U

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

8:05 a.m.

DR. RAO: Good morning and welcome to another day at the FDA. We have one new member on the committee and I am going to ask Dr. Prockop to introduce himself before we start.

DR. PROCKOP: I'm Darwin Prockop and I head up the Center for Gene Therapy at Tulane.

DR. RAO: Welcome, Dr. Prockop. Just a brief reminder. Remember to shut off your mike when you are done and wait for the Chair to recognize you so that we can have a reasonable flow.

We are going to launch straight into the first part which is a continuation of what we were doing yesterday. We will discuss the product issues related to articular cartilage. Dr. Moos will give the update perspective first.

DR. MOOS: Good morning, everyone. Thanks for sticking it out through to the second day. By the end of yesterday I imagine we now know 75 percent of what there is no know about cartilage biology, not only 50 percent as Dr. Luyten suggested yesterday.

Today some of what we may be discussing is the other 25 or 50 percent. I think there are significant uncertainties that surround issues of manufacturing, product characterization, and testing. I am going to hit on a few of those points in a fairly general way.

By way of introduction just to review a bit of regulatory philosophy, while the oversight of things like devices and small molecule drugs is amenable to product testing, entities as complicated as biologicals have needed for a hundred years or so a sort of three-pronged approach where one considers carefully the starting materials, the manufacturing process, and how you take various kinds of precautions to make sure that it is consistent, and testing of the product.

And taking into account all three together allows one to move forward into new areas of endeavor despite significant uncertainties about the nature of the products and the key parameters that are required to determine their safety and effectiveness.

Things that resemble devices or, for that matter, small molecule drugs, are more tractable in a number of ways. They are well characterized. The materials from which they are made are defined precisely. Often we know their structures for small molecule drugs, where the protons are to Angstrom resolution and with devices. All sorts of specifications.

We know what to measure and we have analytical methods that allow us to do that. We know which things that we're measuring actually matter, actually have a major influence on the performance of the product. In contrast as we move to things that are biologically move into a gray area that's a little bit fuzzy and, on this slide anyway for us as well as for you, a little bit hard to read. To try and throw some light on that, these products are very poorly characterized.

We used to be a decade ago concerned about how to characterize a single therapeutic protein. Now we're talking about entire cells or collections of cells or products which have organized themselves with or without the help of an artificial substance or substances into a three-dimensional matrix.

The nature of the product is such that it could be very heterogeneous. The entities are complex in molecular terms. In the case of a cell there may be tens of thousands of difference macro molecules to consider. The distance between what we might want to measure and the capacity of existing analytical techniques to measure them is often quite wide by comparison with other types of products the FDA regulates.

Finally, and I think most problematically there is insufficient scientific data that allows us to relate things that we can measure to what happens in vivo in biological systems.

Now, much of what I'm going to talk about today will not deal with the process. The manufacturing process is generally considered to be something that someone who is coming to us has developed and is their proprietary matter and trade.

Nonetheless, it's worth nothing that issues of source and release testing can help support the process both to optimize it and to take measures to ensure that it's consistent.

Although, as I mentioned yesterday, there is a lot of emphasis placed on getting into initial trials as quickly as possible for a variety of reasons. Our experience teaches us that it may be useful to consider the entire strategic drug development process as early as possible from a global standpoint so that you, as the saying goes, measure twice, cut once.

Sometimes there is a tendency not to spend enough time early in development and making sure that you have optimized to a reasonable extent the product that you are going to carry forward for more expensive kinds of analyses such as preclinical testing or very expensive clinical trials.

It could be that up front investment in looking at your sources and playing with your manufacturing process may prevent a costly waste of time and resources and you may end up after some sort of a confirmatory trial just shy of an endpoint with a p-value that is tantalizing but maybe not quite enough.

Personally, one of my worse nightmares as a reviewer is for the idea to be fundamentally sound and there is something about the product, something that we are not controlling or we don't understand, and there may be a few patients where there is an outstanding success but we don't quite have the data that would support a license application. The trial fails and suddenly the venture capitalists are nowhere to be seen.

So it's my own personal feeling, and I think many of us in the agency would agree, that better characterization of the product early in the development program may make a number of things easier downstream.

The first area is source control. We heard a little bit yesterday about different anatomic sites which may include different sites within a knee as well as starting materials taken from other types of tissue or even other donors.

The question of whether or not a donor site might be involved with the disease process could be construed by some and we did have this discussion at some length internally as a patient inclusion or exclusion criterion but, nonetheless, there are some patients who would meet inclusion criteria.

You go in and you look at the time of collecting tissue and you might see something that would tell you this is maybe tissue that isn't going to work to generate a useful product.

Finally, there was some information yesterday speaking to the issue of cellular inhomogeneity perhaps in the starting material. A graphical illustration of this are these scanning electron micrographs taken by Professor Stanley Erlandsen of the University of Minnesota.

This is a juvenile joint surface. I think you can see that as you go through to different regions within a joint, there's substantial obvious heterogeneity from one cell to another just at a casual glance.

At Dr. Rao's suggestion and with Dr. Luyten's permission I have put in a few of his slides from yesterday by way of review. One very interesting piece of data that we heard yesterday is that even in freshly isolated chondrocytes there is a population of cells that in a model stressed with skeletal muscle injury by cardiotoxin is capable of developing markers for myocin.

This one, and probably the simplest interpretation of this data is, indeed, the starting material is biologically heterogeneous and you can determine this with molecular markers. Clearly even within a single tissue there might be characteristics that need to be considered in terms of subpopulations of cells that would have an influence on the performance of the final product.

The next area is product testing. Now, the general philosophy for developing a set of tests is to do a set of very thorough characterization studies to learn as much about the product as possible in the context of the manufacturing scheme that you have developed.

From that set of characteristics further work is done to try and select a set of tests that could be useful and practical in a manufacturing environment so that you can do the test quickly enough, the assays are robust enough and so forth, that they could be used both to control the manufacturing process and to release the product.

Ideally, you want them to be sensitive enough to changes in the manufacturing process that if there is a change your testing will help you detect it. Similarly if there is something about the product that is either good or bad, your testing procedures will pick it up. And, finally, in an ideal world your set of analytical procedures should predict what happens when the product is placed in an in vivo environment.

Now, data in this field including what we heard yesterday suggest to me and to others on the committee that the focus that has been conventional in many areas of cell therapy on looking at terminal characteristics of the tissue that one desires to see formed after administration in vivo may not be the best ones to measure on the final product or during manufacture.

For example, if we take chondrocytes out of their -- if we take cartilage out of its natural environment, dissociate the tissue enzymatically and put it on plastic, many of the characteristics that are normally associated with cartilage are lost. So looking for those characteristics in vitro may not be helpful in contrast to other types of products where perhaps a terminally differentiated state could be expected.

I think it's equally apparent from a variety of data that many of the terminal markers for several products including these may not predict biological function so there are plenty of examples of products which express type II collagen and aggrecan and don't express type X collagen which, nevertheless, fail in vivo.

So let me suggest that perhaps the real task is not to identify something that has some characteristics of terminal chondrocytes in vitro so much as to identify functional chondroprogenitors. By that I mean a set of cells which may or may not be a homogeneous population that can be qualified according to a meaningful biological response. In other words, let the biology tell you what the tests should be rather than take a guess at what we think we should be measuring and see what works if we're lucky.

Yesterday I promised you an example of this kind of idea from another area of cell therapy and that area is the area of pancreatic islet transplantation.

There is a certain kind of parallel in that in islet transplantation there was a sort of history of a few successes in a few patients who achieved a major benefit and a number of patients who achieved very little benefit and there was this sort of erratic one time to another variability that made everybody think there was something really real going on but that there wasn't a handle on some variable. Work that has been done by pancreatic islet enthusiasts has taken a harder look at some parameters influencing product quality.

The data I'm going to show next is curtesy of Klearchos Papas at the University of Minnesota and also Bernhard Hering at the same institution. At an earlier meeting of this same group Dr. Hering actually showed us this data where a number of islet isolations were performed and by the conventional sort of measure that everybody does which is to look for exclusion of a vital dye, all of the products past. They were all over 80 percent viable.

What the groups then did is to apply a couple of different types of measures of cellular vitality, if you will, that looked at something other than the integrity of the plasma membrane. The first of these that I am going to show is oxygen consumption rate.

Now, you would predict that oxygen consumption rate would be directly correlated with dye exclusion if dye exclusion was really a sensitive measure of how many intact fully living cells you have. In fact, it's not correlated at all.

What is possible to do in this study was to then look at the performance of each of these isolations in vivo and separate groups where you had 100 percent failure or 100 percent cure with a few marginal ones in between cleanly based on this data, a 15-minute procedure that is telling you something useful.

A similar type of test measured ATP levels on the same groups of cells but, once again, could cleanly separate cells which performed well in vivo and cells which didn't. One thing that made this work very well was a clean in vivo assay, the diabetic mice made so artificially in a controllable way. You could qualify these two tests against performance in vivo.

To be clear, I'm not suggesting that either of these tests would be relevant to chondrocytes which might not depend on respiration anywhere near as well or directly as pancreatic islets. Just to illustrate the parallel here between conventional methods not working and perhaps more refined methods and one could consider for chondrocytes looking at other things. Perhaps caspase-3 levels, mitochondrial membrane potential sensitive dyes. There's a whole list of things that could be considered.

It's possible that in vivo models such as the one that Dr. Luyten showed us yesterday might be applicable and we can discuss the applicability of this type of model or perhaps other assays of this sort later on. The idea here is the general concept that some kind of in vivo qualification assay that wouldn't be feasible in a manufacturing environment could be used to help you evaluate analytical tests that would be useful in a manufacturing environment.

Now, the next question has to do with this idea of tests that would identify functional chondroprogenitors. Many cell therapy enthusiasts, especially early in the history of these procedures, had the idea that a cell product could be administered and local instructions would cause the cell product to do exactly what was desired but they would be completely plastic, completely under the control of local environmental influences.

The analogy that I like to use, and people have heard me talk before are probably tired of, was given to us by American cartoonist Al Capp in 1948 who talked about pluripotential entities that he termed the Shmoo. There's a transitive verb that has entered the popular vernacular that you now know the origin of.

The instructor signals that they were subject to were simply the desire of the nearest human. Whatever was needed by the nearest human the Shmoo would transform themselves into the finest butter, eggs, a certain variety of ham, and so forth. This simplistic cartoonist's view, in fact, is one that developmental biologists have known since the 1920s probably didn't work.

In fact, an experiment that was cited for the Nobel prize demonstrated this quite clearly. You could take a piece of an embryo from a pigmented strain and transplant it into a recipient embryo in an ectopic site and far from the expectation that the donor embryo would adopt the characteristics indicated by the surrounding tissue, in fact, the reverse happens.

What you see a couple of days later is an embryo that is almost entirely twin, Siamese twin structure, if you will. If you take a section through the embryo in such a plane, what you'll find is, and I think we have a gamma problem and the colors aren't showing up on this computer, but what you see -- do we have a pointer? Thanks. What you would see if the colors were working is that in the primary axis the pigmentation would correspond to the recipient embryo.

In the secondary axis a small amount of the tissue would correspond to what you would see in the donor but much of the tissue is, in fact, derived from the host. In other words, the host tissues aren't capable of receiving instructions from the donor.

Now, the message is clear, instructions can go both ways. In fact, on another of the slides that Dr. Luyten showed us suggested in some of the discussion following this also raised the question as to whether in repair of tissue what you see is contributed entirely by the host, entirely by the recipient, or some combination of both.

In fact, we know that for another product, and Dr. Tuan mentioned it yesterday, if you inject this product into a joint space, you actually recruit host tissue in such a way that it's clear that its fate has been changed by instructions from these therapeutic cell product. This is a complexity that needs to be considered.

Now, how cells may or may not either convey or respond to these types of instructions is an area of developmental biology that is concerned with what developmental biologists call competence factors. These may be various kinds of monomeric or dimeric receptors or co-receptors.

They are soluble ligands, ligands which may be cell-associated, the signalling machinery intracellulary, and the gene transcription machinery that may represent a more long-term alteration and biological response that leads to something that could translate into an in vivo activity.

Perhaps, may I suggest, that we might need to be looking for our sets of competence factors which could be positive factors or negative factors. Again, from Dr. Luyten's data, we see examples of both of these things that are associated in a positive way with the expression of a stable chondrocyte phenotype and things which are associated in a negative way with such a cellular organization. Perhaps that's the kind of question that we might consider.

Indeed, this final slide from Dr. Luyten suggest that you may have sets of markers. We can discuss his approach as well as alternatives that might be useful. Certainly I don't think anyone suggests that this is the last page of the last chapter but it is, in fact, somewhat reminiscent of the oxygen consumption data that I showed you a few minutes ago.

The last thing I want to discuss is the concept of new regeneration products that we heard some talk of yesterday where the cells are actually organized in three dimensional space. If to the extent that is true, one may need to consider what is responsible for that organization and look into the factors which control that organization.

This bright-field and fluorescent image of a gene that the laboratories of Lee and Kingsley and the mouse and Dr. Luyten and myself and a number of other species is a growth factor associated with forming joint space. I think you can see that it is a remarkably specific marker that separates the joint space from other tissues, one example of the kind of thing to look for in products that may be organized three dimensionally.

To take an even finer look, if one looks at that same growth factor and the molecule that we have found is necessary for its activity, you see in the forming joint space an overlapping region of perhaps one cell diameter corresponding to the forming joint surface. It could be that this kind of a test or analysis might lead to optimizing and qualifying other more simple tests in a manufacturing environment.

So I would like to leave you with a few general questions for thought. What is going on early in the process whether we are talking about acceptance criteria for a homogeneous or mixed set of cells, whether there is selection for or against particular members of that population, or whether instruction is happening.

As I said a bit ago, perhaps competence factors sufficient to define the cells that are useful might be thought about. It's not necessary, may I point out that we actually understand the mechanisms as long as we can qualify them usefully in an animal model.

As I just pointed out, distribution of characteristics in 3-D may be critical. Finally, the theme that Dr. McFarland and I have tried to develop is that there is an interplay between how good your animal models are and how they can be used to tell you something and how easy it might be and how solid your basis will be for selecting particular sets of analytical tests.

The questions for discussion, in highly condensed form for projection, deal with the criteria for obtaining starting tissue: What the characteristics might be for functional chondroprogenitor cells, or if we don't know what they are. What would be useful approaches for finding that out. What might be useful methods, analytical methods to explore for determining these characteristics. What are useful approaches to qualify these tests using various kinds of preclinical models, which need not be disease models.

The special issue that is often neddlesome enough to cell therapists generally that it's worth breaking out separately of potency assays and considerations that might be specific for cells that are contained in naturally produced or artificial matrices.

I will now yield the floor to our chairman so that we can discuss these issues more generally. Thank you.

DR. RAO: Are there any specific questions for Dr. Moos right now? In that case, we'll move on to the questions. I want to make a couple of remarks right in the beginning when we consider this and just remind the committee of some things that already exist in terms of rules and regulations which have come from a long history of sort of cell and tissue sourcing from organ transplants or from bone marrow studies and from other studies.

So the FDA does have a guidance on tissue sourcing and that is available on their website and that provides some general guidelines in terms of consent issues, testing for certain human viral pathogens, etc. That is not something that might be different.

What we really want to consider today in terms of sourcing is what are the unique aspects to sourcing of cartilage tissue or tissue which would form cartilage if you are doing autografts or if you are taking allogeneic tissue.

Dr. Moos really raised some potential issues in terms of just sourcing. Is it how you harvest? Can you ship it? Are there any sort of specific things that one needs to worry about in terms of taking articular tissue?

Is there a sourcing issue in terms of which region of cartilage one takes? Whether if you take articular cartilage from the ankle or whether you take ear lobe cartilage whether there is any difference and are there any ways of measuring quality of the tissue in any sense?

I am happy to have anybody lead off the discussion in terms of making points. If nobody wants to, then I'll ask Dr. High to tell us a little bit about bone marrow just in general in terms of collection so that people can think about it a little bit.

DR. HIGH: Well, first of all, I would like to remind you that I think Dr. Blazar is probably a lot better qualified to talk about harvesting bone marrow than I am. I actually wanted to ask a question about the issue in this first question. Are we only talking about cartilage or are we talking about mesenchymal stem cells or any sort of starting product that might be used here?

DR. RAO: I'll let Dr. Moos answer that question.

DR. MOOS: Everything is on the table. I did want to point out that even if we were talking about cartilage, there might be heterogeneities within even a single joint that could be important whether specific areas of pathologic involvement that might be apparent on arthoroscopy or even characteristics that might appear following collection where you might do some acceptance assays after starting the manufacturing process, arrays or PCRs or something like that.

But, in addition, since there is discussion of using non-cartilaginous starting material if there are specifics there. Now, we saw some data yesterday to suggest that material from synovium could be made to look like cartilage in vitro and totally fall apart in the mouse assay. That is a clue that it may not be so simple to do it from other sources but that is not to say that it can't be done.

DR. HIGH: As I was listening to Dr. Moos talk, what struck me is that the most important thing here, and actually I was thinking this during most of the discussion yesterday, is to attempt to correlate the clinical outcomes with characteristics for obtaining product. Those seem to be the central issues in the discussion here that actually overrode any preclinical considerations that we were spending time on yesterday.

In other words, to attempt to correlate outcomes in the clinical trials with the characteristics of the product that were used. It seemed to me that was something we hadn't heard a great deal about.

DR. RAO: I'm hoping we consider that in the next couple of sections but it's really absolutely very important and I think Dr. Moos has alluded to it when he said that we need to know some way of having a measure of potency. Let's discuss that as potency.

But just looking at tissue, I mean, is there any criteria? When people take pancreatic islets, for example, when Dr. Moos pointed that out, you always worry about the time of isolation because it's a cadaveric source so there's a time window beyond which it is certainly not considered reasonable to expect to get good tissue and that is relatively unique to pancreatic islets in that that is the major source if you were to get cadaveric pancreatic islets as an issue.

When you take biopsies in humans in terms of taking biopsy samples in the nervous tissue, you always worry about the source and what underlying pathological condition was present for which you could be allowed to take a biopsy so that becomes an important consideration in your tissue source material. So is there anything like that as far as sourcing cartilage that one has to worry about specifically when one is considering a source material issue?

Go ahead, Dr. Tuan.

DR. TUAN: I think one issue, of course, is potential donor site morbidity for articular cartilage repair. If you are taking it from articular cartilage we need to be concerned about that. Particularly if we want to use low-passage-number cells we will need to get quite a bit of tissue.

If it's not from the articular cartilage, then where else? And, also, how can we then really -- I mean, it's tied to the second question. We can get whatever we want but if it doesn't work, then it's an issue. I think one of the first concerns ought to be donor site morbidity -- potential donor site morbidity.

DR. RAO: So is it an important criteria the amount of tissue you get? Is that like a really critical parameter that one needs to really know so that when you are sourcing and it's a single person source and it's an autograph that you have to get a minimum amount of tissue per se in terms of having any predictive value and quality or something that one needs to worry about?

DR. TUAN: I think perhaps the orthopedic surgeons can deal with this and how much is an allowable amount with some prediction of acceptable donor site morbidity.

DR. RAO: Go ahead, Dr. Tomford.

DR. TOMFORD: I think donor site morbidity is very important. There are places in the knee, for example, that you can get cartilage that probably does not affect the joint over long-term so I think it's legitimate to consider that. There is some work that shows that chondrocytes in the ankle are different from those in the knee so I'm not sure you should go to another different joint.

DR. RAO: Would there be consensus on that, that if you are taking from the knee people would only take tissue from the knee? If you are working the ankle you wouldn't take cartilage tissue from some other source?

DR. TOMFORD: I don't know. You would have to ask other members of the panel, I guess. It may not be grossly different. In fact, it may be that there is a difference that is advantageous. I'm just saying there is a difference.

For many years anterior cruciate ligaments were taken autologously from the middle-third of the patellar tendon and we now know that there are some problems with that. Long-term there may be some problems with the donor site we don't know about but, as far as I know, not a lot of problems with that.

I think as far as the numbers of cells are concerned we heard yesterday that the Genzyme Corporation actually increases the number of cells in culture so that probably either you have to take a lot of cartilage to get enough cells so that you don't have to expand them or you have to expand them. Of course, when you expand them then you have to submit to other reviews.

I do think we probably need some testing to determine what is the optimum number or the minimum number of cells that you are going to transplant. I'm not sure we have a lot of data on that yet. In particular, depending upon how large the lesion is. In other words, lesions vary up to 4 to 10 sometimes square centimeters so can you get away with 10 million cells or if you get a larger lesion do you have to use 20 million cells? I think there also has to be some evaluation of the number of cells corresponding with the size of the lesion because lesions come in all sizes.

DR. RAO: Dr. Coutts.

DR. COUTTS: I'm feeling very ignorant right now. I don't think there is a lot of data on this which is what Dr. Tomford essentially just said. I know that in the genzyme ACI methodology there's a minimum amount of tissue by weight that they request which I think correlates with the number of cells they anticipate harvesting from that tissue.

Obviously the more cells that you could provide them, the more they would be able to give back to you. As a general principle, I think there's some correlation with the size of the lesion that you are attempting to treat with the number of cells that you ought to have. But I'm not aware that this has been systematically studied and a lot of this has just kind of evolved like trial and error.

DR. RAO: Also if you harvest the tissue is there any measure of the quality of the tissue that one takes in terms of looking at this tissue? Is there any parameter when you look at it and say this is good tissue or is there any worry what you get is what one takes?

DR. COUTTS: I think you are happy with what you can get. You are obviously trying to avoid damaging critical parts of the joint so you are staying on the parameter. The cartilage tends to thin as you go from the main weight-bearing surfaces out to the parameter of the joint. It's quite possible that you are getting some fibrous tissue or fibrocartilaginous tissue as part of your biopsy.

But it gets to this whole issue of how important is the cell that you're providing. All cells start with the same genetic information. We do tend to believe that the site in which it's placed has a tremendous influence on how it performs.

It could be that cells that are not chondrocytes that are provided and expanded in culture and then returned for implantation might not be -- may not have been chondrocytes to begin with but that when put into the joint environment that they will behave like chondrocytes. There is some evidence to suggest this could happen but, again, I think there's a knowledge deficit here that hasn't really been carefully worked out.

DR. RAO: Dr. Scully and then Dr. Mc.

DR. SCULLY: I think that the over-simplification is even more than that. It's not only that joint surfaces differ between ankles and knees but they differ within the joint, locations within the joint as Dr. Coutts has said.

Then the cells themselves differ within the depth of the cartilage. I think all this concept of harvesting chondrocytes and putting them back in whether it comes from an ankle to a knee or from a knee to a knee there is a gross over-simplification. I agree with Dr. Coutts when he's saying that they all have the same genetic information. What we really need to do, I mean, if we had a magic wand and we could make this approach work is to recapitulate the developmental scheme so that we get normal articular cartilage back. That magic wand hasn't been identified yet.

DR. McILWRAITH: We have done some work in the host with looking at donor site morbidity and use in sort of the equine equivalent of MACI taking 300 milligrams from just basically a Ferris-Smith rongeur from the lateral trochlear ridge and then doing follow-up pathroscopies up to 12 months. There's no progression of the lesion. There's minimal healing. It varies but it's off the abaxial side. Like Dr. Coutts was saying, getting off the nonarticular portion so we don't feel that we've got any clinical morbidity but it's always a feel thing. You're basing that on going back to your question about quality or how do you assess it. That's what we gave the company to process it because that's what they wanted, 300 milligrams, for instance.

You can look at the quality arthoscopically but it still doesn't take into account the points that Dr. Scully and Dr. Coutts are raising about difference in cell numbers. But it does -- you know, then you've got the process of whether you culture or whether you put it right back in which is a newer technique.

Obviously if you put that cartilage right back into a new environment, a new location, then hopefully you are going to be able to modulate those cells effectively but we don't know for sure. We just think about it.

DR. RAO: Dr. Moos, do you have something to add?

DR. MOOS: Yes. Dr. McIlwraith raised one point that I would like to address explicitly when he said that you might see something when you scope a patient. Is there something that might not be apparent before you scope that when you go in would tell you I'm not going to take this piece? That's part one. Part two, is there any data to support that your impression is correct or not.

DR. McILWRAITH: You mean to some predictor before you do the orthoscopy that you are going to have defective cartilage at the donor site?

DR. MOOS: Right. Suppose you have a chronic knee with some degenerative changes. The injury was seven years ago just for the sake of example and the patient is a candidate by inclusion criteria and you look and you see the surface of the knee and there is something there. You say this is not going to work. Let's just forget it and not charge the patient what it's going to cost for this second procedure and all the rehab and so forth and not even take the biopsy.

DR. RAO: Go ahead, Dr. Coutts.

DR. COUTTS: I could argue actually on the opposite site of that, that some pathologic tissue might actually be a better source of cells. Studies of arthritic cartilage harvested at the time of total knee replacement shows that these cells are actually more response to TGF-beta, for instance so that their receptiveness to stimulatory growth factors is upregulated.

In fact, cells in fibrillated cartilage frequently are cloned which would suggest that they have been making an attempt to replicate and to try to heal the lesion. These cells actually may be stimulated cells that potentially would be better repair cells than cells from a more quiescent area of cartilage.

This is all hypothesis and conjecture. I have absolutely no idea if what I've just said has any veracity to it but I'm just saying that you can go that line of thought if you want to.

DR. RAO: Dr. Harlan and Dr. High.

DR. HARLAN: I just want to make a small point. I think everybody here is aware of it but the central paradigm of biology for so many years that all cells have the same genetic material and, therefore, that they all should have the same potential. The Dolly experiment has shown us that's not true necessarily, that cells do have epigenetic changes that occur at various stages of their development that may be irreversible.

The thought that not only an ankle chondrocyte might be different than a knee chondrocyte but even within a knee they may be different. Those changes may not be irreversible. They may be but they may not be.

DR. MOOS: That was the point of my slide with the frog embryos. Indeed, as you pointed out, it's a gross oversimplification to suppose that cells always respond to their environment rather than the other way around.

DR. RAO: Dr. High.

DR. HIGH: Just to start with a process that is already in place, the autologous chondrocyte implantation, what parameters are used now? For example, does the material that comes in is it characterized as sort of wet weight of cartilage and then the final product is characterized as total number of cells?

DR. RAO: Can we get to that question after I get an answer to the sourcing question? Maybe we can ask Genzyme to comment on that since they are doing this as a process. To take this sort of sourcing issue is there any age at which you would say, well, I'm not going to harvest cartilage for this because it's a growing child and the epiphysis has infused and I don't want to touch the joint? Is there an age at which you wouldn't do this at all?

On the other end, is there an older age at which you wouldn't take this because we know that cartilage ages and we contrarily get a large number of cells even if we take a biopsy of a size that we can take in terms of being any viable use? Is there a range that there is consensus in the field which people do or don't do?

DR. COUTTS: I think I would willingly take cartilage from a child because a child has the capacity to heal it. Because of this fact that they are still in a growth phase, that imparts to them a repair capacity that we lose when we reach adulthood.

It's rare that children present for this. I think probably because they do have this healing capacity, although the issue of osteochondritis dissecans borders on the child/adult age range.

The issue is more on the age side. It's clear that the ACI technique and the microfracturing technique work better the younger the individual. We know that there are significant changes that occur in cartilage with aging. The cells die off. The cartilage becomes thinner. The cells are, for lack of a better description, less robust in terms of their intercellular machinery and manufacturing capacity and their responsiveness to various cytokines all declines with age. I hate to say this because it's happening to all of us.

I think that the current methods of cartilage repair are pretty much reserved for the young middle-aged people and that for the elderly it hasn't been an option and total joint replacement really is the option for them. There's an arbitrary cutoff of 50, although the definition of middle age keeps changing.

Either you personally change your own definition or society is changing it. It's clear that people age 50 today typically are much more active than they were a few decades ago. That may impart some degree of preservation and may slow the aging process but clearly there's an age factor.

DR. RAO: Bruce.

DR. BLAZAR: It would seem listening to this that if there's an analogy to the bone marrow transplant field, we take biological materials. They have some heterogeneous characteristics but they are from generally single site. What has happened in the field is really to look at the outcome data first and then try to look at correlations with cell dose or CD34 content, etc.

But it's only been going from the biological outcome to correlative parameters to try and find surrogate potency assays that we are then able to determine the answers to some of the questions that are being posed.

Unless you have a correlate that you know will predict outcome whether it's cell number related to metabolism or phenotype, these at best, I think, are going to be best attempt speculations but are really not going to be able to answer the questions in the absence of those clinical outcome correlates.

We'll get into that discussion later but it would seem that it is trying to almost answer the question before you have the outcome data to allow you to back correlate and determine what makes the most sense.

DR. RAO: We'll try and get a little bit into that but we should keep that thought. Before we lose that, maybe I can ask Genzyme to comment on when you get soft tissue, what do you do? Do you look at wet weight and do you have a clinical prediction on number of cells in terms of getting --

DR. WILL: Hi. Jackie Will, Senior Scientist in Manufacturing at Genzyme. We asked the surgeons to give us a full-thickness biopsy so that we capture all of the layers of cartilage and we are looking for a beginning weight of at least 200 milligrams. We say aim for 200 to 300 milligrams. We will process whatever the surgeons send us so we have started with as little as 20 or 30 milligrams of tissue to grow the product.

Then during the biopsy processing steps our technicians are trained to remove any extraneous tissue from sources other than cartilage so if there's bone or if there's synovium in the biopsy we won't process that. We are aiming to just process what looks like hyaline cartilage. then our process is monitored throughout the growth phase of the cultures. We have certain time frames that we expect cells to achieve certain density markers and the cells are monitored for morphology during the entire growth process. Then the final product is actually tested for trypan blue dye exclusion and our product is based on the number of cells that we are shipping out.

DR. RAO: Live cells or are they shipped as a frozen vial?

DR. WILL: They are live cells. They have a shelf life of three days.

DR. RAO: So a flask of live cells.

DR. WILL: A small vial of a very concentrated cell suspension.

DR. HIGH: And what range of numbers of cells are typically released?

DR. WILL: Our average is somewhere in the range of 2,000 to 3,000 cells per milligram of tissues that we process.

DR. SCULLY: If you get a small sample coming in, not the 200 milligrams you asked for, do you go through more passages to end up with a larger product or do you do the same passages and they just get less product out at the end?

DR. WILL: We only go up to three passages so what happens typically in that case is that the cells are in culture a little bit longer so they take a few extra days to get to the confluencing markers that we are looking for.

DR. RAO: So that's an important point. They may go through more population doublings even if they don't go through passaging.

DR. WILL: Right. They definitely do go through more population doublings.

DR. BLAZAR: What surrogate studies do you do at Genzyme or what studies are done on the site to look at differences in the type of products? You have a cell number but what are the attempts being made in the field to correlate the product itself with outcome? We didn't get a good feeling for that other than --

DR. RAO: Let's hold that for a little bit later.

DR. BLAZAR: Okay.

DR. PROCKOP: Point of information. How many population doublings do you go through?

DR. WILL: We typically see four to five population doublings per passage so 15 all together.

DR. PROCKOP: And how long does it take?

DR. WILL: Three to four weeks.

DR. McILWRAITH: Maybe my question is premature, too, but you mentioned markers so it's probably a little bit the same as what Dr. Blazar said but do you have some markers you use?

DR. WILL: We have done process validation where we have taken the cells in our final product and put them into suspension cultures in agarose and alginate. Then we look for collagen II and aggrecan expression.

DR. RAO: Let's hold that, though, and we'll ask this again maybe if you can come back for that. I still want to try to focus on tissue sourcing so if you have a question specifically on sort of the size, amount, patient sort of issues, then let's get that through first before we get to all these other really important issues that Kathy and Bruce and everyone has raised about markers and how you qualify the number of cells that you get.

Rocky.

DR. TUAN: Yes. So my understanding was that we are not only just talking about chondrocytes. Is that correct? So there are these other sources that we need to also think about such as bone marrow, such as adipose and other tissue sources, placenta and so on, so forth.

I guess the placenta is not as critical in this discussion but in terms of the adipose and the bone marrow I think those need to be considered because there are reports, for example, when you