using

15 puromycin to select your recombinants, those are

16 actual cells that you are looking at, so some of

17 those, is that an amplification of a single\--so if

18 you count 400 cells, could that one recombinant

19 cell that has grown out to 400?

20 DR. KAPPES: Very astute observation, that

21 is correct. That is exactly right. It could be

22 one recombinant-containing cell that mobilized 400

23 puromycin markers.

24 DR. ALLAN: I was suggesting that it was

25 one puromycin, it was one introduction into a cell

> 344 1 that was puromycin resistant, and then that cell

2 grew out.

3 DR. KAPPES: Each colony was a cluster of

4 cells.

5 DR. ALLAN: Okay, fine.

6 DR. SALOMON: So, recombination between

7 vector and wild-type HIV, just to sort of continue

8 on this roll, again, we have touched on this

9 several times, is there more to say about that?

10 DR. MULLIGAN: What is the difference

11 between (b) and (c)?

12 DR. SALOMON: They are related, but I

13 assume the concept in (b) is not mobilizing it, but

14 basically just recombining.

15 DR. ALLAN: It should be (c), (b) then,

16 because mobilization leads to recombination.

17 DR. MULLIGAN: I don\'t understand. What

18 is the context where we would be looking at\--

19 DR. SALOMON: I am thinking of the

20 transgene could be mobilized, right, in the HIV

21 vector, just packaged as an RNA transcript,

22 whereas, recombination would actually create a de

23 novo lentivirus.

24 DR. MULLIGAN: I guess I would agree with

25 the gentleman who said (C), then (B) maybe.

> 345 1 DR. SALOMON: I don\'t have any problem

2 with the order here.

3 John.

4 DR. ZAIA: In a way, I think (b) is more

5 important than (c) in the sense that we know

6 already that recombination can occur and make

7 things worse. I mean there are model systems for

8 that. The question is will that ever occur. So,

9 that probably means, who knows, but we should set

10 up the human experiment, so you monitor for that.

11 So, up to now we have not monitored with

12 MLV, who we probably should be. Had we been doing

13 that for the last 10 years, we would have a

14 database now. I know I am not doing that, maybe

15 some other people have been doing it.

16 So, as we go to these newer vectors, I

17 think it is going to be important to give more than

18 lip service to 1(b) because we will at least learn

19 how to progress.

20 DR. SALOMON: I agree with that, and I

21 think one of the suggestions that came up on some

22 of the discussion was that periodically, we ought

23 to be taking blood from patients in these studies

24 as part of this evaluation, and sequencing some of

25 the species specifically for recombinations between

> 346 1 the packaging vector\--no, I am saying the wrong

2 thing actually\--the lentiviral vector, the

3 transgene, delivery vector.

4 DR. MULLIGAN: So, you are talking about

5 (b) is in the context of an in vivo case where

6 there is wild-type, but still I think we ought to

7 address (c) first, because the issue is whether or

8 not, I think fundamentally, how high the bar we

9 ought to set for allowing mobilization to occur,

10 you know, a situation where recombination can

11 occur.

12 DR. SALOMON: I am just trying to be

13 practical. My feeling here with respect to the

14 recombination between the vector and the wild-type

15 is we have got to watch for it, and the only other

16 question that one would follow here, and I am not

17 comfortable with it, but let me just pose it, would

18 be saying you can\'t use a certain vector class in

19 patients that have wild-type HIV because of the

20 higher risk of recombination between, let\'s say, an

21 HIV lentiviral vector, a human lentiviral vector,

22 and a simian or a non-primate.

23 I am not sure that anyone wanted to go

24 there yet.

25 DR. MULLIGAN: But I agree that is the

> 347 1 relevant issue here.

2 DR. SALOMON: Can we have some comments on

3 that? Dr. Allan.

4 DR. ALLAN: Going back to what John is

5 talking about, I mean to me, the greater issue is,

6 okay, you have got chunks that are not

7 replication-competent, and they go into the

8 patient, and then the wild-type rescues it, that is

9 where the issue is. To me, that is a major, major

10 issue here, it is not a small issue. I think it is

11 a major issue.

12 DR. SALOMON: I am sorry, I didn\'t mean to

13 say it was a small issue, it\'s a big issue.

14 DR. EMERMAN: Can I agree with Dr. Allan

15 there? I think the major issue with going into

16 HIV-infected patients is generating new viruses

17 within those people. Even though the vectors are

18 derived from HIV, if they have LTRs, they will

19 recombine and make new LTRs, that weren\'t in the

20 patients originally, which is I think an argument

21 for using the SIN vectors in HIV-infected patients.

22 DR. SALOMON: Just for the transcriber,

23 that is Dr. Emerman, correct, that just commented?

24 DR. EMERMAN: Yes, that\'s correct.

25 DR. MULLIGAN: So, if we trace how that

> 348 1 might occur, basically, you will have an HIV

2 infected cell, that will be like your packaging,

3 then, you infect with your vector, and you would

4 have something that would have greater or lesser

5 capacity to be packaged, RNA to be packaged, and

6 then whether or not it has 3-prime end sequences to

7 be then at some frequency turned into a two LTR

8 mixed-up something.

9 DR. EMERMAN: The vector has two LTRs, and

10 it gets into an HIV-infected cell. They get

11 co-packaged at an incredibly high frequency, the

12 recombination occurs. That is not a rare event.

13 They get co-packaged, the recombination occurs just

14 as a normal process of reverse transcription in the

15 next cycle.

16 DR. MULLIGAN: But several of the vectors,

17 people are going to test, are going to be vectors

18 that specifically should not have mobilization

19 capacity, so they would be SIN vectors, that

20 wouldn\'t make a lot of RNA. They may be\--Sue

21 didn\'t toot her horn about some new vectors that

22 truncate the RNA transcript in the center of the

23 transcription.

24 So, I think this now brings us directly to

25 relating vectors, specific kinds of vectors to the

> 349 1 issue of mobilization, and if we don\'t want

2 mobilization, if we think that is an undesirable

3 feature, then, we have to actually begin to set a

4 high bar for the characteristics of the vector as

5 it pertains to mobilization.

6 DR. SALOMON: Earlier, I had commented

7 that, you know, just as a premise for discussion, I

8 don\'t think that the first clinical trials of

9 lentiviral vector therapy in HIV-infected patients

10 should have any mobilization. I am not saying that

11 it never should, I am saying I don\'t think the

12 first trial should.

13 DR. CHAMPLIN: I guess some analysis of

14 what components of the vector would, in fact, be

15 harmful should go into this. I am just thinking

16 theoretically if antisense to HIV is the sole gene

17 you are delivering, and there is no new product

18 that would make the HIV more pathogenic, would

19 that, in fact, be a risk if it was, in fact,

20 mobilized.

21 In fact, you could argue that that was the

22 way to deliver your antisense to even more cells

23 and to make it even more effective. So I am just

24 wondering if absolute bans here are appropriate or

25 can there really be a vector analysis of

> 350 1 risk-benefit relationships?

2 DR. MULLIGAN: I would argue that the risk

3 is the unknown of what you are going to generate.

4 I mean, again, coming back to the question can you

5 make something worse than what is already there. I

6 would argue probably, probably. We can do much

7 more sophisticated\--the body can probably do much

8 more sophisticated things than we as vector people

9 might be able to do.

10 So I would say the theoretical\--this would

11 be one case where the theoretical risk of

12 generating something in a patient that is

13 replication-competent, that is not garden-variety

14 HIV is something to be avoided at all cost.

15 DR. SALOMON: I think if I understand also

16 part of this is it is not so much the concern\--not

17 that I am trivializing it\--that the RNA ribozyme

18 transgene will get mobilized albeit I think you

19 could talk about that. But the fact is that in

20 systems in which mobilization is going to occur, it

21 implies that there are higher levels of lentiviral

22 vector RNA present and, therefore, recombination

23 with backbone elements of the delivery vector are

24 much more likely to occur and that is more

25 dangerous than any of issues reflecting

> 351 1 specifically to the transgene being delivered.

2 DR. EMERMAN: I think it is not the vector

3 that we are worried about going about it, the

4 recombination with the wild type is already there

5 making the wild type worse. It is not the vector

6 is making it any worse.

7 DR. SALOMON: I\'m sorry; I was hoping to

8 say that. If I didn\'t properly, correct it.

9 DR. ALLAN: I had a question for Mike

10 Emerman. This is John Allan. Where do you see the

11 greatest threat in terms of recombination using

12 this system with the wild-type HIV co-packaged with

13 the vector? Would you see that as any greater

14 threat than wild-type HIV?

15 DR. EMERMAN: You don\'t know. If the

16 vector has, for example, sequences in gag that are

17 necessary for the packaging, some of those

18 sequences contain parts of the matrix protein

19 although the matrix is truncated in the vector.

20 The matrix has epitopes for CTL lysis. You have

21 got a recombination. You would generate new

22 epitopes if someone had already had the existing

23 epitopes. It is a mechanism of generating escape

24 mutants, for example.

25 So, it is the recombination between the

> 352 1 existing elements in the vector and the endogenous

2 HIV, and just making that one a little bit worse.

3 DR. SALOMON: So, trying again to come to

4 some sort of committee statement to the FDA or

5 agreeing that we can\'t make the statement, that\'s

6 okay, too, can we say\--I say that you shouldn\'t do

7 a mobilization protocol in patients with wild-type

8 HIV infection in this first phase of trials.

9 DR. ZAIA: Because?

10 DR. SALOMON: Because of mobilization and

11 because of the danger of creating a novel species

12 of wild-type HIV or I guess now modified HIV, that

13 would have properties that might be more dangerous

14 than the current available crop.

15 DR. DELPH: I guess my question is would

16 you limit it to patients with wild-type HIV,

17 because as I have been saying earlier, you cannot

18 assume that people who don\'t have wild-type HIV

19 today won\'t get it tomorrow.

20 DR. SALOMON: Fine, that is an interesting

21 point. Why don\'t we take that as a (b). It was

22 good, I won\'t leave them one alone, but am I just

23 saying can we agree or disagree that we shouldn\'t

24 have mobilization?

25 DR. ALLAN: I guess my question is, is how

> 353 1 can you prevent mobilization? I mean you are going

2 to get mobilization, how do you prevent it?

3 DR. EMERMAN: The SIN vectors would

4 eliminate the mobilization.

5 DR. SALOMON: Whatever we have said is

6 basically the self-inactivating vectors, the SIN

7 vectors, or otherwise hobbled vectors that have

8 very little RNA that would get mobilized.

9 DR. TORBETT: I guess with the SIN

10 vectors, the LTR is upstream of basically the

11 promoter, so unless you inserted somewhere that had

12 an upstream promoter and get a full-length

13 transcript, that would be very difficult because

14 you would get a package of something that would be

15 much smaller.

16 DR. MULLIGAN: The only thing that is a

17 little complicated is if you look at how people

18 have addressed the question of mobilization in

19 tissue culture, and I think one of them will talk

20 about the VIRxSys. It is not satisfying, I mean it

21 is not a clean picture of things that really should

22 mobilize don\'t do it efficiently.

23 So, I think we ought to leave that just as

24 an issue that we are aware of, that the

25 measurements for mobilization, which are

> 354 1 undoubtedly going to be in vitro, not in vivo, will

2 have to be given careful consideration. That is,

3 we need the same sort of talk about mobilization

4 assays as we have had on the helper virus.

5 DR. SAUSVILLE: I would agree that the

6 information we have been presented today does not

7 present me with confidence that we can pick out

8 ahead of time with confidence the type of assays

9 that would permit mobilization, and I also am drawn

10 to the point of view that one could imagine

11 situations where that is actually a good idea

12 depending on what you are trying to achieve.

13 Finally, since no one is never without

14 risk of HIV infection, if you make a prohibition,

15 then, we might as well go home, because we are

16 never going to reach\--I mean the ultimate never in

17 absolute is I think going to be very difficult to,

18 in practice, realize.

19 So, I think it needs to be addressed on a

20 case-by-case basis. I think it needs to be

21 tracked. I think it needs to be considered what

22 the goal of the construct is, and beyond that, I

23 just don\'t think we have enough information.

24 DR. SALOMON: Is that a position the

25 Committee is comfortable with?

> 355 1 DR. MULLIGAN: I think it would be okay if

2 we did have a disagreement on the committee, and

3 not a consensus.

4 DR. SALOMON: I agree.

5 DR. MULLIGAN: We don\'t have a consensus

6 then, it sounds like.

7 DR. SALOMON: That is what I am getting

8 at. Let me try and articulate this, and this is

9 going to require some modification from my

10 colleagues.

11 In terms of Question 1, generally, we feel

12 that we still should put the emphasis as a first

13 cut at safety on more of a classic RCL assay. We

14 didn\'t quite define the ultimate sensitivity of

15 that, but I thought our discussions that we had

16 when Dr. Kingsman presented her material was pretty

17 good in terms of defining a sensitivity, and the

18 fact that it seemed like nobody disagreed with the

19 concept that sensitivity ranges for detecting RCR

20 in Moloney retroviral vector systems was acceptable

21 to the group unless someone wants to chime in now,

22 in other words, experience, 10 years of experience

23 defining sensitivity of RCR assays seemed to be

24 acceptable to be transferred to assays for RCL.

25 So, that would deal with that. Now, I

> 356 1 think that the group followed the very interesting

2 molecular biological reasoning for additional kinds

3 of assays, as suggested by the two speakers, but I

4 think that my impression, and again the sense I get

5 from the group, is that none of these newer albeit

6 very interesting assays, are (a) very easy to do

7 perhaps, maybe not that as much as not really

8 validated, and it is really rather far from clear

9 to me at least right now how a positive result in

10 one of these assays would relate to a negative

11 result in an RCL assay.

12 In other words, I don\'t think in the end,

13 aside from just a very good scientific line which I

14 follow, that I would be comfortable if I was the

15 FDA saying oh, the RCL assays are consistently

16 negative in this prep, and I think the field is

17 pretty sophisticated now about that, and yet we

18 keep getting a positive in a gag-pol puromycin

19 assay, and therefore, you can\'t do your trial.

20 I don\'t think anything I have heard today

21 would really make me comfortable with that. So, I

22 think there has to be further research and

23 validation of it, which would be a good objective

24 perhaps as follow-on studies for these first

25 trials.

> 357 1 In terms of mobilization, putting (c)

2 before (b), I think that we don\'t have an

3 agreement, so I think there are some people on the

4 Committee that are saying there are methods like

5 self-inactivating vectors that reduce significantly

6 the amount of mobilization, might eliminate it,

7 might not, and that those would be preferable in

8 the first trials of patients with HIV.

9 There are others on the Committee that say

10 that\'s probably not a good idea because there may

11 be situations in which mobilization has a

12 therapeutic benefit, there may be situations in

13 which patients could get infected afterwards or

14 prohibition on mobilizing vectors might lead to a

15 necessary limitation of patient groups that could

16 get these kind of therapies and therefore hold the

17 field back, and that wouldn\'t be fair because the

18 proof of a detriment of mobilization is far from

19 clear, if it can even be of therapeutic value.

20 That recombination between vector and

21 wild-type HIV, well, nobody is comfortable with

22 that, but the question is what is it that we could

23 do besides what we have already talked about to

24 reduce the risk of recombination between vector and

25 wild-type HIV.

> 358 1 I guess I don\'t think that we had any

2 clear answer for you there either, and that may

3 reflect the state of the field.

4 DR. MULLIGAN: That is coupled to the

5 mobilization question, so I mean I think that the

6 point of view that it is okay to have mobilizable

7 has to be coupled with an articulated point of view

8 about events between wild-type and vector.

9 DR. SALOMON: So, what Dr. Mulligan is

10 reminding me is that one line of argument is if you

11 allow mobilization to be a part of the therapy,

12 then, you potentially increase the risk of

13 recombination between vector and wild-type HIV, and

14 he is pointing out the logical flaw in that it

15 seems everyone\'s consensus is that that is not a

16 good thing.

17 DR. ALLAN: If you put in the SIN vectors

18 to reduce mobilization, you are going to reduce

19 recombination.

20 DR. SALOMON: So, that would be an

21 argument to favor protocols in this first phase of

22 HIV studies using lentiviral vectors that would

23 reduce mobilization, but there are people on the

24 Committee who are not comfortable with that. That

25 is the cycle here.

> 359 1 DR. ALLAN: But I think the people who

2 aren\'t comfortable, maybe it is because you are

3 interested in efficacy, and the first studies are

4 safety studies, so I think you have to separate

5 those two.

6 DR. SALOMON: Dr. Sausville, you were the

7 one who articulated the mobilization argument.

8 DR. SAUSVILLE: Right, but the limit case

9 of the safe vector will be one that doesn\'t work

10 when you eventually look for efficacy. There, you

11 have it.

12 DR. SALOMON: I was pointing out to Dr.

13 Mulligan, just teasing him, that that was one of

14 his comments yesterday, was the safest retroviral

15 delivery system was one that didn\'t work.

16 DR. CORDOVA: I would just like to follow

17 up on RCR assays versus this type of vector. The

18 one difference is that this is a human pathogen,

19 and that we can see at least what would be the most

20 likely type of recombinants that are undesirable,

21 and that would be potentially a similar vector or a

22 similar virus that has a broader tropism, for

23 example, so the typical RCR assays would not pick

24 up a partial recombinant, that only takes the VSV

25 gene, for example, into the next step.

> 360 1 Even though we don\'t want mobilization and

2 we don\'t want recombination, that may, in fact,

3 occur with the wild-type HIV. Then, if we take the

4 next step where we think that anybody may become an

5 HIV-positive, of course, we can take it to the

6 extreme, but all the same, I think some discussion

7 would be warranted on the possibility of creating a

8 new lentivirus that now does incorporate the VSV as

9 the envelope.

10 Although it hasn\'t been detected in vivo,

11 it hasn\'t really been looked for that hard in vivo

12 either as to natural pseudotypes that then just

13 occur.

14 DR. NOGUCHI: Dan, if we could pursue a

15 point related to that, the statement has been made

16 that surely with so many HIV-infected individuals,

17 we \"haven\'t seen this sort of recombination that we

18 are worried about.\"

19 I would like some sense of the Committee

20 as how valid a statement do you think that really

21 is, has it been looked at, by what means, is it

22 something to give us any comfort at all.

23 DR. EMERMAN: I think the point that the

24 recombination is going to occur in the fermenters

25 was the relevant point, you know, in populations,

> 361 1 these viruses aren\'t in the same cell types, and

2 there are not mixed deliberately, so in testing the

3 culture brews is what you are meaning to do.

4 DR. NOGUCHI: Excuse me, but yes, but in

5 the in vivo experiment, you have everything that

6 you need to package and recombine including non-SIN

7 LTRs, so I agree. I think we all agree we should

8 know as much about the synthetic product that is

9 going in as a therapeutic, potential therapeutic,

10 but the larger question is really whether or not

11 our experience with patients who have HIV infection

12 gives us any reassurance at any point that there is

13 either a lack or that if recombination with

14 endogenous sequences or elements, or even other

15 viruses like herpes, takes place or doesn\'t take

16 place.

17 It has been suggested that it\'s a means of

18 evidence that if recombination of that sort were to

19 occur, we would have seen it, but have we actually

20 looked.

21 DR. SAUSVILLE: I don\'t think we have

22 actually looked, but on the other hand, I really

23 wouldn\'t use the lack of detection or any clinical

24 phenomena that would rise to the fore as being

25 comfort in this regard, because the constructs that

> 362 1 are being talked about, particularly with VSV and

2 the making of these things, are very different than

3 what is running around out there in nature.

4 So, no, I don\'t have any comfort that the

5 clinical experience to date has anything to do with

6 this.

7 DR. CHAMPLIN: I would agree with that.

8 You are putting in artificial genes in viruses now

9 that have been optimized in the vector, and so this

10 really is nothing like what is there in nature, and

11 could obviously result in something very bad.

12 DR. ALLAN: The other thing is, is that

13 HIV is a highly evolved virus, and it has a

14 selective advantage over almost everything, so you

15 really have to do something or genetically engineer

16 something that is going to have a selective

17 advantage over HIV wild-type.

18 It is possible if you have got a VSV-G

19 envelope in there, maybe it would have a selective

20 advantage over HIV, but you look at patients, even

21 if they were rescuing bits and pieces of viruses,

22 whether herpesviruses or other endogenous viruses,

23 they are certainly selected for because people keep

24 getting HIV-1, they don\'t get weird recombinants,

25 and they keep transmitting HIV-1.

> 363 1 So, at least in nature, I think HIV-1 has

2 such a selective advantage over anything else.

3 That experiment has been done.

4 DR. MULLIGAN: I would disagree. I would

5 think that all the other pathogens, herpes,

6 whatever, the route of entry is different than it

7 is going to be via gene transfer. If you compare

8 the difference between a natural VSV infection in

9 someone who works with horses, cows, to having a

10 template in an retroviral RNA that has VSV-G

11 sequences, the chances for an event to occur that

12 could cause HIV to pick up the VSV-G, is far

13 greater than it would be, I think, from a natural

14 infection.

15 So, I think I would echo several other

16 points, that there is no reason to think that

17 because we haven\'t seen these things, that we

18 couldn\'t get these things.

19 DR. SALOMON: I think just for clarity, I

20 think Dr. Allan and Dr. Mulligan absolutely agree.

21 You disagreement was with what Phil thought.

22 DR. DELPH: I think that looking at this,

23 1(b) and (c), since we will be going into Phase I

24 clinical trials, which are as was pointed out

25 earlier, primarily safety trials, what we would

> 364 1 want to go in initially with would be a vector that

2 has potential for efficacy, but has the greatest

3 potential for safety that we can guarantee while,

4 at the same time, having some potential for

5 efficacy.

6 DR. SALOMON: I just think that as long as

7 we are trying to make sure that the FDA hears all

8 points of view, then, I would, as uncomfortable as

9 I am with this, try and represent the HIV

10 community, you know, as I have experienced them in

11 several different venues, and one of their

12 responses would be, my God, guys, you know, you

13 take a heart, you know, failure HIV patient, they

14 are going to die. These are young people, you

15 know, this is unacceptable to do anything based on

16 such theoretical safety issues, that, you know, you

17 wouldn\'t move forward in this community, and it is

18 insulting to suggest that you couldn\'t ask these

19 patients not to use safe sex or to refrain from

20 primary contact and participate in all the safety

21 features.

22 I think that, you know, it doesn\'t seem

23 like there is anyone here from Act Up or any of the

24 HIV communities, so I am not trying to represent

25 them here at the table for a second.

> 365 1 DR. SIEGEL: Just a comment about your

2 comment about safe sex, could we not presume that

3 if an HIV type vector wound up getting packaged in

4 VSV, then, its transmission might be other than

5 sexual? VSV is not sexually transmitted.

6 MS. KNOWLES: I would agree. I think you

7 have to make sure that every safety issue is

8 addressed, so that there is no repercussions down

9 the line.

10 DR. SALOMON: As long as we realize that

11 that is the ultimate super safety issue might

12 prevent this from ever coming to clinical trial in

13 the next decade, you know, at some point I think

14 the FDA is very well aware of this, that you have

15 to draw a bar somewhere and decide how high that

16 is.

17 DR. EMERMAN: Isn\'t the question about the

18 VSV-G recombinant addressed when you are just

19 looking for RCL?

20 DR. SALOMON: I am sorry, I didn\'t get

21 that.

22 DR. EMERMAN: We were talking about VSV-G

23 recombinants, but those are going to be looked at

24 when you look at whether there is

25 replication-competent viruses in the production

> 366 1 stock, whether they are VSV-G recombinants or some

2 recombinant with some endogenous envelope. Either

3 way, those would be picked up.

4 DR. SALOMON: I guess they would be

5 providing you sequence the RCL that come out of

6 these assays, right?

7 DR. EMERMAN: I guess that is one argument

8 to use the gag-pol PCR as an endpoint.

9 DR. CHAMPLIN: The wild-type HIV is not in

10 the production cells. This is an in vivo

11 consideration to mobilization end or interactions

12 with wild-type HIV.

13 DR. EMERMAN: The VSV-G isn\'t in the

14 patient, it\'s just the VSV-G protein. The VSV gene

15 is all gone, and they are testing for the absence

16 of DNA in the production lot.

17 DR. SALOMON: That is not true if a

18 transient transfection system ends up packaging a

19 certain amount of VSV-G transcripts in the vector

20 that you then deliver to the patient.

21 DR. MULLIGAN: This comes back to the

22 mobilization business, of course, which is that if

23 you don\'t test for something that transfers, but is

24 not replication-competent, VSV-G, in a case where

25 you have wild-type HIV, and that is a

> 367 1 mobilizable\--packageable VSV-G, then, you might

2 well get something that you wouldn\'t have been able

3 to detect before because it didn\'t exist, didn\'t

4 have the substrate, whereas, in the patient, you

5 might have the other helper sequences.

6 DR. EMERMAN: Maybe we could design a test

7 for that, though. I mean you could make a test

8 where you use your supernatant to infect

9 HIV-infected cells, and then look for something

10 with an increased tropism to grow out, bypassing

11 293 cells.

12 DR. MULLIGAN: In fact, someone raised you

13 could also look in the patient, I think you maybe

14 mentioned you may want to look at samples from the

15 patient to get a sense of what weird things might

16 be happening.

17 DR. SALOMON: Correct. I had one question

18 that I kind of want to end on, hopefully, we are

19 ending on this one, so we can go on Question 2,

20 that we earlier kind of touched on this and then

21 left it, that there are going to be patients with

22 HIV that are going to be candidates for lentiviral

23 gene transfer vector therapy and patients without

24 HIV.

25 If you talk about patients who don\'t have

> 368 1 HIV for a second, that are potential candidates for

2 lentiviral gene transfer vector therapy, would it

3 be appropriate to ever give a vector supernatant

4 that had even one RCR? I mean could you ever be

5 sure that your vector supernatant was absolutely

6 negative?

7 DR. SAUSVILLE: You are referring to HIV

8 based vectors.

9 DR. SALOMON: Yes. I could handle giving a

10 1 to 2 RCR risk to a patient with wild-type HIV. I

11 think we could get our heads around that, but I

12 just wanted, before we ended, to ask the question

13 of the group about to a non-HIV-infected patient.

14 Dr. Zaia.

15 DR. ZAIA: Well, there is no certainty in

16 clinical research, and that is the purpose of doing

17 it. So, you would set a bar, and you would see

18 whether your bar had been set high enough by the

19 clinical experience.

20 DR. SALZMAN: I am speaking I think on

21 behalf of the non-HIV patients, actually children

22 that have fatal disease, that they don\'t have a

23 long time to live, and what I have learned here and

24 from my own background is nothing is 100 percent,

25 and when you are talking about 1 RCR and whatever

> 369 1 that may be or may not be, because we just don\'t

2 know, and how long it is going to take to further

3 explore that versus a fatality within 12 months,

4 you definitely have to weigh it out.

5 So, again, on behalf of the non-HIV

6 pediatric fatal disease community, we see things a

7 little bit differently in terms of our approach

8 towards safety. While we believe it is obviously

9 paramount, you can never be 100 percent, and we

10 don\'t want to spend the next 10 years getting to

11 100 percent. It\'s not worth it in some cases.

12 That\'s all.

13 DR. CORNETTA: This is Ken Cornetta.

14 DR. SALOMON: Yes, Ken, go ahead.

15 DR. CORNETTA: I think partly just to

16 follow up the last statement, I think you never can

17 get to 100 percent. You would have to test every

18 virus particle that you gave back to a patient. To

19 get to say that this is absolutely free is just a

20 non-attainable goal.

21 I think the challenge for the

22 recommendation from the Committee, and what the FDA

23 and investigators combined will try to develop is a

24 thing where you are as assured as possible that

25 there is no RCL, but you can never, without going

> 370 1 again through every virus particle, assure that

2 there isn\'t an RCL in that product.

3 DR. SALOMON: Fair enough. I just really

4 asked that question to get that on the table as we

5 sort of left this particular section. You could go

6 another step farther just for purpose of

7 discussion. You could say\--and that\'s what I think

8 the person from the audience addressed\--you could

9 say, okay, then, you should only do this. Now,

10 getting to this question, should the first trials

11 be done in patients with HIV because we would have

12 no idea about the possibility of an RCL, and that

13 would be so terrible potentially to give it to

14 someone who didn\'t have HIV.

15 I am not saying that is my bias. I am

16 just putting it on the table.

17 DR. CHAMPLIN: You obviously can\'t answer

18 the question whether it is safe in terms of

19 transmission of HIV. I agree with the comment that

20 in very high-risk patients with end-stage and fatal

21 diseases, where again the risk-benefit relationship

22 would be on the side of going forward with the

23 test, that it would be a place where that could be

24 assessed.

25 DR. CORNETTA: Dan, this is Ken Cornetta

> 371 1 again. I think when you are talking about initial

2 studies here, you are talking about Phase I

3 studies, and I think the risks and possible side

4 effects that you might see in an HIV-infected

5 individual may well be different from what you

6 would expect in someone either with a normal immune

7 system, or just because of these are lentiviral

8 vectors going into an HIV-infected versus

9 non-infected.

10 So, the outcomes and the potential risks

11 may well be different, and we may need to think

12 about clinical trials in these populations

13 separately rather than as a single entity.

14 DR. SALOMON: I like that. That is a good

15 point.

16 DR. DELPH: Would it be possible or is it

17 more difficult to detect RCL in somebody who has

18 wild-type HIV than in somebody who doesn\'t?

19 DR. SALOMON: What you mean is not detect

20 wild-type RCL, but you are talking about

21 vector-derived RCL.

22 DR. DELPH: Right.

23 DR. SALOMON: Would it be harder?

24 DR. SAUSVILLE: This does touch on the

25 issue of what goes into the construction of the

> 372 1 input, as it were, HIV-derived strains. I mean one

2 might imagine that the RCL that would be derived

3 from them might have some marker or might have some

4 tag, might even be constructed with the pol and the

5 protease or with sensitivity to drug.

6 I mean there is all sorts of ways that one

7 could conceive of following in some way or another

8 the input, and we might encourage, if this is on

9 the table for a particular case, the design, so

10 that we could actually follow them easily.

11 DR. SALOMON: Fair enough.

12 DR. KINGSMAN: I apologize if I got

13 confused or lost the plot. I think there are two

14 issues that may be getting pulled into one. One is

15 using a SIN vector to prevent mobilization in the

16 target cell, and the other is using a SIN vector to

17 reduce the amount of LTR that comes through in the

18 components, and if you have got extended bits of

19 LTR flanking VSV-G, that could be bad.

20 They are two separate issues. You could

21 reduce the amount of LTR, but not have a SIN vector

22 because you could put a tissue-specific promoter

23 in. So, I think I would like to just ask that the

24 word \"SIN\" is not seen as just another way of

25 naming mobilization. It is one particular type of

> 373 1 vector.

2 So, that is one point. The other point is

3 this VSV-G issue. If we take VSV-G and create it

4 as an issue, then, this applies outside of the

5 lentivector field. It applies to anyone who is

6 trying to make MLV-based vectors, which are

7 packaged with VSV-G, which may have useful

8 properties.

9 So, I think the VSV-G issue may be get rid

10 of a native type of LTR that could possibly

11 recombine with an HIV LTR and deal with that

12 separately from mobilization in the patient. I

13 just wanted to say that.

14 DR. SALOMON: That\'s fine.

15 Yes, Carolyn.

16 DR. WILSON: I just wanted to briefly

17 address your last point, which is that up to now

18 there are no clinical trials with MLV-based vectors

19 using a VSV-G glycoprotein, and we do except that

20 the concerns regarding VSV-G would be the same with

21 an MLV vector.

22 DR. SIEGEL: I just want to put on the

23 table just an issue, not for discussion, but just

24 for background and comment. It has troubled me

25 about some of the recent comments. That is the

> 374 1 notion that although the risk of perhaps a certain

2 toxicity, such as having an RCL in a HIV-negative

3 individual, given that they are very small, they

4 would well be counterbalanced potentially in

5 high-risk patients by the prospects of benefit.

6 I mean we are talking about the context

7 now is the first introduction to people, and I

8 think any honest view of the first introduction of

9 any therapy to people, including the first

10 introduction of something this highly experimental,

11 would say that minuscule as our concerns are about

12 any particular toxicity, our hopes for benefit of

13 the first patient who will probably receive an

14 extremely low dose as a single time in something

15 that has not been studied, and without any dose or

16 route optimization or vector optimization, are

17 surely minuscule, and the notion that that patient

18 would consent to that therapy thinking that they

19 had a substantial incidence of benefit, suggests

20 that there is a problem with consent.

21 So, I think as you look at product

22 development in the bigger sense, you could say

23 these small risks are well compensated in a

24 population by the fact that the research may lead

25 to important therapies, but from the perspective of

> 375 1 the individual patient who I guess would probably

2 not be a child, who is going to first get this, I

3 think we realistically have to look at risks and

4 benefits from a little bit different context.

5 MS. A. SALZMAN: Amber Salzman. Maybe I

6 can give a perspective as a mother of a child who

7 potentially could be saved from this. I sort of

8 feel like why not at least give him a chance to

9 live. I mean I understand that the efficacy may be

10 low, and there is a question about the safety, but

11 if you do due diligence and you think maybe there

12 is a shot, I just would hate for a room of these

13 people to say that you wouldn\'t give my son a

14 chance to live even though it may be low.

15 DR. SIEGEL: I appreciate that, and I

16 understand that, and I would hope what your doctor

17 would tell you is that this could be very important

18 in terms of developing a therapy, and it may well

19 be the only chance for your child, but I think if

20 your child is the first person to receive this

21 therapy, it is likely not going to be even given in

22 a way that holds out any substantial hope of being

23 curative. That is just the way new therapies are

24 developed, and I think that is the context we have

25 to consider this in, in these early experiments.

> 376 1 DR. SALOMON: I think that is a good

2 point. I guess my response to it and to the mother

3 is the purpose of the Committee is to define these

4 sort of general safety issues. I think that if a

5 sponsor can find a group of patients, I don\'t think

6 that the Committee here wants to particularly

7 specify any group is inappropriate. I didn\'t think

8 you were saying that, Dr. Spiegel.

9 DR. SPIEGEL: No, let me clarify because

10 this is very important. I am not disagreeing with

11 the prospect that we want to make impossible

12 potentially valuable therapies for serious diseases

13 because of theoretical risks. We don\'t want to do

14 that.

15 I understand and I agree and support that

16 principle completely. I just don\'t want to be\--you

17 know, some are suggesting that, well, we will

18 introduce these into high-risk patients because

19 they will stand more to benefit than lose, and you

20 run into a significant concern in those patients if

21 you haven\'t informed them that their chance of

22 benefit is either zero or extremely close to zero,

23 you haven\'t gotten appropriate consent probably for

24 the type of research that we are talking about, and

25 when new products are introduced to people, they

> 377 1 can consent and often will consent recognizing the

2 importance of the research and even based on such

3 small chances of benefit.

4 But, you know, there are important ethical

5 questions in vulnerable populations in particular

6 in getting consent based on the fact that somebody

7 is desperate because they have a seriously ill

8 disease, and have the potential to believe that

9 there is chances for benefit which far exceed those

10 that are there.

11 MS. A. SALZMAN: I guess I would say I

12 work for a major pharmaceutical, so I am very, very

13 familiar with clinical trials, and that really

14 comes down to a very good consent form, and I know

15 with all of the hoopla over the last year or so, we

16 are getting much better consent forms.

17 DR. CHAMPLIN: Needless to say, the people

18 in the middle of the Phase I trial, are now up to a

19 meaningful dose, may well benefit, and what you

20 said certainly is true, and consent is a

21 complicated process, and one tries to give just the

22 message that you indicated, but there is hope,

23 hopefully, that when you get into the meaningful

24 doses, that even in the Phase I trial, that there

25 will be some benefit.

> 378 1 So, I think we want to just be sure that

2 the patients selected, where if, in fact, there is

3 an adverse event, we aren\'t going to make them

4 worse in a much more substantial way that would

5 have been the natural history of their disease.

6 DR. SAUSVILLE: I certainly agree with

7 those comments and also I agree with Jay, but on

8 the other hand, I think when this has been looked

9 at in study after study, patients go on Phase I

10 trials despite the protestations of lack of benefit

11 because they think they are going to benefit. I

12 mean that is the way it works.

13 So, I think, as you state, the point of a

14 consent form is to highlight or balance both

15 aspects of the science to be gained, and the

16 theoretical, although perhaps low, notion of

17 benefit.

18 I can understand the scenario where 1 RCL

19 of HIV in a given population might actually be

20 acceptable. I can certainly imagine populations

21 where it would be absolutely unacceptable, and I

22 think that has to be factored into this.

23 Also, just as a final point, the notion of

24 dose is applied to biological therapies in general,

25 and in particular, this type of therapy, I don\'t

> 379 1 know what the middle of a Phase I means, because I

2 think we are definitely treading new ground here.

3 DR. DELPH: I think as we are talking

4 about safety, in trials, we need to consider that

5 when we are looking at safety for most

6 pharmaceutical agents, you are talking about safety

7 as it relates only to the person who is taking that

8 pharmaceutical agent.

9 In this instance, where recombination may

10 be a possibility, and you may get

11 replication-competent virus, you may be involving

12 the safety of others, as well.

13 DR. SALOMON: I think that is a good way

14 to end this. I think if we started with Dr.

15 Cornett\'s comments and kind of follow the loop that

16 followed, I don\'t think that the Committee\--again,

17 if someone doesn\'t like this, jump in, but I don\'t

18 think that the Committee is coming down one side or

19 the other on whether the safety risks to an HIV

20 patient population with a lentiviral vector versus

21 the safety risks to a non-HIV, they are different,

22 as Dr. Cornetta started us off with very clearly,

23 but I am right now fairly neutral.

24 I mean I think it is going to be a

25 case-by-case basis and I think we would look to the

> 380 1 sponsors and look to the vector and look to the

2 data that they have, and make those decisions, but

3 I don\'t see any really compelling argument to say

4 no, no, yes, yes. I think it is fairly balanced

5 right now, which is an interesting place to be.

6 The quiet I assume is that we are okay

7 with this?

8 DR. HIGH: I wanted to make one other

9 point, and just to echo something that Eduardo said

10 earlier, because I bears emphasis, that although it

11 may be adequate to just leave this at RCL assays,

12 since this is a new therapy, and since there are

13 other methods we have heard about for looking for

14 helper sequences in other ways, I think it would be

15 good to encourage sponsors to incorporate that into

16 their design of the trial.

17 DR. SALOMON: I agree with that. I have

18 tried to capture that in my statement of saying

19 these would be really valuable follow-on assays to

20 be added on. In fact, that would be something even

21 to lobby the NIH to support these sort of clinical

22 assays added on to clinical trials. I agree.

23 Question 2. What should be the

24 appropriate species for in vivo, preclinical safety

25 and toxicology evaluation of lentivirus vectors?

> 381 1 Specifically, consider the following:

2 Wild-type HIV-1 does not infect monocytes,

3 lymphocytes, or other target cells in rodents nor

4 in cynomologous or rhesus macaques and will only

5 poorly infect CD4 T lymphocytes from chimpanzees,

6 so mobilization studies will be complicated.

7 Lentiviral vectors pseudotyped with

8 different envelopes, VSV-G, but also rabies and

9 flaviviruses, and I guess flaviviruses include the

10 ebola virus, may have expanded cell tropisms, but

11 the infection may be limited, for example, mouse

12 cells have multiple blocks to HIV replication.

13 DR. ALLAN: I haven\'t been following the

14 gene therapy vector field very much, but I mean the

15 premise here is that there is no animal model

16 system essentially. That is what this basically

17 says is HIV doesn\'t infect rodents, it doesn\'t

18 infect monkeys, so let\'s go to humans.

19 I spent the last 14 years working on SIV,

20 and we have good monkey model systems to study

21 recombinant SHIVs that replicate extremely well in

22 monkeys and kill monkeys, and function almost

23 exactly like HIV-1.

24 You have to reduce and redesign, but it

25 would just be a proof of concept. Everything is

> 382 1 theoretical. I mean basically, what you are saying

2 is here is the needle, close my eyes, hope for

3 okay. I mean that is what you are doing, I mean

4 essentially, because you are betting on what we

5 know scientifically, and you have heard some people

6 that have more concerns than others.

7 So, if you took and designed these things,

8 you could take a VSV-G recombinant S, whatever the

9 vector is, and pop it into monkeys, you could shoot

10 the virus directly into monkeys. You could do the

11 studies where you took the cells out of monkeys,

12 you could infect them with the SHIV virus, and then

13 put your CD4 cells with the vector in afterwards,

14 beforehand. There is all kinds of studies you

15 could do in monkeys that seems to me have been

16 totally underutilized, but like I said, I haven\'t

17 been following this field, and maybe some of those

18 studies have been done, but I think that if they

19 haven\'t been, I think people have missed the boat,

20 because the model system is just sitting there

21 waiting to be used.

22 You can look for recombinants very easily

23 if you pop a monkey, you have got a whole

24 ecosystem, and whatever pops back out, you may see

25 it. I am sort of perplexed why that hasn\'t

> 383 1 surfaced.

2 DR. SAUSVILLE: I guess my concern is that

3 while there is, as you indicate, a whole biology

4 that could be explored, I guess one has to have a

5 balance between closely mirroring the clinical

6 application of a proposed product and the doing of

7 ultimately toxicological research in a very

8 interesting model.

9 I would come down on the side of

10 recognizing the difficulty here as an intrinsic

11 part of the biology, attempting, how imperfectly it

12 may be, to pick a system that most faithfully could

13 replicate something of the human biology, but

14 really focusing on the safety testing on a close as

15 possible mimic with the product to be used to the

16 proposed clinical study, and let it go at that.

17 I think that to go beyond that, certainly

18 to engineer things that might look for effects, I

19 wouldn\'t know how to extrapolate them back to the

20 intended clinical use.

21 DR. MULLIGAN: I would say that you would

22 have to look at this on a case-by-case basis, but

23 Jeff\'s talk was a revealing talk in terms of the

24 kinds of questions you can ask about, certain tox

25 questions, you know, immune consequences of CNS

> 384 1 gene transfer. I think that is always going to be

2 the case here, you know, you are going to do the

3 best you can. You are going to be able to ask some

4 questions in some reasonable system, and obviously,

5 when people develop their preclinical information,

6 I think there is going to be an expectation by the

7 FDA that they address the obvious things that

8 people would think you could address in that

9 system, but I think the tone of this is, is there

10 any, you know, this is probably back to the old

11 monkey, you know, you have got to do monkeys or

12 something, and clearly, many of the issues we have

13 just been talking about are so complicated that

14 there is no easy answer to look for mobilization in

15 the context of a monkey or something like that.

16 Just to make it appear that we are moving

17 ahead, on the third point, I do think in vitro that

18 there is a lot of assays, in fact, I think the only

19 thing we can grasp probably over the last couple of

20 hours are some of these mobilization issues could

21 be better addressed in vitro, obviously, the ICR

22 assays, and so I think that there is no reason not

23 to do as much as you can in each of those systems.

24 DR. SALOMON: It seems to me you could

25 break it down a little bit, right, and I think that

> 385 1 Dr. Sausville kind of pointed that out to us, would

2 be a toxicology or a toxicity, direct toxicity of

3 the gene delivery, you could do in a monkey. You

4 could even do it in a rodent model.

5 I think as Dr. Kordower showed, I mean if

6 his strategy of putting GDNF in a monkey, you know,

7 and then the monkeys got better and they didn\'t

8 have strokes and, you know, he followed them for X

9 number of months, and I don\'t want to say all the

10 obvious things to everyone here.

11 That\'s pretty good. I mean I was pretty

12 impressed frankly, almost to the point where my

13 only comment, if you remember, was I find it hard

14 to validate a primate model when the guy is telling

15 me there is no toxicity. I would rather hear that

16 there was some toxicity and you avoided it.

17 So, I think, to me, it always worries me a

18 little bit when there is absolutely no toxicity and

19 everybody is cured.

20 But with that said, I think there would be

21 some things then that we could feel comfortable

22 modeling in rodent and non-human primate models.

23 Would we at least buy into that first part?

24 DR. CHAMPLIN: I think the animal models

25 in general are a useful proof of principle, but

> 386 1 obviously, they are different reagents, they are

2 different drugs, if you will, and different

3 biologics. It is great to do it as a proof of

4 principle, but when you come down to the individual

5 agents being proposed for human trials, and once

6 you have proved the principle, now you are still

7 left with doing it in man.

8 DR. SALOMON: What he did, he actually

9 used his HIV GDNF vector in the monkeys, so that

10 was his product at least to my understanding.

11 DR. CHAMPLIN: But as you are looking at

12 recombinant events and those kind of things, which

13 is what we have been talking about, you know, you

14 need to do it in a parallel system using SIV, which

15 obviously hasn\'t been applied to the HIV vector

16 that is ultimately going to be used in humans.

17 DR. ALLAN: I think when you go to monkeys

18 and you start talking about SIV, you say, well,

19 gee, I am not going to redesign all these vectors

20 and redo all this stuff, and, you know, we are not

21 using this in humans, so, you know, that seems like

22 a lot of work.

23 Well, you know, I mean for some of these

24 trials, you know, you can use most of the same

25 vectors. You have already got the same VSV gene,

> 387 1 you have got the same whatever therapy you are

2 trying, I mean you have already seen that it is a

3 human gene, you put it in the monkeys, and you

4 don\'t have to redesign anything there usually.

5 Sometimes, you know, maybe they are only 95 percent

6 related, but you are talking about if you are doing

7 gene therapy, I mean the monkey is 95 percent or

8 whatever, the chimp is 99 percent, so there is not

9 a lot you have to do.

10 Even with the difference between HIV and

11 SIV, when you are just looking at gag and pol, the

12 studies that have been done so far, and they

13 haven\'t really\--people haven\'t spent enough time on

14 this, which is unfortunate, but there is only a

15 small region in the gag gene that doesn\'t work in

16 terms of packaging between HIV and SIV, it is just

17 a small piece.

18 So, you are not talking about all these

19 things you have got to redesign, it is just very

20 little, and I am not saying that that has to be

21 done before you go on to humans, I mean I sit on

22 these xenotransplant committees and I know that

23 that is not going to happen anyway, but I think

24 that you have really got these model systems and

25 they are sitting there and people ain\'t using them,

> 388 1 and those are the model systems that are going to

2 be able to tell you, I think, you know, in terms of

3 safety.

4 Certainly in toxicity, but I think also in

5 safety and also in terms of recombinational events,

6 I think that is the model system.

7 DR. NOGUCHI: Jon, would you expand a

8 little bit on that, would you be comfortable with

9 creating a VSV envelope SHIV as an example, to try

10 to get to some of this?

11 DR. ALLAN: No, I wouldn\'t do that. What

12 I would do is create the same system.

13 DR. SIEGEL: Are you suggesting

14 specifically that a SHIV, you could take a VSV

15 product developed, packaged product developed for

16 human use, perhaps with a SIN vector or without a

17 SIN vector, and use a SHIV model as a useful way of

18 exploring mobilization, how much occurs, which

19 vectors mobilize more and which don\'t, and where

20 and what time course, and things you would want to

21 know for human use, and you could do that with

22 vectors designed for humans in that model, and

23 perhaps that would model behavior of use in

24 HIV-infected humans.

25 DR. ALLAN: It should especially in

> 389 1 HIV-infected humans, especially in HIV-infected

2 humans in terms of mobilization and recombination,

3 and I think that is really the critical area, but

4 we would have to redesign like gag-pol vector. I

5 am not saying it\'s a small thing, but\--

6 DR. JOLLY: This is Doug Jolly. Are you

7 saying that you think HIV vectors rescued by SIV

8 would be a suitable model?

9 DR. ALLAN: What I am saying is depending

10 on what you are looking at, you can use a SHIV

11 virus in the monkey and you can make a SHIV

12 packaging vector, essentially an SIV packaging

13 vector with a SHIV challenge.

14 If you use SHIV, any kind of study you

15 want to do whether it\'s an antisense, HIV, envelope

16 or whatever else, you could use a SHIV. You could

17 also use SIV certainly, so it just depends on what

18 you are going to use as a therapy and tested in the

19 monkey.

20 DR. JOLLY: But the vector itself, the

21 backbone of the vector would have to be SIV, right,

22 so it is a different vector?

23 DR. ALLAN: Just the gag-pol

24 DR. JOLLY: So the actual vector genome,

25 the 900 nucleotidase of HIV that are left in the

> 390 1 vector, you would use that vector, not an SIV-based

2 vector.

3 DR. ALLAN: I don\'t think it matters. You

4 could just plump in the gag gene and that probably

5 would take care of it. I mean Mike Emerman may

6 have some more insights than that, but I think you

7 could probably do that very easily.

8 DR. EMERMAN: I don\'t think you could do

9 it very easily I think you are talking about a

10 much different kind of experiments using SHIV. The

11 kind of recombinations you will get are going to be

12 different, acting vectors are going to have to be

13 much different.

14 I think it is an interesting exercise, but

15 I don\'t know that it actually tells you about the

16 product that you are actually going to be using.

17 DR. ALLAN: I am just talking about proof

18 of concept and trying to examine the issues

19 recombination and mobilization. I think that model

20 will give you that information, but not on a

21 specific product.

22 DR. EMERMAN: Dr. Allan, it\'s a five-year

23 grant. It is not a straightforward simple

24 experiment.

25 DR. SAUSVILLE: I think we have got where

> 391 1 we want to be because while I agree that it would

2 be a very interesting, intellectual, and biological

3 exercise, I think to make that a product-related

4 matter, you know, each product somehow has to go

5 jump through a hoop would be problematic.

6 DR. SALOMON: I think we all were

7 thinking, you know, you take this and that would be

8 your background and significance for your RO1 or

9 your program project probably would even be more

10 appropriate here, and I think it might enhance the

11 field, but I think we all agree that that wouldn\'t

12 be advice to the FDA to hold sponsors to that at

13 this point.

14 But I do think that we have articulated a

15 very important problem in the field and we spent

16 the whole day articulating it. I doubt it\'s not

17 clear to you by now, right? I am sure it is very

18 clear to you that we are concerned about

19 mobilization and recombination and

20 replication-competent alteration.

21 This is the kind of thing, you know, to

22 OBA. This is where NIH leadership to gene therapy

23 could come out of these kinds of discussions. I

24 mean these are really important questions and maybe

25 this is the kind of thing there should be for an

> 392 1 RFA.

2 DR. MULLIGAN: This stuff haunts the gene

3 therapy field, how do you get the basic research

4 that most directly supports these things. We were

5 talking about the assays, our interest in having

6 better assays for gag-pol, and so forth, we ought

7 to really make the case, it is very key.

8 DR. SALOMON: Any other comments? I think

9 we have answered basically all your questions, but

10 if we haven\'t, this is a good time to tell us.

11 DR. WILSON: I think the Committee has

12 done a really commendable job going through some

13 very difficult territory today, and we really want

14 to thank everybody on the committee for their very

15 thoughtful and thorough discussion of all the

16 issues that have been raised today. Thank you.

17 DR. SALOMON: I also want to thank the

18 committee, this is a lot of hard work, to our

19 speakers, to the audience who actively

20 participated. I think it really contributed to the

21 whole balance of things.

22 Tomorrow morning we begin at 8 o\'clock

23 sharp mainly because it is so important, but a

24 number of us are going to have to make planes, and

25 I don\'t want to decimate the committee without

> 393 1 really getting to the meat of tomorrow\'s sessions.

2 So, tomorrow morning we will definitely start at 8

3 o\'clock on the money, although we were pretty good

4 this morning and we did finish at 6:03, so I guess

5 we did pretty well today, as well.

6 Thank you all very much. See you tomorrow

7 morning.

8 \[Meeting recessed at 6:03 p.m., to

9 reconvene at 8:00 a.m., Friday, October 26, 2001.\]

10 - - -