A F T E R N O O N S E S S I O N

DISCUSSION OF HUMAN GENE TRANSFER

PROTOCOL #01007-488 ENTITLED:

A PHASE I, OPEN-LABEL CLINICAL TRIAL

OF THE SAFETY AND TOLERABILITY OF

SINGLE ESCALATING DOSES OF

AUTOLOGOUS CD4 T CELLS TRANSDUCED

WITH VRX496 IN HIV-POSITIVE SUBJECTS

DR. MICKELSON: If we could get started then, please.

We are now moving towards discussion of protocol No. 488, which is a Phase I clinical trial of the safety and tolerability of single escalating doses of autologous CD4 T cells transduced with VRX496, which is a lentiviral based vector in HIV-positive subjects.

The reviewers for the committee were Dr. Aguilar-Cordova and Dr. Markert and Nancy King, and we have multiple ad hoc reviewers, Dr. Zaia from the Beckman Research Institute and City of Hope and Dr. Yee from also the Research Institute and City of Hope.

Also, in addition, Dr. John Coffin, who is director of the HIV Drug Resistance Program through NCI is present as sort of a special ad hoc member for the committee for this afternoon's review.

And we will be following the same format. Dr. Dropulic from VIRxSYS will be doing a 20 to 30 minute presentation on the protocol itself and then we will go through the committee reviewer's comments, ad hoc reviewers and then open the floor for discussion and comment from the public as well as the discussion with the investigator, with the result that hopefully what will come out of this are recommendations from the committee that will be transmitted to the sponsors and the investigators as well as their local oversight committees.

So with that, Dr. Dropulic, thank you.

DR. DROPULIC: Thank you.

First of all, I would like to thank the reviewers for, you know, reviewing our protocol. I think that overall it has made it that much a stronger proposal.

What I would like to do today is basically give an overview presentation of VRX496 and the transduction procedure for a Phase I clinical trial in HIV-infected patient subjects.

And during the course of the presentation I would like to touch on basically the questions that were raised by the reviewers. What I have provided today is a booklet there which I think you should have. It is basically answering in detail in writing the reviewer's questions that were not answered in time for the submission because we had received the reviews late. Okay.

(Slide.)

So this is our proposed protocol. It is an ex vivo gene transfer of VRX496. This is the name of the HIV-1 based vector that we are proposing for Phase I clinical trials in HIV-infected patient subjects.

VRX496 is an HIV vector that contains an anti-HIV antisense sequence. This antisense sequence is about 1 kb. The protocol involves a patient subject coming in, undergoes leukophoresis, the T cells are isolated, and then the T cells are exposed to VRX496. The cells then are expanded at the University of Pennsylvania cell processing facility and then undergo rigorous QC testing. Importantly, one of the rigorous QC testing points is that we would perform a Taqman PCR assay on VSV-G DNA and show that the final cell product does not contain any VSV-G DNA present in the product. After QC release the cells are then basically infused back into the patient.

(Slide.)

This is the structure of VRX496 and its analog vector 494. This is the laboratory grade version of 496 and the difference between 496 and 494 is that 494 contains a GFP coding region so that cells transduced with 494 mark up green and we can use it for laboratory analysis.

In contrast, clinical grade vector does not encode for any proteins and the only sequence that is not derived from wild type HIV is a small 186 base pair marker sequence that does not code for anything derived from GFP that we use to determine cells that are marked with the vector by Taqman PCR.

So the vector, VRX496, is derived from the prototype molecular clone, pNL4-3, and the components from which the vector was constructed are shown. We have a 5' region, a region from the central polypurine tract. The antisense region is derived from the 5' end of the env, which is flipped in the reverse orientation into the vector construct. We also have a region from the RRE that also contains a splice receptor site. A disposable GFP marker sequence as well.

What is important to note is that the antisense is expressed only -- it is tat and rev dependent. That means the genomic RNA is only expressed in cells that are infected with wild type HIV because it is located upstream of this major splice receptor site. This is what we have found that makes thes vectors very, very effective.

(Slide.)

Okay. Some of the safety features of VRX496 for gene transfer. We believe it is the safest approach for gene transfer in HIV infected patient subjects using this type of vector because no new sequences are introduced into the patient. The vector, except for that small disposable marking sequence, is entirely derived from wild type HIV and patients that would be treated during this protocol are ladened with wild type -- with the wild type virus.

VRX496 cannot produce a novel pathogenic virus since the vector is a whole derivative of wild type HIV. Any recombination event that would occur between the vector and the wild type could not produce a virus that is more pathogenic than the wild type virus itself.

What I am talking about here is the vector itself and I am not discussing about any VSV issues which I will talk about later. Okay.

Also, in addition, our vector antisense payload is expressed in a targetable manner. The antisense payload is both tat and rev dependent and thus is expressed only after wild type HIV infects vector containing cells.

In addition, our vector does not contain any heterologous vol promoter sequences. It is entirely derived from wild type HIV. The bottom line is that we are not really introducing any new sequences in the patient other than what is already there and the vector is entirely derived from the wild type virus.

Also, what we found very recently is that the antisense payload appears to decrease vector mobilization to cells and I will show data for that in a little while.

Expression of the anti -- envelope antisense results in decreased infection of mobilized vector genomes.

Also, our vector contains not a triple, a single stop codon in gag, which basically creates a friendship mutation and a stop codon downstream. So if recombination with wild type or helper should occur downstream from this stop site a nonfunctional gag pol open reading frame would result.

(Slide.)

So this is the possible -- we have just schematically drawn the possible events that could occur between the vector and the wild type in terms of recombination. For example, as its well known, recombination occurs at the RNA level where reverse transcriptase makes the jump in order to recombine with a co-packaged strand of RNA.

One example of a noninfectious particle that is produced through recombination between the vector and the wild type is reverse transcriptase makes the jump at exactly upstream of that gag stop codon, resulting basically in a nonfunctional gag pol open reading frame in the recombinant. That means that this recombinant would not be infectious.

I am not going to take you through all this but really the overall conclusion here is that basically the result of recombination between the wild type and the vector results either in a noninfectious recombinant or wild type HIV. Even if the vector -- even if the wild type virus could somehow pick up the antisense payload and reverse orient it back in, still the result is wild type virus. It is not a new virus of unknown pathogenicity.

(Slide.)

So let me tell you a little bit about the packaging construct, which we call VIRPAC. It is also -- the actual plasmid is called VRX170. We use a two plasmid system rather than a three plasmid system which is commonly used in the field. We have found that our two plasmid system produces three -- at least threefold higher titers than the commonly used three plasmid system in 293 cells.

This is important because during our discussions with the FDA the history of the cell line became very, very important. So we decided that because the history of 293 cells can be readily established in contrast to 293 T cells, we have opted to use a transfection procedure that uses 293 cells and not 293 T cells. So in that situation we get better titers with VIRPAC.

So instead of physically partitioning the envelope and gag pol structural open reading frames, what we have included in VRX170 is a transcriptional partition of structural envelope genes. What we have incorporated are core sites both upstream and downstream of the two, you know, determinant open reading frames for envelope and gag pol. So we feel that this helps alleviate concerns of safety that one may expect from a two plasmid system because we are transcriptionally partitioning the envelope away from the gag pol open reading frame.

Also, what we have done in VRX170 is codon degenerated various sequences in order to decrease the likelihood of recombination but what I want to stress here is what is important in the use of HIV-1 vectors in HIV-infected individuals, what is really important is whether VSV-G recombines and forms a VSV-G RCR.

This event where gag pol is linked to the ltr is already amply present in HIV infected individuals. So the event that we really have to be worried about and we have focused a lot of our attention is whether VSV-G basically can become incorporated into RCR and whether we can in our final preparation show that there is no VSV-G sequences available for that putative recombination event to occur.

(Slide.)

So there was a question by one of the reviewers is where are the plasmid raw materials produced. We produce both the plasmid raw materials and the purified VRX496 vector at VIRxSYS clinical vector production facility using C-GMP conditions.

The cell processing on the other hand is performed at the University of Pennsylvania hospital's clinical cell processing facility, also using C-GMP conditions, and that is under the auspices of Dr. Carl June.

(Slide.)

When we produce these vectors, and I am not going to go in -- there is no time to really go into the production on protocol in the manufacturing process but basically, in brief, it involves cell factory production of a bulk harvest that then undergoes ultrafiltration, difiltration, benzonase treatment and then final column chromatography before it is formulated in a bag and it can be stored at -20, for six months at -20 degrees C.

The vector basically that we have used here is VRX494 and we can show that this vector can transduce primary human CD4 T cells with very, very high efficiency. The way that this transduction was accomplished was a single dose of vector at an MOI of 20 in conjunction with immobilized CD3 and CD28 antibodies. Then once the cells were transduced, they were cultured in these antibodies and IL-2 and two weeks later we performed FACS analysis to determine the percentage of transduced cells. We found an extraordinarily high level of transduction of these cells. We can routinely get anywhere between 90 and 98 percent transduction efficiency with this class of vector.

(Slide.)

So one of the advantages -- there was a reviewer's comment regarding the use of murine leukemia viruses instead of HIV vectors. One of the advantages of using HIV vectors is that with a single dose of vector, right, at one time you can accomplish this high level of transduction efficiencies. My understanding of the literature is that you either have to multiply transduce or prestimulate the cells extensively to achieve that level of transduction with an MLV based system.

After transduction of the cells we analyzed various parameters of the cells to look for stability of vector transduction and were there any toxic effects on the cells. This is an arbitrary scale here but it -- and it designates depending upon what we are looking at here. Cells transduced at a very high level of transduction efficiency are EGF positive essentially almost to the 100 percent level and remain so during the course of the ex vivo expansion period, which in this case is 29 days.

When we looked at the vector copy number by Taqman PCR, we found that the copy number in these cells also remained very, very stable during the course of the experiment, about nine or so during the 29 day period. And this stability is really remarkable when you think about it because this copy number is remaining stable even when these cells are expanding over 1,000-fold in culture. So this is the fold level of expansion of transduced cells in blue here compared to untransduced cells in red and you can see similar levels of expansion occurring and no real appreciable differences between the expandability of cells that contain the vector and cells that do not contain the vector. So the vector does not appear to be toxic and it can transduce primary human T cells with very high efficiency.

(Slide.)

Now we take these cells directly. We do not select for these cells at all and then simply challenge them with wild type HIV. In this case we use an L4-3 strain and here we use an MOI of .001 but we used various MOIs. And as you can see here is that while control cells that do not contain the vector are not transduced replicated wild type HIV very well. There was one, two, three log inhibition of wild type HIV replication in the cells that were transduced with the vector. When the cells are transduced to sufficient levels we do not see any breakthrough occurring.

(Slide.)

Also, what is very interesting is that when you look at the frequency of CD4 expressing cells in these cultures, while cells that do not contain the vector downregulate CD4 expression during the course of the culture period, this downregulation is a result of productive HIV replication that results in the expression of nef, VPU and gp120, resulting in CD4 downregulation. So the frequency of CD4 expressing cell is a marker, if you like, for the number of cells that are productively infected with HIV and in this case more cells here are productively infected with HIV because they are downregulating CD4 as compared to cells that were transduced with the vector.

As you can see here, there is no significant decrease in the number of CD4 expressing cells in the transduced cells compared to the control cells.

(Slide.)

We have also tested various strains of HIV. What we have done is transduced primary human T cells and then challenged them either with prototypic X4 and X4 strain of HIV, an R5 prototypic strain, and R5 primary stain of HIV, and then looked for the ability for the virus to replicate during the course of, I think in this case, about 19 days. These are all the same time points and the same scales.

And so what you can see here is that while the mock transduced cells that are depicted here in red replicated the wild type virus to predictable levels, the vector containing cells strongly inhibited the replication of the virus no matter whether it was an X4 type or strain of virus or an R5. Interestingly, we did find that protection against an X4 type of strain was better than R5 strain and this would be predictable based on the antisense sequence since the antisense payload that is present in the vector targets an X4 strain and not an R5 strain but as you can see here still because the antisense is about 1 kb it still inhibits the R5 strains at least two logs.

(Slide.)

As we move forward towards the clinical trial we did a comparability test between VRX494, which is the laboratory grade vector that expresses GFP, and also VRX496, which is the vector that basically has only that small 186 base pair sequence as a marker sequence, and we transduce the cells of various MOIs, T1 cells, and then challenged them with wild type HIV. As you can see, the wild type HIV cultures grew to predictable levels while both the VRX494 and 496 comparably inhibited wild type HIV replication.

(Slide.)

The next thing that we did was in collaboration with Carl June and Bruce Levine at the University of Pennsylvania, is that we produced the vector at patient scale at the level that would be used for one whole leukophoresis transduction procedure.

So we made patient scale vector and transduced the cells and then looked for various parameters of toxicity that may indicate that the vector was toxic to the cells. In red in each of the slides those are the mock cultures. These are cells that do not contain the vector. While the blue squares are cells that contain the vector.

As you can see, when the mock and the transduced cultures were compared for doubling time, there were no appreciable differences. When the viability was compared during the course of expansion there were no appreciable differences.

When we looked at the cell size difference between the mock or the transduced cells during the course of expansion there was no significant difference.

Also we looked at various cell surface markers and what you have to do here is you have to compare the first blue bar with the first red bar that would be day seven transduced compared to day seven mock and then you compare the day 11 transduced compared to the day 11 mock.

And if you look at each doublet you will notice that there is no real significant difference between the expression of these surface markers when you compare mock transduced cells or cells transduced with VRX496 at the clinical scale.

(Slide.)

What we then also did is took this clinical level scale transduction and then took a sample of it and then challenged it with wild type HIV to see if the cells could resist wild type -- productive wild type HIV replication. And as you can see here, all the control cells replicated wild type HIV very well. Over two logs of inhibition of wild type HIV was seen with the vector transduced cells. We were rather happy with that because when you look at the copy number of the vector in these cells it was an average copy of about six per cell, which falls within our specs. Our specs are between one to ten.

(Slide.)

So what we have now done more recently is taken CD4 T cells from HIV infected donor, transduced the cells with VRX496, and looked for various parameters. In this case we are looking for toxic effects and toxic effects is measured by the level of cumulative cell expansion when we compare mock transduced cells compared to VRX496 transduced cells.

As you can see during the period of expansion here there was no significant difference between mock and vector transduced cells. There was a question by one of the reviewers asking about the relative transduction efficiencies of normal CD4 cells compared to HIV infected CD4 cells. We have a very small n here but we have seen an average three copies versus two copies, which we do not think is an appreciable difference given the very small n size that we have.

(Slide.)

Okay. Now what we did is took that cell that were -- these cells that were transduced -- those cells from an HIV infected donor that were transduced with VRX496, expanded and then all frozen down, right, just like in a procedure that would occur into the clinic. Then we thawed the cells and grew out the cells in immobilized CD3, CD28 and IL-2 and looking at it for the endogenous virus to replicate in these cells. Right?

And what we found is that while the mock cells, the virus -- you know, this is the endogenous virus from the patient -- grew extremely well after the four and the growth in vitro there was a two log inhibition of virus replication from the vector containing cells. We do see this bump occurring, this breakthrough occurring, and I will describe that just in a moment.

We generally see this bump when we basically have copy numbers in the one to two range. When you have copy number -- an average copy number in the whole range, four to six or so, you do not see this breakthrough effect.

(Slide.)

What we also did on these cells that were transduced with the vector derived from the single HIV infected individual, basically we looked for CD4 expression on the cells. And while in the nontreated control cells we saw 40 percent of the cells expressing CD4, almost twice as many cells were expressing CD4 in the cells that were treated with VRX496, indicating a selective resistance to productive HIV infection by cells that are transduced with the vector. So we were rather excited about this.

(Slide.)

Okay. So now we wanted to look at more closely about this breakthrough phenomenon and the way that we analyzed this is by taking the supernatant from various time points from both the transduced cell cultures and the mock cultures and then looked for duplex RT/PCR for the types of RNA that were present in the supernatant.

As you can see here, for example, this is the mock of day one and this is the vector containing cells of day one, right, mock vector, mock vector, mock vector all the way through until you increase to day 16.

Now the duplex PCR involves two sets of primers. One set of primers specifically detects wild type HIV, right, and the other specific set of primers detects vector, right. And as you can see in the mock cultures during the course of virus replication we are detecting wild type HIV. But what is interesting is that during this viral breakthrough we are seeing the selective packaging of vector in these cells.

So what we are seeing, in fact, is that qualitatively most of this p24 that is coming out here is vector genomes being packaged into the supernatant. Okay. Now the question is does this vector -- mobilizable vector -- does this packaged vector, does it mobilize into cells? Can it effectively transduce naive T cells? And we have found that it is very difficult. It is very inefficient to transduce T cells. I will show you the next slide.

(Slide.)

We have done similar RT -- DNA PCR now looking at cells that were transduced with the vector. So these are the cells that have HIV and vector, right, and these are the mock HIV cells and this is a DNA PCR of the cells that were transduced with a representative of that supernatant. And while you can detect wild type HIV, you can see that by this assay we could not detect the vector.

So what we did is by this gel PCR method because we could not detect vector, we undertook to take Taqman DNA PCR on these cells and what we found was is that, in fact, there was a very low level of mobilization and it revealed that 40 copies of the vector mobilized into primary CD4 T cells per 10,000 cells analyzed.

So the conclusion is from these experiments is that this vector VRX496 can mobilize but very, very poorly. Okay.

(Slide.)

We looked more extensively about the mobilization of this vector into a more sensitive cell type, MT4 cells, and what we did here is we took either primary CD4 T cells or T1 cells and took cells that were either untransduced, right, or transduced with a vector that did not contain the antisense payload or cells that contained VRX494, which is the same vector that contains the described antisense payload. We challenged those cells at an MOI of .2 and then took the supernatant and assayed them on MT4 cells and, as I said, it is a very sensitive cell to pick up HIV replication.

And what we found is that the control shows there was no mobilization events. The -- while the VRX430 that did not contain payload, you could barely detect some level of mobilization. What was interesting was that when the vector did contain the antisense payload, the mobilization level went down and we have done this experiment many, many times, and this data remains very consistent.

(Slide.)

Okay. We further looked at the mobilization events in vivo and this was in negotiation with the FDA to look at two questions, whether mobilization occurs and what type of mobilization occurs. Are there any adverse mobilization events occurring? Right? For example, if the vector mobilizes weakly, does it mobilize just the CD4 cell or can it now infect another cell type?

So what we did is we undertook this experiment. We isolated human CD4 T cells and divided it into two lots. The first lot we transduced with VRX494, which is the vector that expresses EGFP. Then we constructed another vector which we swapped out the EGFP for EYFP. So we could discriminate between cells transduced between the yellow fluorescent protein vector and the GFP vector. And then mixed in back CD -4 PBMCs, which include B cells as well. And then injected these cells intraperitoneal back into mice.

Now if a mobilization event occurred from CD4 to CD4 cells then you should see doubly positive stained cells. Right? However, if an adverse event occurred, say mobilization from a CD4 cell of a green or yellow vector to CD19 cells, for example, these cells, then you would see either green or yellow fluorescence in these cells which you could discriminate by FACS.

(Slide.)

So had groups of five animals and this is a representative of the data. And we showed that basically in vivo in this mouse model that VRX poorly mobilizes from CD4 cell to CD4 cells. So these are cells that are expressing GFP or YFP but are not challenged with wild type HIV. Right? And as you can see there are no significant double positive events. This is the level of background that we typically see. Right? While in the cells that are challenged with wild type HIV at a high MOI of .2, we find that we can detect some mobilization events, double positive cells, indicative of some mobilization is occurring.

(Slide.)

However, when we looked at CD18 cells, right, whether there was an adverse mobilization event occurring, in the noninfected cells, this is the background here, we do not see any double positive events. And what we are looking for here is CD19 and EG -- or EGFP or EGYFP double positive cells. No events here and no events here. It is actually lower than the background.

So what we can clearly say is that VRX496 is a vector that mobilizes poorly but it does not mobilize adversely.

(Slide.)

Again in negotiation with the FDA we have performed some safety and biodistribution animal studies using SCID-hu mice. We think that the SCID-hu mouse system is really the best animal system to look for potential adverse safety events and the reason for that is that you have the ability to inject human cells that contain your candidate vector into a mouse that is not immunocompetent and these cells then can survive for a long period of time and die off naturally, right, giving ample time for any adverse event to occur.

Now the adverse event that we are really looking for here is RCR autonomous mobilization into mouse tissue. That means is that if there is some sort of strange event that would occur between the VSVG and the vector to give mobilization events into mouse tissue, this assay would pick it up. So let me describe to you the assay.

We isolate human T cells and then we transduce those cells with our candidate vector. These cells are then injected i.v. at very high dose into the mouse. The cells then distribute throughout the animal. We then kill mice and isolate over 10 organs and then look at those organs at day two, day 30 and day 91 for the presence of vector in the various tissues.

Now at day two obviously you would expect that all the tissues would contain vector because the cells are there as well but during the course of time these cells die off, right, and so if you see the presence of a vector sequence -- so the adverse event would be is if you would see the presence of a vector signal and not the presence of a signal to a human gene. That means that the vector has mobilized into the mouse tissue so let me repeat that again. The RCR event would be indicative if you would see a positive vector signal in the mouse tissue and not a positive human signal, which would be indicative of residue of human cells in that animal.

So to detect human cells we are using PCR primer specific to the gene. It is a homeodomain type of gene. It is a housekeeping gene present on chromosome 12. The reason why we chose this gene over beta actin is the homology of beta actin between mouse and human is 100 percent. You need to have something to discriminate between mouse and human cells and so this -- the primers to this gene that would effectively discriminate between mouse and human.

(Slide.)

So the first thing that we did is we wanted to see whether mouse cells to the point of the reviewer's comment could be transduced with our vector because if the mouse cells could not be transduced with the vector there is no point in doing this assay. So what we did is we took murine hemopoietic cells and basically transduced with our vector. These are the controls and these are the cells transduced with the vector and we analyzed them 13 days after transduction, with incidentally a very low MOI of 2 and we find a very high level of transduction, over 70 percent of the cells.

There is no question that murine cells can be transduced with these class of vectors and so if an RCR is present it should have the potential to infect mouse cells.

(Slide.)

So what I am going to do now is I am going to show you representatives of the data. First for day two and then I am going to show you a summary table and for day 30 a summary table and then day 90 in a summary table.

But first I am going to tell you about -- a little bit about the assay. The assay is a DNA PCR. It specifically identifies the G tag sequence, that 186 base pair sequence that I mentioned previously, and basically the sensitivity of this assay is 50 copies per microgram of DNA. So, for example, in this animal here we took the spleen and we have three reactions here of one microgram each. And in this third sample here we spiked in 50 copies of our control DNA into the sample and the same here.

(Slide.)

So what we can show is that we can amplify 50 copies. This validates the sensitivity of our assay. However, in this group of mice, these are the control CD4 T cells, that means that these are mice that are injected with cells without the vector, right. There is no signal present in the unspiked samples. This is our positive control for our positive PCR control right here and these are the markers. Okay. So in the control group animals basically they do not contain -- the cells that do not contain the vector, we do not see any positive vector sequence.

(Slide.)

However, when you analyze the mice day two post-injection of cells that were transduced with the vector, the mice injected with VRX transgene CD4 T cells, we find that, you know, a great majority of the samples light up so these are the spiked controls. These are the no spiked samples and you can see a very strong positive signal for vector. This is the control.

Now whenever we see a positive signal for vector, we then look for huCART expression to see whether that signal is due from vector that is mobilized or is a signal due to just the T cells that contain the vector, human T cells.

(Slide.)

So again we have a huCART PCR primer set that effectively discriminates between mouse CART and human, right, and when we take those samples we get a positive band. That is the sensitivity of the assay.

(Slide.)

So now a summary of the day two data is as follows: We have four groups of animals. Group one animals contain -- are injected with medium only. Group two animals were injected with cells that did not contain the vector. Group three animals were injected with a low dose of VRX496 transduced cells. And group four were injected with a high dose of VRX496 cells.

As you can see in the various tissues that we tested, we have here a panel of ten tissues, heart, testes, ovary, liver, lymph node, blood, tail, spleen, lung, you can read that for yourself. Basically you can see in every case we saw a positive signal for vector and you would always find a positive for huCART, demonstrating that that signal that we see there is due from the human cells and not due from an adverse event. In some cases we find that PCR from blood was a little bit problematic mainly because of tissue sample size.

(Slide.)

Now the next set of animals are the mice taken at day 30 post injection. So these human cells were injected and 30 days later we then killed the animals. And a pattern is starting to show. We are starting to see that, in fact, the cells -- the human cells that are in the animals are starting to die off. So you do not detect vector signal anymore in some of the animals. So in this animal, this tissue sample of the tail you can see that this is the spike control but there is no vector signal. The human cells have died off and with it the vector signal.

In some animals you still see the vector signal and so what we do in this circumstance is analyze these samples for huCART to see whether that signal is due from the human cells containing the vector or an adverse RCR type mobilizable event.

(Slide.)

And again in every case that we find our vector signal we find that the tissue sample always amplifies the huCART human cell band.

(Slide.)

And the summary of the day 30 data is as follows: Now you are seeing many more cells are negative for the vector, right. Some cells are still positive for the vector. But in every case where the vector signal was seen the huCART signal was seen as well. Again we have problems with blood in terms of sample size but the results are, you know, very, very consistent.

(Slide.)

And, finally, now the day 91 data and basically by this time most of the human cells have died within the animal and so this is a typical result that we see here. These are the spiked controls again. These are the samples and you see no signal present.

(Slide.)

And the summary of the data is that everything was negative except for four independent tissues in four different animals that were positive and these bands were extremely light. What we did then was again we took those and performed huCART analysis and again we could detect the huCART gene.

So what we have seen is that we have seen no adverse mobilizable events occurring in all the animals studied to date from the day two to day 90. That is the summary of the data.

(Slide.)

So now a brief summary of the proposed clinical trial. Our proposed clinical trial is now that we are selecting patients that are failing or discontinued HAART therapy and we -- what we have now done is to say that if a patient is showing virologic failure and can enroll into the study, he can enroll -- he or she can enroll into the study and keep on the same regimen that that patient subject is on as long as they do not change their regimen. So to avoid any ethical issues.

So the patient has no opportunistic infections. We have now at the suggestions of the reviewers narrowed down the CD4 count range from 200 to 600 but we have still maintained that viral load of greater than 500, which is demonstrating virologic failure.

We are going to enroll up to 24 patients, 12 will be, you know, in the actual study. The patients will come in. Their cells will be isolated. And then the cells will be exposed to a vector. The vector has been previously produced by the methods that I have described incorporating benzonase and chromatographic and ultra filtrating/difiltration schemes, which will then be QC'd prior to transduction of the cells. And we have an extensive panel of QC tests both on the vector and on the cells.

After the cells are expanded and they are released by QC, they will then be introduced into the patient in a dose escalating manner. The trial is divided into four escalation doses, 109, three by 109 and 1010 and three by 1010. And what we would like to do is to start off at the lowest dose with a single patient, run that patient all the way through the 28 day cycle, and then if there is no adverse, everything looks fine, then we would enroll the next two patients. And then after that we will enroll concurrent three patients at each dose.

Both the vector and the cells undergo extensive QC testing but the only one that I really want to describe today is the testing for RCR, for VSV, because I think that is the pertinent issue.

(Slide.)

So here is RCR testing that we will perform and we have performed on transfected 293 cells and our vector product. This is before transduction of the cells. So we would test both the end of production 293 cells, that means the cells that were transfected with the vector and the helper, and the bulk harvest. That is the supernatant that is taken from the cells after transfection.

The RCR assay is that we would take, let's say the bulk harvest here, infect H9 cells, 300 mls of vector of supernatant will be tested, and then we would passage those cells for six passages and then in the final sixth passage we will use Taqman PCR on the supernatant to detect for any potential RCR using HIV gag and VSV-G primers.

Okay. And what we have found during validation of this assay is that we can detect by Taqman PCR a wild type HIV that is 100-fold less fit than wild type. So what does that mean? We can take one infectious unit of HIV, take it through three passages and detect it by Taqman PCR.

We will not only do three passages. We will do six passages and from that final amplification passage then use Taqman PCR to detect where there is any virus present in the supernatant. The sensitivity of our assays for HIV gag is one copy per 10,000 cells or one copy per reaction, which is generally 10,000 cells. For VSV-G it is ten copies per 10,000 cells.

We also at the same time test the end of production 293 cells and again we are co-cultivating on H9 cells because there is no wild type HIV here. That is why we are using H9 cells. And we will take 108 cells and basically co-cultivate it with H9 for the first passage and then take the H9 cells through six passages before also taking that supernatant and then assaying it by Taqman PCR for gag or VSV-G.

So if the results are negative there are no VSV or gag detection, we will release the vector for transduction pending other QC tests. There is a whole battery of them. Although if there is a positive result, obviously we would not release it. We would go ahead and characterize what is going on.

(Slide.)

The cell processing will be performed at the University of Pennsylvania and a rough scheme is depicted here. Basically the patient subject comes into the clinic and undergoes leukophoresis and then T cell selection. The cells are then transduced with the vector in the presence of immobilized CD3 and CD28 beads. The beads are removed by a magnet. The cells then are washed and concentrated, formulated in a bag containing DSMO and frozen.

During the period of freezing the cells undergo QC testing. If the cells pass QC testing with the cell tests then they can be released for infusion into the patient.

(Slide.)

And so this is the RCR testing for the transduced T cell product. What we will do is we will take our ex vivo transduced and expanded T cells, take the supernatant and then do basically two assays. The first assay will be a biologically RCR assay where we now take the cells and infect 293 cells. We do not infect H9 cells because these T cells are already infected with HIV, right, so we are infecting a cell line that is not permissive for wild type HIV but would be permissive for VSV pseudotype version of HIV because of the broadly tropic nature of the VSV envelope protein.

So we would take the required amount, infect the cells, passage it for six passages, and then perform Taqman RT/PCR on the supernatant. We know that the 293 cells are readily infectable with the vector. We think -- we have chosen 293 cells because we know that these cells are readily transduced with a vector. We know that we can produce the vector from these cells so the entire cycle of viral replication can be accounted for with 293 cells. That is why we use them.

Okay. In addition to the biological RCR tests, we will also take the supernatant directly from the expanded cells and then directly do RT/PCR, right, and looking for VSV-RNA. If there is any residue of VSV-RNA that is present in that supernatant, if we detect it, we would not release that vector product.

Now for the transduced cells we would take the transduced cells, co-cultivate it with 293 cells, and then again for six passages, then look by Taqman PCR on the amplified supernatant by VSV and gag primers. We will also at the same time take the transduced cells and then do a DNA PCR using VSV-G primers, right. If we detect any signal here, our sensitivity here is very sensitive, one copy per 10,000 cells, we would not release that product for clinical trial.

So the way that we address the VSV issue is that our final product will not contain any VSV sequences that will be capable of recombining either with the vector or with the wild type virus.

(Slide.)

Okay. So patient monitoring. In your booklet you have got the updated protocol. I am just giving you a snapshot here. Basically at day 28, which is the important date for dose escalation, we will do such studies as T cell counts, differential viral load. What that means is looking at the plasma for both vector and wild type HIV genomes. We will do immunological assays. We will look in the RNA for VSV-G RNA in the plasma. We will for VSV-G antibody response. At the advice of Dr. Markert we will perform also a TCRV-beta diversity analysis to look for the repertoire diversity. And also we will do various hematological and chemistry assays.

The dose escalation scheme is as follows, and one reviewer asked about the difference between 28 days and six weeks. What we will do is the patient will be monitored periodically during this 28 day period and then when these samples are obtained they will be assayed and then reviewed by the data safety monitoring board in a 14 day period and then they will decide if they will authorize dose escalation.

The reason why we chose 28 days, and this is referring to another question, is because CD4 T cells -- there are two types of T cells, long-lived and short-lived. The ones that are short-lived are the activated cells and they generally survive for 14 days. We would predict that if there was any real adverse event that would occur as a result of infusion of the vector containing cells that, you know, it would occur sooner rather than later, and that is why we chose this type of dose escalation scheme.

(Slide.)

Okay. So for patient subject monitoring looking for the potential adverse events and toxicities, these are some of the points to keep in mind. We will -- it would trigger an event if a patient subject experiences a precipitous increase in viral load of .5 logs or greater. If this occurs, the viral load will be followed for up to seven days to determine if the increase is a sustained result. If it is, then we will enroll another -- we will expand that dose level to see whether it can be seen in two of the patients.

The same thing for CD4 T cell count. If the patient experiences a 50 percent or greater decrease in CD4 count we will again follow it and then this will be reported to the DSMB and then they will decide whether to stop the trial or to expand the dose.

However, in the case of VSV-G RNA, if we detect sustained detection of VSV-G RNA, we will then stop that patient. The patient will undergo aphoresis and then we will look for that patient, whether there is a virological RCR present. If there is a single biological RCR depicted in any patient, we will immediately stop the trial.

Also, here this is about the grade 3 greater or toxicities and we will monitor for those and again everything will go through the DSMB which we are presently instituting.

(Slide.)

Again this is a little bit about the dose escalation scheme. You have a patient. If there is one patient that demonstrates toxicity in the group of three then we will treat another -- not another, three patients. If there is toxicity in two or more of those patients then we will stop the trial. If there is not, then we will proceed to the next dose level and that is how we will proceed through the trial.

(Slide.)

So, in summary, we have shown that our vector can attain very high transduction efficiency in primary CD4 T cells. The vector transduced cells can significantly inhibition HIV replication in these cells. We believe it is the safest approach for the use of HIV vectors since the patients are already ladened with wild type virus.

Importantly, we will have very stringent release testing criteria. No VSV-G sequences will be present in the cell product that could recombine to form some sort of RCR.

We have found that our VRX vector weakly mobilizes to CD4 T cells in vitro in a SCID-hu mouse model but it does not mobilize adversely. It mobilizes from CD4 to CD4 and not to another type of cell.

We have seen no adverse events in our safety and biodistribution studies in our SCID mouse models. Our clinical protocol is targeted to HIV patient subjects that are failing HAART. And our clinical trial is a Phase I clinical trial. Safety is the endpoint here and it will be complete when we demonstrate no adverse events. No precipitous sustained increase in viral load, no precipitous sustained decrease in CD4 T cell count, no RCR or other significant toxicity associated with the vector.

(Slide.)

So I would like to thank my collaborators. First of all, I would really like to thank all the team at VIRxSYS. They really are a bunch of talented people. They have really pushed a lot of this research within a very, very short period of time and I feel very grateful to have them on board.

Particularly I would like to thank Yung Chang who has been with me from day one and also Tony Pascarelli, our CEO.

Also, I would like to thank our collaborators at the University of Pennsylvania, Rob Roy MacGregor, who is the PI; Carl June, who is also a co-sponsor of this protocol; and I would like to mention Bruce Levine, who has been great in terms of cell processing; Richard Carroll who has helped us with the primary challenge experiments; and also Peggy Bennett who has been interacting with TheraSolutions, which is a company located in Rockville, which is helping us coordinate the clinical trial.

So that is it. Thank you.

DR. MICKELSON: Thank you, Dr. Dropulic.

While everybody is readjusting to the light, Dr. Aguilar, would you like to start with your comments, please?

Thank you very much, Dr. Dropulic. Just take notes.

DR. AGUILAR-CORDOVA: So I guess I would like to start by commending the investigators for taking the plunge and obviously going through a tremendous amount of work in developing this new vector platform.

I will limit my comments or concentrate my comments primarily on the product. That was the major reason that I thought that this was novel enough to warrant full discussion even though the investigators have come previously to get some ideas on this forthcoming study.

And the -- some of the issues that I will bring up just highlight the difficulties that come about in following with this particular lentiviral product. I would, first of all, caution the investigators that with the full statement that no new sequences are included into this study since not all HIVs are identical. Clearly there are some that are macrophage trophic, some that are lymphocyte trophic, and even within those caveats there are differences between species, thousands that have been demonstrated throughout the country. NL4-3 is particularly virulent in vitro although we do not really know what its in vivo phenotype might be.

And certainly recombinants in the envelope section as you mentioned as a possibility would not necessarily generate the same phenotype of virus as had been previously found in whatever patient might be enrolled in this protocol. And one cannot predict what the addition of a novel viral phenotype in a particular patient will bring.

Thus one can also, only with great difficulty, say things that one can produce virus that is more pathogenic than the wild type found in that patient. In fact, one can produce a virus that is more pathogenic in a particular patient by recombining with another virus.

Now the mobilization studies and the difficulty with this whole process is that one can easily assess by doing the RCR assays a full VSV pseudotype lentiviral construct. What is more difficult to assess is a partial chimeric vector, one that would have only the VSV envelope but not the gag pol and thus not be mobilized by itself but may be carried through and then mobilized in vivo and recombination in retroviruses has been well documented at the RNA level like you said especially since they are deployed inside the virion. I think Dr. Howard Temin had showed that there is as high as say 10 percent recombination frequency inside the virion.

So in your original proposal you had shown that in 32,000 cells you had been able to detect 27 copies of VSV-G given the detection limits of your assay. And that was dismissed because it was not detected in bioassay and thus believed not to be an RCR. However, the bioassay is less sensitive and, as I mentioned before, one may have partial recombinance without having full RCRs in your product but that would still potentially generate a de novo recombinant in vivo.

The mobilization studies in the SCID mice are also not necessarily at the same level of sensitivity as your PCR so when one says that you can detect 50 copies and you can standardize that to the human DNA that you have, even though you can transduce the mouse cells, even a full gag pol VSV vector or virus in a mouse cell may not necessarily replicate because it is not just the entry that gets inhibited in the rodent cells for the replication of the gag pol portion of it and the ability to form a full virus inside the mouse cell that may be inhibited as well. So the sensitivity of using the SCID mouse model may not be sufficient.

I noticed, also, in your Taqman PCR, for example, in table 3, when you were detecting number of copies of plasmid per bacterium, you detected only 65 or 133 copies per bacterium, which seems rather low for plasmid copies inside each bacterium, and I was wondering whether that would correlate with your yield from those bacterium or if that implies something about your sensitivity of the Taqman assay.

The degenerate gag, rev and tat, which might ultimately also end up in your -- in any potential recombinant vector, it was not quite clear to me whether your PCR detection systems that you were proposing for release criterion and for assays, whether they would be at all affected by the degenerate sequences of that gag, pol and rev PCR.

And even though you do give some justification as to why you are using the only two plasmid vector system rather than the multiple plasmid vector system that is currently used with various other studies, it appeared to me that you could have cross over between the pol sequence and that only one illegitimate cross over at the 3' end of the VSV with the consequent flanking of the ITR would yield to a virion that would have a full context of a degenerate gag, pol, rev and the VSV construct in it with an internal promoter.

Most of your challenge experiments that I saw were using very, very low MOIs, which are called MOI, and I am not sure what volume you are using, et cetera, but the MOIs of your HIV challenge is in the level of .001, whereas your vector was we are saying about 9 copies per cell.

I was wondering if you had done those same challenge experiments at higher concentrations of HIV and also how does that concentration of HIV relate to the -- to what one might expect in vivo not only in the serum of the patient but also in the reservoirs that are found in lymph nodes and other sites.

In figure 13 you show no dose response in that situation so do you see a two dose response if the challenge concentration is greater? And in -- and I will just mention the figures and perhaps you can follow that.

In figure 19 you show that there was no detection of the RCRs but what is the level of detection because there were no positive controls and I realize that there is a very -- that is one of the difficulties of this whole system is what is your positive control and one will not easily go and make -- purposefully make a lentivirus with a VSV pseudotype as a positive control but then that just raises the bar of how to set up how many controls you must set up into this and I am not sure that going into a SCID mouse, that really increases your detection limits.

And in the detection of your RCRs in figure 22, as well as those before, you show that in -- with the definition of one that you get for your positive control, and that is based on the TCID50 of wherever it is that you purchase that virus, the NL4-3 from. With that definition of one you were able to detect it after three cycles of cell passages and you say that you are increasing your detection limit by going an additional three cycles.

But I would caution you that really the limiting factor there is the ability to have infected a cell in that first passage because if you did not infect the cell in that first passage you can passage 20 times and you will still not detect it.

So the conclusions from figure 26 which was no detection in the mice is not a strong conclusion based on the fact that you have not shown that replication can actually occur within mouse cells. You have only shown that it is able to transduce it.

In the ones that you have shown -- like, for example, in figure 37 you showed that there is some mobilization. I think it will be of great importance to know what do those mobilized genomes look like. So do they contain exactly what the vector was originally or do the mobilized genomes show some rearrangements that would be perhaps not expected.

And that might give you some idea of what is happening in vivo since within the patient one of the strengths or one of the justifications of -- one of the few justifications perhaps of using an HIV lentiviral vector would be you would have some mobilization that would give you greater efficacy since the high level of transduction that you are seeing in your CD4 cells is actually in the pseudotype VSV-G vector and you have not -- or I have not seen the comparison that you might have if you were to use VSV pseudotype C type vectors.

And you showed us right now some data with fairly low mobilization using your env antisense but in figure 39 I see that you had as much as 1.89 percent with a double color mobilization after HIV challenge. And that is quite significant, I would think, and certainly sufficient to perhaps evaluate what kind of genomes are in there.

DR. MICKELSON: Dr. Markert?

DR. MARKERT: I would like to commend the investigators for making a number of changes subsequent to the submission of our comments to right now. There have been many changes in the protocol. I will go through a variety of my comments where I would like to have them in the record.

On the preclinical data I really did enjoy seeing all the animal data included, in particular the mice data, and I had a few questions that I still am not clear as to the answers. And just the one for a little bit of humor, I do not understand -- I understand the animals who were weighed on day two all weighed less and these, of course, were all the animals that were sacrificed. So I do not know if there was a sign over their cage that they were going to be sacrificed and, therefore, they weighed less. I do not know why. I do not know why all the animals that were looked at weighed less than everybody else.

But the data -- it will be very nice -- I mean, seeing as safety is so much of what one wonders about is based on preclinical models, there are a lot of holes in the animal data and it does say that more data is being accumulated, whatever, but there were a variety of liver enzymes and other studies that were sort of rather fluctuating in this. So it will be nice to get the complete or for the investigators to look over all the data when it is all available because that can give clues as to what to look for in the patients.

With respect to those white focal splenic lesions and the pulmonary lesions that were judged to be incidental, it would be nice if the -- I do not think one needs to put anything in the consent right now because what would one put in the consent but it would be nice to know what those incidental findings were under the microscope. I mean, are they T cells or what? It would just be nice to know as opposed to someone just saying, "Oh, they are incidental and I did not look under the microscope at them."

Under protocol design and methods, with -- so there has been quite a bit of change here. The -- with respect to my concern about T cell diversity in these patients, one -- and I note in your responses that have come in the table that this method of expanding cells should not decrease the T cell diversity. Therefore, the patient should not or the research subject should not be put at risk by losing their diversity.

But in just looking through now there is not an evaluation of the T cell diversity prior to entry of the research subjects into the protocol. These are patients who have been on HAART and have "failed." They may have a very limited T cell receptor diversity and I might suggest -- what I had suggested in my comments was doing a study prior to the research subject receiving this gene transfer and then about six months later.

The way the protocol has been revised now is the only testing of the T cell receptor diversity is at day 28 and I do not know that the single time point will reveal anything. I would think it would be nice to have -- for safety, to have the research subjects have some reasonable diversity by immunoscope prior to entry and then make sure it has not decreased through the study.

Another issue with the research subjects -- I have seen the addition of the proliferative responses to tetanus just as an example and if one has a choice it could be nice to use research subjects who have a proliferative response to tetanus just to show that proliferative response remains after the gene transfer through the next six months or so as opposed to it disappearing at any rate.

So that would be moving it to being a screening test as opposed to just prior dosing. I am not so stuck on either of those but I do think both the T cell receptor diversity should be done prior to the gene transfer.

Now with respect to the adverse events, the -- you had a description of what would happen, and I was so glad that that was included, if a research subject has an increase in the plasma HIV RNA or a drop in the CD4 cell count but the description up on the board was that it would be checked -- the lab would be repeated and see if it goes on for another seven days or something along that line.

It would -- I would like to have the Data and Safety Monitoring Board look at that. In the protocol it says that the Data and Safety Monitoring Board will meet after the first patient 28 days and then after the first cohort is finished, second cohort is finished, third cohort is finished, and I would feel better if the -- and it does say that for other things the DSMB may meet but these are -- that is the sort of other thing I worry about, would be changes in the plasma HIV RNA and the CD4 count that I would want them to be meeting about.

Sort of continuing along on some of these issues, with respect to lot release there is the LAL testing is -- let's see. It is not clear. Is it done? Is the result -- does the result come back prior to giving the cells? And the -- I appreciate the response that gave the EUs per ml. Of course, the dose allowed to a patient depends on how many EUs per kilogram of the patient but in calculating out what a typical patient would be, you come well within what would be allowed if that is your typical response. I was just wondering if that was a lot release criteria.

Let's see. Then -- okay. Now with respect to the issue of failing HAARt, it would seem that it might need to be a little tighter in the protocol about what is -- is there some other physician, for instance, who looks at the patient and says, "This patient really has failed and there really is not -- there is not some set of medications I would like to switch this patient to right away and that my first choice definitely is to go with treating this standard way because they failed this HAART regimen and I want to switch to this one."

Because the way it is written, it would seem that a patient being followed in some clinic might fail the first regimen and then be told, "Oh, we should go directly to this research protocol," whereas all the rest of the HIV doctors in the country would have said, "No, the standard of care would be to do something else." I would like some comment on how do you decide that you are not just going to switch to another standard regimen as opposed to coming on this protocol

There are issues of the risk to the research subject of just holding on to a protocol that is not working very well but the way you have worked your protocol now that you are going to be looking more closely if the plasma RNA goes up or the CD4 count goes down, but I like standard therapy to be protecting the thymus and, you know, my favorite organ, if at all possible.

Let's see. And I think -- oh. With respect to the safety of infusing these numbers of activated T cells into humans, and I do understand that activated T cells have been infused into humans in other protocols, I would wonder are they -- have they been activated in the same way and then this is dose escalation so we will see what the adverse events are as we go along.

I made the comment about IL-6 and not -- it is not necessary that this be done real time but it might be nice to save some samples. You never know what later on could be helpful in trying to determine what went on in an adverse event.

Okay. So with -- I guess the -- so that -- those really are my comments and the issue that I had initially was, just so that people know where I was coming from initially, was that could we be letting the virus just be out of control and destroying great T cells -- the T cells they have and you put in one little population. If it is oligoclonal that would be a problem, which actually brings up the other comment.

You might want to check as much as other people have done this, although maybe it is the same group, done this amplification of the T cells in culture and not seen a diminution of the repertoire. You might want to just check with what you are doing just to be sure that again -- that the repertoire stays fairly robust because it would do -- be a very great disservice to the research subjects if they had virus sort of go off up -- knock of their own -- the T cells that were not protected and you put in T cells that have a limited repertoire. But if they have a good -- if the research subjects come with a good T cell receptor repertoire and you can maintain that then the risk is less in my opinion.

I thank the investigator team for making lots of the changes that were discussed in my comments. Thank you.

DR. MICKELSON: Thank you, Dr. Markert.

Ms. King?

MS. KING: Well, I also want to thank the investigators for doing so much work with -- between the time that they received our comments and the meetings. It is really great to see a lot of positive changes and I guess most of my questions and comments have been pretty well addressed. I think I have got two left.

One, I think, I would like to echo Dr. Markert's comment about ensuring that there is some kind of independent assessment of the potential subjects not having reasonable standard alternatives that either are likely to have a good effect or that are acceptable to them given that some people might be failing on their regimen but also might be finding the side effects unacceptable and that sort of thing.

So it would be good to have an independent determination of that and related to that there needs to be more discussion in the consent form. Right now the consent form sort of reads like this is standard treatment but there are other standard treatments available to you so the alternative section needs additional work.

All right. I still have some lingering concerns about the -- in the consent form again because these are sort of key to potential subjects understanding of the study, the purpose section and the benefits section, but I do not -- you know, I have a fairly conservative perspective on what should be described in the consent forms and I do not want to micromanage it at all but I do have one suggestion.

In your revised consent form, on the first page of it you have got three paragraphs in the purpose section. That middle paragraph is really your benefits section and that should be lifted out of the purpose section and labeled "possible benefits" rather than benefits and just placed in the appropriate place on the consent form.

That is it.

DR. MICKELSON: Thank you.

Dr. Coffin, did you want to make a few comments and then we will open it up?

DR. COFFIN: Yes. I have a number of comments. I would like to go back a little bit to the -- although this does not directly perhaps affect the safety of this particular product. I would like to go back a little bit to the basis, the rationale for -- do you want to go to somebody else first, Claudia?

DR. MICKELSON: I apologize. Yes.

Dr. Yee and then Dr. Zaia and then John. I apologize.

Yes, Dr. Yee?

DR. YEE: I probably overlap a little bit several other reviewers comments. First, I am a little concerned with the vector production system using two plasmid. I think that severely compromises the safety issue.

In the regular vector production system we use, in general you have four plasmids instead of two plasmids. You have a GABA expression plasmid, you have a VEGF expression plasmid and RIF expression plasmid, and then the vector. In most of the systems people use they do not use tat because they use CMV promoter to produce the virus.

So with four plasmid vector production systems it is much safer than the two plasmid in terms of recombination to generate RCR. I guess the reason you use two plasmids is because you use 293 cells for vector production so you can get higher vector titer. And with four plasmids people use 293 T cells and again can get a very high vector titer.

So I like maybe if you can elaborate a little bit more about what is the problem with 293 T cells because this is a cell line everybody uses and that probably is the cell line people are going to propose for the next HIV vector clinical trials so that, I think, is a very important issue.

The second problem is the original idea used the functional ARTI in your vector system because it can be mobilized by wild type HIV. But since your preliminary data shows that it cannot be mobilized very efficiently by while type HIV, my question is can you go to the third generation HIV vector that is seeing vector without any functional ARTI so it cannot be mobilized by wild type HIV. That again increases the safety of using this vector in this particular clinical trial.

And the third question: I am not particularly concerned with RCR because with RCR you can detect with your current system. The p24 assay is a very sensitive assay and it can detect anything above seven picogram per milliliter of p24. So that is a very sensitive assay.

I am more concerned with the recombination event that generates a vector containing only the HIV GAT protein or VSV-G genes. And it is clear from a publication from University of Alabama that this kind of recombination happens and happens quite frequently. It depends on what kind of assay you use to detect this kind of recombination.

And I am sure -- unless your transfection method is different from anybody else, I think you and everybody else all experiment using a plasmid cotransfection in 293 T cells probably will generate a recombination event which generates either VSV-G gene recombined to a vector or GAT protein recombined into a vector.

And in this case you probably will not be able to detect those because those viruses are now replication competent. They can be delivered into target cells but they cannot spread. So you probably will not be able to detect by p24 assay or DNA PCR or RT/PCR assay.

You mentioned that if you have a vector with a VSV-G gene that integrates into the host cells and then you have an incoming wild type HIV, then the VSV-G gene will get activated -- the expression VSV-G gene can get activated and pseudotype the wild type HIV. Then you have a wild type HIV that now can infect not only the CD4 cell line but any other cell types. So this is a potential problem.

So I think assays should be established to detect this kind of problem. Again this is the G gene and if you have a GAT protein this is derived from NAO4-3. If this GAT protein is delivered into target cell and then recombined with endogenous wild type HIV it may generate a different HIV strain which can give you higher toxicity. So I think it is very important to establish an assay to detect this kind of recombination event.

And again related to this issue in table 15 you actually can detect VSV-G genes by DNA PCR and you explained that. That is obviously important. Why you can detect VSV-G genes even after several passages of the transduced cells in culture?

In terms of animal studies, again I think for mobilization in vivo it depends on HIV replication, while type HIV replication. And again we know that wild type HIV does not replicate very efficiently in animal -- in mouse cells so I wonder about the sensitivity of this kind of an in vivo assay in mice. Is it necessary to have this kind of assay because what is the sensitivity of this assay?

So these are some of my comments.

DR. MICKELSON: Thank you, Dr. Yee. I apologize again.

Dr. Zaia?

DR. ZAIA: Thank you. I would also like to congratulate you for bringing this to public discussion.

I want to address a different area that I think is most important as we begin this kind of a discussion and that is what is the best design for this kind of a study. The dose escalation study proposed here is the kind that Dr. Greenblatt and his colleagues are so expert at and that is for cancer drugs you want to protect the patient and you want to make sure that the dose -- that you know what the toxicity is so that the dose you give can be observed. And during that period when you expect to see that toxicity you can then make an adjustment and you may have to adjust the dose and de-escalate it.

So the question here is what is the toxicity that we are expecting to see? Well, there are certainly patient related toxicity but there is also -- let's call it societal safety. That is this talk we are hearing about -- from the virologists about a recombinant event really relates to society outside the patient -- I mean, safety outside the patient.

So there could be, in fact, close contacts of that patient, research participant. People how have intimate relationships with that patient may be part of that same safety profile. And it may even be larger than that. I do not want to make it any more complex but you can imagine. But if you do see that one patient who does have a recombination that could have put a new envelope on to the virus, it could infect its sphere of infectivity.

What do you do with that person? I think you need to be prepared to address that issue.

But what I am really driving at is this choice of a dose escalation after 28 days of observation. You are -- I know your rationale and it is reasonable but I do not think it is completely correct when you look at the broader aspects of safety.

Now what would be the best way? Would it be 28 months or would it be 28 years? You know, who knows? But you could imagine that if there is from your data in vitro -- you see the spread of virus after about two weeks and so I think you use that two week scheme. And in nature you get a new infection with HIV and probably in two to six weeks you see detectable HIV. Maybe two to four weeks. But it is possible that T cell that you have put in there is going to need to be activated by influenza next winter and once it is activated, at that point it is going to then allow up growth of a recombinant virus.

So I do not know what the best time is but I do not think it is 28 days but I think my advice to the committee in terms of the recommendations would be that you not have a dose escalation scheme but that you have a scheme that uses a single dose with a period of time of observation that allows you to capture the data that you need for safety.

Okay. Going to another part of the study design, study number two is really kind of efficacy related. You want to see a change in the viral load and stability of CD4 cells, which I think is, you know, fantasy that you will ever expect to see with an infusion of this number of cells an effect on the virus.

Maybe you will see it but you certainly will not see it. I would not think you would see it in six months. Maybe you would see it in six months but my guess is that you are not going to see in a person who is failing HAART therapy the infusion of these cells correcting change in the virus load. The CD4 count may be stable anyway in these patients even with HAART failure.

So the question is what else can you really do? I think that if there is going to be one thing to do that is going to help the field, it is to demonstrate that the cells that you have put in there that are so-called protected actually survive for a period of time.

And that may require that you go back to the old scheme that has been shown in the past, namely a controlled vector. So it does change the equation of risk because now you are going -- now the possibility is you will put in cells transfected with two such vectors so you double the potential problems for the sponsor of the study and also for the reviewers but that at least will answer the critical question that you have posed here in a definitive way.

I will just comment briefly on the choice of vector from my own standpoint. I think that your rationale is to look at the issues of homologous sequences in part in the construction of your vector and yet you leave the LTRs untouched. And I have a problem with that. I think you should -- if you think homology is so important, I think you should go the extra mile and make these LTRs safer for mankind.

And then the concept that Dr. Yee referred to. I think philosophically I like the idea of multiple site in packaging systems to minimize the possibility of some kind of a recombination and putting everything together I would think limits that strategy.

In terms of the preclinical data, I just have one comment that concerns me. I have seen your comments on the others but one that still concerns me is this outgrowth of virus after two weeks in vitro at a time when your cells are showing predominance of the correct phenotype. That is the survivor phenotype.

So what is going on in that virus? Has that virus made an envelope in the presence of the antisense and outgrown? And maybe that envelope is different. Have you actually looked at that? And if the envelope is different, what does that mean? Does that mean the pathogenesis of the virus is going to be different? A theoretical possibility.

In terms of the protocol itself, I do not have many comments. I think the pulmonary tox -- I think the issue of the infusion is minimal in terms of our concerns of toxicity but pulmonary toxicity I think is the one thing that may occur -- I mean, fever of course but there may well be pulmonary toxicity. I am not sure that was dealt with that thoroughly. I could be wrong about that.

And finally -- oh. I still -- I think there is the perception of conflict between the person who is responsible for the quality assurance and release testing of the cells if that person has a proprietary interest in the method that is used to expand the cells. And I notice in your response you said that you did not think that there was a perception of conflict there.

That is all. Thank you.

DR. MICKELSON: Could you just go over that last point again? You think that if a person who has the authority for lot release criteria --

DR. ZAIA: Carl June discovered the method and he is --

DR. MICKELSON: Okay.

DR. ZAIA: -- I think expanding the cells at the University of Pennsylvania. Presumably his lab will do the release testing.

DR. JUNE: That is not true.

DR. ZAIA: No, that is not. Okay.

DR. JUNE: We have an external --

DR. MICKELSON: If you could can come to this mic here.

DR. JUNE: This is Carl June so I would like to just clarify that. At the University of Pennsylvania we have established a quality assurance program that is external to the cell production but we do GLP based QC release criteria and there are five of them that will be in place for this protocol with an external quality assurance that has been established over the last year.

DR. ZAIA: That was not clear. So you are not responsible for signing off then on the --

DR. JUNE: No, we have a quality assurance that does not report to me.

DR. ZAIA: Okay. That is all I wanted to know. That is all. Thank you.

DR. MICKELSON: Thanks. Great. Now, Dr. Coffin, thank you.

DR. COFFIN: Okay. I have a number of issues, many of which echo what some of the previous reviewers have said but I will repeat them anyway. And, also, some that get at some of the basic science underpinning your approach here.

The history of retroviral vectors is a history of argumentation that certain kinds of adverse events cannot occur and then discovery that in the face of the right kind of experiment those things, in fact, do occur and I sense a little bit of that in here so I would like to be sure that we root out as much of that as possible.

I mean, the first question that occurs to me is what is, in fact, the mechanism by which this vector is inhibiting HIV replication. In vitro it certainly seems to do so. It is quite impressive in that respect. I do not know what the mechanism of antisense inhibition is in these cases and I am not sure that anybody else does and given that one has to be very cautious about making assumptions about properties of what is happening.

For example, you make a statement that hundreds of mutations would be required to make the virus resistant to suppression but I see no experimentation that supports that and, in fact, relatively small numbers of mutations between some of these different subtype E sequences do seem to make some difference and some more experimentation. Perhaps you have done it but I did not see it in here. Using more diverse viruses, for example, or subtypes or things like that would certainly go a long way towards supporting the -- what appears to be an unsupported claim to that respect because I am concerned that, in fact, resistance of the resident virus to this might, in fact, be able to evolve in some straight forward way, although perhaps straight forward but unanticipated way during the course of your treatment.

In fact, there may be -- in your figure 15 where there is a little bit of breakthrough virus coming up -- may, in fact, be exactly that. You do not carry those experiments out far enough to see the appearance of breakthrough mutations according to the standards that people have done when they have put in specific mutants and looked for reversions and so on and so forth or odd ball recombinants.

So certainly some experimentation along the line. By the same token, you do not propose in your follow-up to do any real virology to see if the virus is changing, if resistant virus is, in fact, emerging in these patients. I would think it would be a very important thing to do since you will have created this base of where you have this virus trying to replicate against this inhibitory sequence at least in some small fraction of the cells that are in the individual.

So I think significant follow-up on the phenotype of the virus regarding its ability to replicate on transduced cells and the appearance of odd ball variance would be called for, I would think.

I am not convinced. I am puzzled that the vector mobilizes poorly when, in fact, it works fine when you transduce it with the help -- with your helper construct. You are not in a sense doing anything different and I -- you do not give the details of the design of that experiment or at least I do not know them or I did not see them.

This may be somewhat of a -- of really what is an old phenomenon in virology. What you have done is created what used to be called "defective interfering" virus that when you go to low multiplicity rapidly disappears from the population only because it does interfere with virus replication and because it is incapable of mobilizing itself.

So at the high multiplicity -- in what are called high multiplicity double infection conditions when you have your high levels of transduced cells you pick up a lot of this and it mobilizes well in the virions and, in fact, it does get transduced in cells efficiently but because I might guess not knowing what your protocol was the virus has been replicating for some -- is allowed to replicate for some period of time and the wild type virus simply out replicates the initially transduced virus and it looks like the ratio has changed a lot. Now I may be misinterpreting the experiment but that is the way it looks from the way I saw it presented here.

To change the subject a little bit again I agree strongly with the issue that Dr. Aguilar-Cordova raised and that is how you define the wild type. You call this virus wild type but what is in any given patient might be quite different. A virus that is in that patient might be of a kind that because we know the genetics of the virus have a lot to do with the eventual outcome of infection and how long that patient lives, by introducing new sequences there is a chance within that individual patient you will improve the quality, if you like, of the virus that is there by inadvertently repairing some defect in the LTR, for example. And that could have -- at least in a theoretical sense that could have important negative consequences for that particular patient.

I am not, I must admit, as concerned about what happens in the context and so on and sort of to society because you are not creating things that could not have -- except with the exception of the VSV-G issue as far as recombination between your vector and the resident virus, you are not creating things that could not have happened naturally and probably in a sense may well have in some patient or another somewhere in the past at some time. NL4-3 after all did come from -- originally from a combination of naturally occurring viruses.

I think you need to do -- to repeat -- some serious experimentation to show that recombination of wild type HIV does not occur either in your growth experiments -- both your growth experiments in vitro and even if that means, for example, infecting cells with a virus that has been deliberately crippled by reducing -- by making some mutations in the LTR to help transcription factor binding sites or something to make it replicate a little less and then see if you can pick up that LTR again from -- see if you can pick up that LTR from your vector or looking directly in patients to see.

The obvious issue that might more seriously arise is if you can switch the phenotype of what -- of the -- of an R5 virus in a patient to an X4 virus. And I do not know whether you have -- whether your envelope sequence includes everything that you would need to do that. For example, the V3 loop was unclear what -- most of the envelope sequences, gp120 sequence, is there although not all. And whether that includes all the sequences that you need to do that I could not tell.

Getting to the patients, the relationship of your protocol to HAART therapy is still somewhat unclear to me. I guess from what you said, patients can be continued on HAART during -- on preexisting HAART therapy during the time that their cells are taken and are treated. That would seem to raise a problem of how you can be sure you wash out all the drugs from the cells so you can actually effectively infect them with your vector.

And the confusion that could arise during your analysis if the therapy is changing or if the patient status is changing would seem to be something you have to worry about a little more carefully than you do.

Also at least in the protocol that I saw originally was -- there were some discordance between what kind of sampling you were going to do when. On one page the virus load assays were being taken on different days than they were on -- on page 15 there were different virus load assays than there were taken on different days than on page 18 and following.

You -- I gather from what you said now that all patients will have been treated, which means presumably that there will be no patients in this group who would have been classified as long term nonprogressors or patients with very low load who have very low risk of progressing because I would be extremely concerned about treating a patient where there was a good reason to believe that his virus was genetically crippled in some way and then inadvertently improving that virus, that virus' fitness in that particular patient.

In the follow-up for the cells, one of the things that might be worth looking for or perhaps should be looked for just on the off chance adverse things were being done to the cells, is for the possibility that surviving -- a few surviving clones of the transduced cells grow out and that can be detected either by looking at T cell repertoire or actually better by looking for clonal integration of the vector -- for the appearance of clonal integration of the vector.

And I think that is just about all of the specific questions that I had.

DR. MICKELSON: I know there are lots of questions. I hope you have written them down. I have written some too. If you want to maybe try to group some of them because a lot of them did overlap in some ways.

DR. DROPULIC: Yes. I was wondering, Carl, did you want to -- did you want to handle the clinical questions? Do you want to do that now? First group that together or shall I just go forth with some of the virologic questions first.

DR. MICKELSON: Yes. If you want to go up front. Could you just be sure to introduce yourself.

DR. MacGREGOR: My name is Rob MacGregor. I am the PI for the clinical trial in the Infectious Disease Division at Penn.

And as I recall the comments relating directly to the clinical protocol seemed mainly to be dealing with the selection of the patients and I would agree there are ways that we could make it more explicit as to the kind of patients we are looking for. But to answer that question, the kind of patient that we want to offer participation to is the patient who has been on treatment with several different regimes and has not been able to maintain or to gain control of their virus production so that in the face of ongoing treatment they have had continued virus production and loss of CD4 cells down to but not below 200.

In our clinical group we estimate that we have 15 or 20 patients in that category at our place and if we think of the whole city of Philadelphia, we think that there are enough patients who would fit in a category like that who might be interested.

The plan would be to let people know that this approach for people who have failed treatment in terms of -- the definition of failure being the ongoing production of virus despite antiviral treatment. Patients of that nature who are desirous of trying to do something more. There are some patients and some doctors who would say in a setting like that if I am not losing ground I will wait until more antiviral agents come along and hope I stay alive long enough to have that happen. That is a reasonable alternative.

But there are a number of patients who would say that I am unhappy that I am continuing to make virus and that my CD4 count was higher and now is lower, and I would be interested in participating in this in hopes that (a) we would learn something more about this approach and (b) as all patients who participate in any trial think this might possibly benefit me as well. Although we would say that we have no guarantee, of course, that that would happen.

DR. MICKELSON: Dr. Markert?

DR. MARKERT: I would like just to address a question here. This is the reason made from the immunological perspective that I felt patients should be screened by immunoscope prior to enrollment because here would be a patient who is failing HAART and they may be down along to the 200 CD4 cell count. If their diversity is low then the patient is -- all I see is increased risk for the research subject participating because taking T cells with a low diversity and putting the antisense vector into them and expanding them up, you give back to the research subject T cells with a limited diversity, which are not going to be all that helpful. And if there were that, I just do not see can -- a limited diversity will not help the research subject and you can end up -- I mean, it just -- I mean, it just -- so you have a lot of cells of a limited diversity but it will not help with infections.

DR. MacGREGOR: Yes.

DR. MARKERT: And so I think that the only way that this -- I mean, aside from all the virology issues -- that this can be helpful is if the patient starts out with a reasonable repertoire because you cannot generate a repertoire from a very oligoclonal situation.

DR. MacGREGOR: That sounds like a reasonable thing to do. It would probably limit the number of patients that we would have who would be eligible and I guess you would have to balance the problem of not having patients that we could include versus the benefit of wider diversity.

DR. MARKERT: Yes. But I do believe that putting patients -- research subjects on who have limited diversity, what is the point? And that should -- I mean, if -- I mean, I certainly feel that it would not make any sense and I would put a whole page in the consent form that here is a problem. If you have no repertoire, this cannot possibly make your ability to respond to a variety of infections any better. And then who is going to sign that?

DR. MacGREGOR: Carl, do you want to comment?

DR. JUNE: Carl June from the University of Pennsylvania. My role would be in the cell manufacturing but I do have experience on that with some of the trials with Cell Genesis that we have done with CD4 zeta and in patients similar risk profile that was outlined.

So you can look at it, I think, in two ways legitimately. One is, yes, you have patients that are going to have a skewed CD4 beta spectro pipe, you know, and some of them more so than others in this failed group and you can say those are cells that are left overs and not useful and that is why they have not been killed because they never saw an antigen. Or you can say that those are cells that are keeping that patient alive and, in fact, you would like to clonally expand those clones because they are the ones that while -- you know, in order to enroll in this protocol those subjects have to have no OIs. So these are people failing HAART but that are free of OIs.

So you could say that what is left in these people is a good pot of cells, although with limited diversity, and why not expand them if they do provide antiviral. You know, if they render a cell that is infected with a protease resistant virus if it renders it resistant to that.

So I think there is a rationale both for a subject who has a gausian distribution of V beta T cells as well as someone who has a skewed distribution. Maybe it should be separately specified in the protocol but I think scientifically at least I do not know the answer to what would happen there.

But as long as we evaluate that I think your comments that we need to evaluate the V beta gausian distribution, you know, not just at the end of the protocol but at baseline and then a real outcome is comparing within that baseline -- within the intra-subject comparison of baseline to end of study and that we should have as you suggested sequential evaluations, baseline, day 28 and then six months off study.

DR. MARKERT: Your point is well taken. The -- if the initial study is done prior to actually enrolling the patient in the gene transfer, then whoever is looking at it at least can look at it because it is awfully hard just a priori to say this is what it would have to look like to enroll someone. I have taken your point here. But at least for someone to look at it and be thinking about it at the beginning.

DR. MICKELSON: Dr. Ando?

DR. ANDO: I would just like to comment since I have worked in this area with both interleukin 2 and with the gene therapy but basically if you are failing HAART and your CD4 count is 50 or 150, you are losing cells at 50 per year, and 100-150, you are basically at the point -- that is the point where you get the opportunistic infections, et cetera.

Cliff Lane here has done a lot of work and the beta scope narrowing seems to be associated with a really clear sharp increase in OIs. And, in fact, what Cliff has done is been using -- trying to find patients who are responsive to HAART, low CD4s and using interleukin-2. Even though they have a limited beta scope, if you can get their CD4 counts up, they do better.

So that data suggests that it does not necessarily preclude -- it would not preclude patients who had a limited beta scope. They are actually better off at 150 with a limited beta scope than at 50. An absolute count of 50, you are definitely going to have a limited beta scope and you are definitely worse just from the numbers game. So the reason they choose 200 is that below 200 it is a very narrow slope before a very aggressive pathway of OIs and, you know, the actual outcomes.

DR. MICKELSON: Go ahead. Yes, Dr. Zaia?

DR. ZAIA: Yes. Dr. Carl, this is another topic that we did not really touch on that I think is important. We touched on it but we did not go into detail. That is your sampling from the peripheral blood, which I think represents about one percent of the total body CD4s, so that there is 99 percent that are below the surface. And the question then is do you have -- really have a reasonable chance of affecting the virus outcome with just modifying that one percent?

I realize you cannot predict the future but is the -- I mean, there is the possibility that the rationale is flawed unless you can address that question.

DR. JUNE: I think the ways to address that are, as you know, I mean that there is not -- I mean, there is a pool of circulating T cells and there is a pool of secondary lymphoid resident cells but there is interchange. I mean, maybe 30 seconds is maybe the average resonance time of a T cell in the peripheral blood before it goes back in the lymph node.

So if we sample at any one time it will contain a mixture of all the various types, whether called mucosally derived T cells and lymph node derived, and memory and so-called naive cells. So it is the only practical way we can do it outside of infusing stem cells.

There are direct estimates of that and I was not here this morning to see if Dale Ando showed some of the studies from even your institution with CD4 zeta where there have been biopsies of the gene marked cells in the case of CD4 zeta that I am aware of and also in a few other gene marking studies where people have looked at the frequency at least of the cells following infusion and there is a good correlation in both tonsils and lymph nodes in the CD4 zeta studies that I saw that were sponsored by cell genesis.

I think Dale could probably talk more about that but I think that is the persistence and the time of appearance that supports the rationale to do that.

DR. DROPULIC: Okay. So I would like to address some of the questions that were posed. I am just going to go through the list that I have here.

Regarding the X4 strain and potentially changing the phenotype, antisense is, in fact, derived from N04-3. It does include the V3 loop and so that is an X4 strain.

What we could do is restrict the patient population that is more advanced, that is demonstrating X4 strain, so that there would be no issue regarding switching a patient from R5 to X4. We have discussed this previously. We can -- you know, we can do that if that is what is required.

Regarding partial VSV recombinants -- well, the way that we are handling the whole VSV issue is that our release testing criteria will demonstrate no VSV DNA or RNA sequences in the final product. If there are no VSV sequences in the final product then in the case of patients that are infected with HIV, we do not see how we could -- how a detrimental RCR could evolve with recombination. We have very sensitive assays. We are using Taqman PCR.

Regarding the 27 copies of detected VSV-G DNA in our pilot clinical lot, that was using a manufacturing protocol that is now being improved. We can now show that we do not have any DNA or VSV-G DNA present in the final cell product.

And what we found was is that this residue of VSV DNA, the 27 copies, was present outside the cell. It was not in the cell because when you wash the cells you could remove it, okay, by multiple washings. So it is not some one type of event, one single event, and it is integrated into the cell. It is residue of VSV hanging out on the outside.

Regarding the mouse SCID studies, and we are not saying that those studies, the biodistribution studies amplify any potential RCR. They are only detecting a single event. Obviously there are issues of sensitivity but all we can say is that there is no significant RCR type mobilization in these animals. We feel it is the best possible animal model used to look for such adverse events.

Regarding a question regarding higher MOIs of challenge. We have done that and the cells have been protected. We can provide that data if required. That is not a problem.

The question was regarding low mobilization and saying that 1.89 percent was relatively high. Our background for those assays can be anything up to one percent so in reality, you know, it is really in the .89 percent. It is on the limit of detection by FACS for us. We can barely detect it.

Regarding the question on the mechanism of the antisense, what we have shown is that when -- first of all, the reason why you do not affect production is the antisense is against wild type HIV env, there is no -- there is no wild type HIV env that is targeted during production because there is no wild type HIV in there.

To your point in terms of the mechanism --

DR. COFFIN: That was not my question.

DR. DROPULIC: That was not the question. I would like to get at your question.

DR. COFFIN: The question was how -- since you did show that you had a lot of -- you know, at least in one case you had a lot of pseudotyping of the vector with virus that was -- with HIV that was in the culture at the same time and you are highly -- in one experiment where you had the naturally infected cells and you transduced those and then you got this breakthrough virus coming up.

DR. DROPULIC: Right.

DR. COFFIN: Most of the genomes in that virus you showed were vector and not --

DR. DROPULIC: Yes.

DR. COFFIN: -- and then you claim that despite their -- your claim then was, I think, that despite the fact that there was a lot of vector in there, you were getting --

DR. DROPULIC: There is not a lot. There is not a lot of it.

DR. COFFIN: There is a lot more -- but there is more vector than virus.

DR. DROPULIC: Yes, but there is a --

DR. COFFIN: But you are getting --

DR. DROPULIC: It is a small amount.

DR. COFFIN: -- but your experiment then appeared to show much less mobilization of vector of the genomes -- the majority of the genomes in that particular --

DR. DROPULIC: Right. Those genomes --

DR. COFFIN: And then you concluded from that this was mobilized very poorly.

DR. DROPULIC: Yes.

DR. COFFIN: Despite the fact that it was apparently taking the virions reasonably well at least in that one -- at least in that one setting. My suggestion was that may have been due to allowing multiple rounds of replication before you did the analysis.

DR. DROPULIC: I see.

DR. COFFIN: Maybe you did not do that but I could not tell from what you said.

DR. DROPULIC: Okay.

DR. COFFIN: I understood perfectly why it did not interfere with the --

DR. DROPULIC: Okay. Fine.

DR. COFFIN: -- that was not the --

DR. DROPULIC: But in terms of -- yes, that is right. But in terms of the mechanism just as a side note, we do have a construct that contains an antisense against gag, right, and env, the titers for production do go down. So it shows that the antisense is having an effect because you had a question.

But I think that the effects are not purely just the payload. I think there are competition effects just to your point regarding defective interferon particles in competition. That effect is also occurring.

DR. COFFIN: But there are a lot of other possible ways in which antisense can have these kinds of effects. They can be double stranded RNA and can be directly toxic to one mechanism.

DR. DROPULIC: Okay.

DR. COFFIN: And other kinds of things you can imagine.

DR. DROPULIC: Okay. With regards to Dr. Zaia's question of measuring the survival of the cells, I think in this report here I have now said that we will be looking for direct survival of the cells. I do not know whether it answers your issue regarding control cells versus antisense containing cells but it was not in the original protocol and now we have included using the unique 186 base pair fragment.

A lot of questions.

DR. MICKELSON: I think there were a number of general sort of questions on the vector production that might deserve comment. User of higher numbers of plasmid systems.

DR. DROPULIC: Yes.

DR. MICKELSON: 293 versus 293 T.

DR. DROPULIC: Right. Yes.

DR. MICKELSON: LTRs remaining.

DR. DROPULIC: Right. So let me take the functional LTRs. First of all, I do not understand if I understood your question but we do not degenerate the vector. Right. That is not what is being degenerated. And in the helper, right, construct, where we are doing the degeneration, there are no HIV promoters. They are heterologous promoters.

So in terms of promoters there is no homologous regions. Right? You cannot really degenerate the vector at all because you need the elements in order for it to do its function, transduce and compete with wild type HIV. So I suppose I really -- I did not understand exactly that question.

But the bottom line is that we are not saying that recombination will not occur between the vector and the wild type virus, for instance, but the outcome of that recombination is still that you get wild type. You may change the phenotype but if we restrict the phenotype to patients that are already demonstrating X4 where you are putting in more of a homologous vector, if you like, against the endogenous HIV strain.

Regarding the trace-ability of 293 --

DR. AGUILAR-CORDOVA: Just a second. Just to follow-up on the degenerate sequence.

DR. DROPULIC: Yes.

DR. AGUILAR-CORDOVA: The question was that if you took that degenerate sequence into the partial recombinant and then it became part of a new replication competent vector or virus, given the assays that you were describing, whether your PCR fragments or anything else, and whether the biology of that vector would then be changed because you were taking a degenerate sequence that was no longer the wild type sequence, it was again addressing the issue of whether you could bring in new sequence into the vector types.

DR. DROPULIC: Okay.

DR. AGUILAR-CORDOVA: Even though you do not have HIV LTR driving it. The sequence is still there and it could be imported into a retrovirus.

DR. DROPULIC: You are talking about which sequences are still there? I am sorry.

DR. AGUILAR-CORDOVA: Your helper vector has the degenerate sequences.

DR. DROPULIC: Yes.

DR. AGUILAR-CORDOVA: Those sequences then could be imported into a partial recombinant.

DR. DROPULIC: Yes.

DR. AGUILAR-CORDOVA: Would your assays detect those sequences?

DR. DROPULIC: We are focused on now detecting the VSV-G. That is what you are saying in the final vector preparation. We have not got an assay to detect any of the gag pols because -- no, we have not got an assay, right, to detect those degenerate those sequences but we could do that. That should not be a problem.

Okay. So let me take on 293T versus 293. It is -- I think, Dr. Yee, you asked that question in particular. We have switched to 293 because of trace-ability issues, right. 293 was derived from Frank Grimes' laboratory at the University of Toronto and you can get the 293 cells directly with a sublicense from Microbix. And so the trace-ability of the cell line, what serum was used to passage the cells is clearly established. That is important because you want to use serum from non-BSC countries. Right? I am not so sure if that trace-ability you can obtain for 293. We found it was just easier to go from 293 and so that is what we have been using. It is a trace-ability issue for the cell line and what serum was it passaged in.

My understanding is Michele Kalos from the West Coast developed the 293 T cell line but I am not so sure of the trace-ability in terms of what serum was used to passage it.

Okay.

And the four plasmid versus three plasmid versus two plasmid systems. Again I do not know if there is any direct evidence showing that one is better than the other. What we have done is we are using a two plasmid system and we have included other things into that plasmid. These pore sites to prevent read through that could give rise to, you know, a gag, pol, tat, rev, VSV, you know, that would be co-packaged and recombined.

If you have documented evidence to show that four is better than three or two, you know, I suppose I would like to see that. I do not know of any and I may be wrong but I do not know of any.

That is all.

DR. MICKELSON: Are there other comments here? Dr. Noguchi? I think there are some comments from the audience as well but, Dr. Noguchi.

DR. NOGUCHI: Just a technical thing on the VSV-G detection. I am not aware of any system that can detect down to the absolute molecule. I think that the issue being brought forward is that in any production system there is an inherent limit of sensitivity to whatever you are trying to assay for and the possibility, maybe not the probability but the possibility exists that VSV-G gets through the whole process.

And I think to say that it is not in the final product is not quite answering the question about the potential for recombination where VSV-G could then pseudotype an endogenous -- not an endogenous but a naturally existing HIV in an HIV infected person. So I do not think it is easily addressed because it is a hypothetical question but I do not think that saying that it is not there is the right answer.

DR. DROPULIC: Yes, I suppose it is not detectable by the techniques that we are using, correct.

DR. MICKELSON: Are there any comments?

Dr. Zaia?

DR. ZAIA: What would you do then if a patient did have VSV-G virus isolated and if there were damages to that patient or to his environment would you be prepared to pay for those damages because they are unknown. I mean, if South Philadelphia has an outbreak of VSV HIV, VIRxSYS is not going to be able to solve that problem.

DR. DROPULIC: Well, obviously, right.

DR. ZAIA: It is kind of a rhetorical question.

DR. MICKELSON: Are there other questions or comments from the audience?

Yes, you have to come to a microphone and introduce yourself, pleas.

DR. HUTCHINS: Hi. I am Beth Hutchins and I am -- although I am associated with a gene therapy company, CAN-G, we do not work on retroviral vectors adenovirus. My question relates to just as a member of the public.

Ms. King raised a question about the description of the clinical product that you are testing in your informed consent and you revised that slightly but although you say it is a gene transfer product, you do not say it is a viral vector. You do not say it is a new vector class. And the risks that you list do not address any of the issues relating to those factors.

And there may be politic reasons why you want to take that approach but I would think that you risk a really horrible PR problem once the patient or the public find out what it is that you are testing and that you would be better dealing with that up front in the informed consent and not just the process orally but the written material, and I would just like to know your rationale and what you plan to do about that.

DR. DROPULIC: No, I think that is a fair comment. We can put into the consent form that it is a new class of vector and, if you want HIV vector, we could do that as well. I would rather have it in -- I will lean on the side that you are saying. I would be happy to do that.

DR. HUTCHINS: I think you would be heading off more problems than trying to appear as though you are hiding something.

DR. DROPULIC: Okay. We were not really trying to hide anything.

DR. MICKELSON: Ms. King?

MS. KING: I just wanted to say I really thank you for making that comment and for following up on that point because I think it is an important one.

DR. MICKELSON: I think certainly that members of the RAC are very willing to help and work with you on the informed consent document. There is a great deal of experience here on this committee that at least those kinds of issues would be something that we would be anxious to -- we have a lot of people who are not scientists who can read for clarity kind of thing to figure out, gee, I did not understand that one at all. So you might as well take advantage of it.

DR. DROPULIC: We would work on that.

DR. MICKELSON: I think in the interest of time I might just go through, and to be quite clear, I am only going to be able to, I think, hit some of the highlights and these points and recommendations that I will bring up will -- I might miss a few and sort of when we come to looking at all of these and coming forward for recommendations, it is with the understanding that if other members of the committee remember things that I have left out that we can notify Dr. Patterson because the letter that will go to the investigators will have all of these things and you will all have seen it so we are sure we have gotten the intent correctly because I might have gotten -- it is quite probably I missed a fair amount of this.

I think some of the initial points -- they certainly always focused on the production of the vector, safety issues as well as then some of the monitoring that would go on for the particular patient population, as well as some of the inclusion criteria and certainly the committee would recommend that it be a little clearer, at least, that for patients that had -- it was not just patients that had failed HAART but that were failing HAART. But that also it was quite clear that alternative -- that there could be enrolled -- or that, if possible, there were other alternative drug regimens available that this be another person or another party involved in making -- possibly making that decision or referring people to enrollment in this trial if they were failing one particular drug regimen to be sure that there was a clear description of alternatives.

I think that the investigators laid out a good outline of certain clinical adverse events that they would be monitoring for. Dr. Aguilar outlined a number of issues with the vector production and that certainly raised the point that what is novel is certainly almost an individualistic thing here per patient and that it is very difficult -- you cannot say that there are no new sequences present but that there are many HIV variants and tropisms that could be novel.

And that even though -- and I think the investigators recognize this to a certain extent and are recommending or saying that they might restrict the patient population to those that demonstrate a narrow band variant so that it would be less likely to -- somewhat less likely to develop these -- I guess genetic drift within the population there.

I think there also was a recommendation that there be development of -- that the investigator include methods and assays to detect survival of the transduced cells, that it not just be the -- by unique -- the unique base sequence in the 186 sequence but that also you look to develop an assay that also -- within the vector stock and then also possibly within the patients for the degenerate gag-pol sequences that you were talking about as well. The investigators agreed to that.

I think -- I am not sure that we came to an agreement on whether the use of higher number of plasmid packaging systems increased safety or not. I think the investigators feel that they have incorporated a number of mutations within their two plasmid system that might offer some level of safety. However, there is at least in some of the basic research laboratories three and four plasmid systems certainly get much significantly lower levels of generation of replication competent viral vectors but I think we should put this in the letter as something that we discussed.

I think you have to understand that each time -- for each recommendation we make you can certainly -- when you respond to the committee can say that -- no for whatever reason and certainly lay it out if you disagree with this point or this recommendation and that certainly that is your position that you can take on that but I would certainly think that that would deserve a response of some sort.

I think, also, Dr. Market raised a number of very good points on inclusion of the focal lesions that might be incidental findings but that the fact that these were found should be put into the informed consent document.

DR. MARKERT: Actually I did not -- well, what I was looking for was just the descriptions so that the investigators would know and not to put something into the informed consent because now we would not know what to put in. It is probably -- I mean, probably not --

DR. MICKELSON: Because you do not know the significance of them.

DR. MARKERT: Yes, but it is just to know what is in these lesions and actually going back to just your last comment, it seemed to me, although I am not the virologist here at the table, that maybe there was not good data out there saying that the two versus the four plasmids. It was -- I mean, which was the way to go. It did not seem to me that there was hard and fast data. Therefore, I would not have that be something in the letter because all it will do is generate a lot of confusion at the receiving end about what needs to be done but it --

DR. MICKELSON: Did you want to --

DR. YEE: This is so theoretical and there is no real data suggesting the four plasmid is safer than two plasmid.

DR. MICKELSON: Okay.

DR. YEE: Again, I would argue that it depends on your assay system. I mean, people use the same method to -- with different assay they can detect recombination and we use p24 assay and PCR assays the same. We do not get RCR detection. But again I would argue strongly that other assays need to be established to detect the recombination event, which those events can lead to -- eventually to pseudotype or wild type HIV.

DR. DROPULIC: So what you are saying is if we have an assay to look for those events that would --

DR. YEE: That would be great.

DR. DROPULIC: All right.

DR. YEE: You can set up a similar assay so you --

DR. DROPULIC: We will do that.

DR. YEE: -- and you show an active result, that will be great. I will be satisfied.

DR. MICKELSON: Thank you.

Dr. Markert also suggested that prior to and after enrollment of patients that they be tested for their T cell receptor diversity and that -- and then that -- whether there was a limited diversity or not, I think, was -- and whether that should be an inclusion/exclusion criteria was not something that we wanted to make a recommendation on but that would be just good to know, immunological diversity -- immunological competence of these patients.

She also recommended looking for a tetanus response before and after.

DR. __________: (Not at microphone.)

DR. MICKELSON: Oh, they did that. Oh, I missed that one.

Also, she recommended just asking the DSMB look -- review results if a patient shows a drop in CD4 counts or dramatic increase in plasma viral load just to be sure that that was clear that that would be looked at.

And archiving some samples from the patients just in case you may wish to look later on for different indicators of some adverse response.

DR. MARKERT: They did that.

DR. MICKELSON: Oh, did you? I am sorry. You had a question about lot being released.

DR. PATTERSON: Lot release criteria, whether LAL assays would be done prior to patient administration.

DR. MARKERT: That was a question.

DR. PATTERSON: Okay. Did you want to --

DR. MARKERT: (Not at microphone.)

DR. PATTERSON: Okay.

DR. JUNE: I can address that. It is fairly standard that the antitoxin LAL will be done. It is done at the time of cryopreservation. In our current negotiations with FDA they prefer that just as we cryopreserve it to take a sample and it is not -- the lot would then not be released unless it met the correct negative result below the specified limits for LAL. The same thing -- mycoplasma is done at the same time and then, you know, the other lot release criteria for this would be looking for magnetic beads, residual in the final product that were used to grow the cells and to look at p24 levels as well as standard culture and sensitivity.

DR. MICKELSON: Thank you.

Ms. King recommended some possible changes to the informed consent document. Certainly some of the paragraphs that were initially in the introduction were, in fact, benefits and might -- the informed consent document would benefit from shifting those.

I think some of the -- Dr. Yee's comments on the vector production system, plasmids, multiple plasmids dealt with those, I think. Is that correct -- certainly -- I think the still remaining question that was brought up and I am not sure what people would like to say. There certainly was a lot of discussion of generation of novel recombinants and generation of potentially pseudotyped virus but that might be something that if I would -- my guess would be that given the lot restrictions that you have that that would be quite unlikely. However, you would probably be looking -- you will be analyzing viral loads in patients. Would there be --

DR. DROPULIC: Absolutely. We also have an assay for VSV RNA so we will see if there is anything there.

DR. MICKELSON: Would there be any use for looking for some of these other unusual recombinants for possible --

DR. AGUILAR-CORDOVA: I would just think that your sensitivity of the assays need to be -- in whichever method that you choose need to be better specified even though you can detect 50 copies per microgram, for example, in some of those assays. That is of the human DNA and that is not necessarily the same as your VSV and you would have to have in that particular copy 50 RCRs in order to be able to detect them because it is not going to replicate probably.

I am just doing this off the top of my head. I think the assay development as described was not really detecting the sensitivity levels that you might at first glance assume because they were replicating in the mouse.

The other thing that we mentioned, Claudia, was the issue of going forward to characterize the mobilized genomes to see what it is that you are mobilizing.

DR. DROPULIC: Yes.

DR. MICKELSON: Right.

DR. DROPULIC: Yes, we can do that.

DR. MICKELSON: Right.

DR. DROPULIC: Actually just as a side point which I forgot to mention, we have actually taken off not the mobilized but the produced vector, transduced cells, and then did PCR on the DNA in the transduced cells and shown that it was completely the identical sequence what was put in so now we will do that for the mobilizable genomes as you suggest.

DR. MICKELSON: Dr. Zaia brought up some very interesting questions about the design of the trial which I think certainly we discussed. I am not sure what we would like to do. Mentioning that the dose escalation which is standard for drugs may not be the most appropriate for this initial study. I think that also the selection of just the dose escalation after 28 days -- he suggested that one might consider using a single dose and then waiting for a longer period of observation at least at this initial time.

I do not know whether -- how -- Dr. Gordon?

DR. GORDON: I think the way that discussion went I feel most comfortable with the letter mentioning that these issues were discussed with some uncertainty about what the best approach might be.

DR. MICKELSON: I agree.

DR. GORDON: I do not think we decided that the approach in the protocol is the worse and I do not think we had a specific suggestion for improving it but nonetheless it may not be the best so I think we should just say it was discussed in that context and leave it at that.

DR. MICKELSON: I think the investigators made it clear that if you chose the 28 day observation period based on the --

DR. DROPULIC: The active --

DR. MICKELSON: Yes, the reactivated mobilizations or the breakthrough seen after 14 --

DR. DROPULIC: Will be activated -- that cells -- activated T cells survive generally for about 14 days and so we said 28 days.

DR. MICKELSON: Right.

DR. DROPULIC: Because those cells would in the majority of cases die and, you know, you have the greatest chance for an adverse event early rather than late.

DR. MICKELSON: Absolutely.

DR. DROPULIC: Yes.

DR. MICKELSON: Dr. Coffin?

DR. COFFIN: No matter the escalation study. I and maybe a number of others here might think that the overall six month follow-up time might be kind of short. If at all possible I would suggest that at some -- there being some ongoing follow-up beyond that time frame.

DR. DROPULIC: In the new protocol it is yearly for life.

DR. MICKELSON: Yearly. Okay. I think that Dr. Coffin brought up several interesting points, which may or may not go into the letter at all. Certainly made it clear that not a -- there are multiple mechanisms by which antisense vector might inhibit replication but also recommended that in some instances where there was breakthrough mutations that these be looked at and characterized to see what the genome structure sequence was for some of these breakthrough genomes?

DR. DROPULIC: Yes, we will be happy to do that.

DR. COFFIN: This goes towards eventual efficacy of the vector. It does not directly address the safety issues. It would seem also in the patient samples that you get out to test the virus that is in those patients for perhaps having acquired some resistance.

DR. DROPULIC: Right. We have done some small meta-studies.

DR. COFFIN: It would be fairly easy to do I am sure.

DR. DROPULIC: We have done some of those studies but we will have a complete study.

DR. MICKELSON: As part of this you would probably be looking at the genotype of the virus for variance in the patient before. You might or not? Would that be helpful to know? Just to know the sequence of the variants in the patients before they put into the trial.

DR. DROPULIC: Yes.

DR. MICKELSON: It might help with what you were talking about, right? No?

DR. GORDON: Also what comes out.

DR. MICKELSON: Yes. And then what comes out.

DR. COFFIN: Also it is present at the end.

DR. MICKELSON: Yes.

DR. COFFIN: I think looking for some change, genetic change in the virus.

DR. MICKELSON: Right.

DR. COFFIN: Would not be --

DR. MICKELSON: Right, so you would do a baseline and then -- right. Okay. I think, also -- I am sorry. Dr. Noguchi?

DR. NOGUCHI: Also Dr. Coffin also brought up the very interesting observation that the -- one of the old mechanisms of defective interfering particles may be a part of what is going on.

DR. MICKELSON: Right, exactly.

DR. NOGUCHI: And I think that is certainly worthy of more future discussion. I am not -- it is not clear to me which way actually would work in this case but it could be in an adverse way.

DR. MICKELSON: Right. But that is certainly something that would be worthwhile. I think certainly Dr. Coffin supported the detection or baseline and post assay analysis for the TCR repertoire and possibly another way to look at that was after the transduction to look for clonal integration sites if there was a limited number of T cells and T cell clones that were grown up and reinfused into the patient.

And it was quite clear that the investigators would not include nonprogressors or patients with very low viral loads so I think that goes without saying because we are looking at patients that have failed HAART.

DR. COFFIN: There is one more point related to that that I forgot to mention that really did not come up and that is one of your sort of failure criteria is a half log increase in virus load but you are not, as far as the protocol goes, collecting any baseline information on the stability of the virus load in each individual patient.

DR. DROPULIC: We do screen and obtain a baseline count but not --

DR. COFFIN: You have just one.

DR. DROPULIC: Yes.

DR. COFFIN: And that does not then address the stability of that. If you have a patient whose virus load is increasing for other reasons you could have --

DR. MICKELSON: John, we cannot hear you.

DR. COFFIN: I am sorry. If you happen to be a patient -- I am trying to --

DR. MICKELSON: I know. It is impolite to have to turn your back.

DR. COFFIN: The issue is the protocol just shows a single virus load time point.

DR. MICKELSON: Right.

DR. COFFIN: But in some patients the virus load may, in fact, be on an increase or in some way unstable, inherently unstable and may report an adverse event of an increase in virus load when there really is not one in this because the patient -- this is an unstable patient and it would seem to their interest to collect some more to be sure that the virus load in any individual patient is put on protocol is reasonably stable at the time that that happens.

DR. MacGREGOR: Sure, that sounds very reasonable. The patients that we are going to be recruiting are going to be well studied patients because they will have had to have a history to show the failure on treatment so what we could do would be to include a trend over a six month period or even a year period as far as that goes.

And as they enter the study there will be two so we will have the screening viral load and we will also have the initiation of viral load so we will have a mean there, too. I think that is a good idea.

DR. COFFIN: Also in patients with very low viral -- your entry criteria goes down to 500 copies, which is very low, and a half log increase in that is only up to 1,500 and I do not think many of us would think that that was a significant difference. I would worry about entering patients with that low a load.

DR. MacGREGOR: Right. I think that is true, too, and I think most physicians and patients who were stable above 200 with a viral load of 500 would not want to be in such a study anyway, nor would I recommend it to any of my patients but we ought to put that in more explicitly. Sure.

DR. MICKELSON: So now given -- yes, Dr. Zaia?

DR. ZAIA: I would also encourage the letter to at least ask the sponsor and investigator to address how the -- what measures will be taken to protect the public health from this experiment in the worse case scenario because I think that still is going to be the most limiting question in terms of implementing the study. I mean, you may, for example, have to isolate a patient for the first 28 days in order to be sure that in the case of a VSV recombination it is not going to get spread before you know about it.

I think that is going to be an important issue to address in the further review of this.

DR. MICKELSON: I certainly think that is something that we should address and comment on public health issues, whether you agree with what scenarios may or may not be feasible or viable and I think that certainly this is something that we would not ordinarily be asking but as the first use of a lentiviral vector. I think one of the things is certainly what you come back with as your response is something that you would have to use your best judgment on but the committee would look at that.

Ms. King?

MS. KING: Just one other thing to add to the list of things that goes in the letter, which is the expanded discussion description of the vector in the consent form.

DR. MICKELSON: Oh, yes, the expanded discussion description of vector. Certainly. But again we would be willing to look at a new informed consent document if you would like -- if you think that would be helpful.

DR. DROPULIC: Yes.

DR. MICKELSON: We would be very happy to try to do that.

DR. DROPULIC: Yes, thank you.

DR. MICKELSON: Given all of those waffling and comments that I think we have probably come to in recommendations, may I have a motion that we incorporate what we have just discussed into both the indications of comments and discussions that occurred as well as recommendations, as well as the recommendations that the investigators had agreed to already in the form of a letter that will go to the investigators?

DR. GORDON: I just want to confirm before moving --

DR. MICKELSON: Yes.

DR. GORDON: -- that we will be able to see this draft before it goes out.

DR. MICKELSON: Oh, absolutely. Absolutely. That is always done.

DR. GORDON: In that case I so move because we touched on some pretty sensitive issues here and I want to make sure that the draft is at least in my --

DR. MICKELSON: That is the standard process.

DR. GORDON: Makes me comfortable, yes. I just want to --

DR. MICKELSON: It is part of the standard. Yes. No, it is good for everybody to know that we do not just get one version of what is going on. All right.

So can I have a motion? Dr. Aguilar. May I have a second for that?

DR. AGUILAR-CORDOVA: I second.

DR. MICKELSON: Excuse me. So it is the other way around.

All those in favor, please, raise your hand?

(A show of hands was seen.)

DR. MICKELSON: All in favor, nine. And there are no abstentions, no votes. So it is nine in favor.

So let's have a ten minute break and then we will come back for the data management report. Thank you all very much. It was very, very interesting.

(Whereupon, at break was taken.)