PR thoughts (Part I)
by: icposse2000 (27/M/Bethesda, MD) 2/2/00 12:08 am
Msg: 15313 of 15539 I hate to make my first post to a group as obnoxiously long as this is (multi-part and all), but I thought that there seemed to be a lot of disagreement regarding what was (and wasn’t) said during Enzo’s press release today. I am by no means an experienced investor, so I’ll have to leave the discussion of whether the stock is overvalued or not to the pros ;) However, I do work in medical research and have worked with both HIV and stem cells before, so I thought I’d throw my 2 cents in on what I thought today’s PR covered.

The first thing to realize is that today’s announcement is an early report of data from a phase I clinical trial. It is NOT a completed phase I trial…which limits the type of information that a responsible company can divulge:
As was mentioned previously today, phase I clinical trials are designed to test a therapeutics SAFETY, not EFFICACY. The biostatistics used in setting up the phase I trial skew the results to magnify any possible safety issues at the risk of allowing potential efficacy endpoints to go unnoticed. This is why many phase I trials have been prematurely terminated due to safety concerns, but (to my knowledge) a phase I trial has never been terminated early because of fantastic efficacy. It is also true that the PR statement by Enzo mentioned neither safety or efficacy, which is completely appropriate, because safety and efficacy data from patients are released at the conclusion of the phase I trial, not before. The phase I trial of the HGTV43 construct is scheduled to end in April (from what I’ve read on the board). Announcement of phase I data before the end of the trial serves no purpose other than to violate the conventions surrounding reporting of medical results and undermine a company’s credibility. Therefore, the fact that no safety and efficacy data has been released should not be cause for drastic concern.
However, if you read between the lines on the PR statement and on the abstract being presented, there are several indications that the initial safety data is good:

PR notes (Part II)
by: icposse2000 (27/M/Bethesda, MD) 2/2/00 12:09 am
Msg: 15314 of 15541 1) As I mentioned above, for a phase I trial to be prematurely terminated is almost always an indication that the therapeutic is unsafe. Therefore, the fact that the trial is ongoing (and going to be expanded) is strong indication that data so far do not suggest that the therapy is unsafe.
2) The PR sentence: "To date, no other trial has reached this point in adult human subjects without first ablating the patient." Ablation therapy in bone marrow transplantation (BMT) consists of a strong course of chemo- and/or radiation-therapy designed to reduce the number of host bone marrow cells and mature T cells. In the case of allogeneic BMT (between non-identical members of the same species) it serves the purpose of increasing the chances of engraftment by helping to eliminate a host immune response (T-cell and NK cell responses) against the graft. In the case of autogeneic BMT (where the donor and the recipient are the same individual, as in this study), it serves a different purpose: by ablating the host’s bone marrow, you increase the proportion of genetically modified stem cells to normal, unmodified stem cells, which in turn leads to a higher percentage of modified ‘descendant’ cells, such as CD4+ T-cells. However, subjecting a patient to ablation therapy weakens their immune system and leaves them vulnerable to opportunistic infections until the new stem cells have engrafted. This is one of the primary risks of BMT. The fact that HGTV43 is efficacious enough to have reached levels without ablation that other agents could only reach with ablation means that it is likely to be CONSIDERABLY safer than previous approaches.
3) On an slightly related tangent…did anyone pick up on the fact that these trials are being conducted at UCSF? I noticed a lot of panic about a Washington post article that mentioned several unreported gene therapy deaths at Harvard…I have serious doubts that the article (even if it’s true) has any relevance to Enzo.

PR thoughts (part III)
by: icposse2000 (27/M/Bethesda, MD) 2/2/00 12:09 am
Msg: 15315 of 15541 So what DID the company announce today? In simplest terms, Enzo’s PR stated that they have something to run phase I trials on. Now I realize that doesn’t sound very impressive, especially for the investors who are used to traditional pharmaceutical companies. The difference in Enzo’s case is two-fold.
1) Transfection of CD34+ stem cells is quite a difficult task. The transfection efficiencies reported in the abstract presented at the meeting are (in themselves) a breakthrough: "In all instances the cells were successfully transduced as measured by in situ RT/PCR and the presence of total RNA in a population of cells…" Similar comments were made in todays PR: "the high rate of transduction in cells from the HIV-1 infected subjects has been consistent throughout the study." For those of you who have never tried to transduce immune cells (esp. stem cells), this is quite impressive. To make these results even more astounding, they managed to achieve these rates in the absence of feeder layers and in less than a day. The absence of feeder layers is advantageous b/c it reduces a source of possible contamination as well as making the transfection efficiency more predictable between lots. The duration is of critical importance b/c stem cells tend to differentiate rapidly into cells which can no longer give rise to all lines of blood cells. The short version of all of this is that Enzo has developed an unbeliavably good system for delivering genes into blood stem cells. If they own the patent for this technology (does anybody know?) it alone could make them a LOT of money.
2) Antisense DNA technology is still in its infancy. However, two statements, one from the abstract and one from the PR, tell me that it’s growing up fast: "The amount of antisense RNA per cell of the population of transduced cells was greater than 1000 copies." And " In the trials, antisense RNA was observed in circulation for more than four months." Again, this is nothing short of a breakthrough in antisense DNA technology.

So today Enzo reported that they have overcome two of the technical hurdles standing between their therapeutic and a potential treatment for HIV infection. They did not mention any information on the safety or efficacy of their study, as is appropriate. The question remains…will it work? I have a few thoughts on that subject as well, but I’ll save them for tomorrow (since this post is way too long already). Thanks to all who made it through this. I’d be happy to try to answer any questions/comments/clarifications…etc.

-icposse2000

HIV-1 trial (part I)
by: icposse2000 (27/M/Bethesda, MD) 2/2/00 8:57 pm
Msg: 15470 of 15538 Thanks for the positive response to yesterday’s post…BTW, yes, I do work at the NIH.

I thought I’d post today about a bit of the science behind the HIV-1 human study, so those of you that aren’t scientists will have a little bit more info on which to judge this company. This discussion tends to get a little technical at times, so I’ve put in background material for the non-geeks among us. There are 3 supplemental sections which cover basics of the immune system and HIV-1 biology. I think they are pretty useful in helping to understand the analysis.

Again, this is a monster post, but I hope those who read it will have a better idea of what Enzo’s up to. Please feel free to post comments/questions/criticisms (be nice!)

-icposse2000

HIV-1 trial (part II)
by: icposse2000 (27/M/Bethesda, MD) 2/2/00 8:58 pm
Msg: 15471 of 15538 I will agree that Enzo needs some PR people to help them out with their releases…not because they should be shouting about a ‘cure for AIDS’ (announcing that at this point in the game would make the cold fusion reports of the late 80s look excessively substantiated by comparison)… but rather b/c the language they tend to use makes it difficult for the educated layman to understand what they have done. As an example, this entire post will be about the following statement from yesterday’s press release:

"Our objective with this particular application of Enzo’s gene therapy program is to successfully modify white blood cells to render them resistant to HIV-1 infection using the principle of genetic immunity and to reconstitute the patient’s immune system with these white blood cells."

The problem with this sentence is that it is basically a mission statement for the entire HIV-1 project, but with so little information that whoever reads it must know quite a bit of molecular biology in order to fill in the gaps. I will try to dissect that statement into its core parts, with a particular emphasis on the risks and benefits at each stage, and where Enzo’s technology is at each stage. For those of you who are unfamiliar with the biology of the immune system and the HIV virus, I strongly suggest that you read the supplemental sections (there are 3 of them) before continuing….

HIV trial (part III)
by: icposse2000 (27/M/Bethesda, MD) 2/2/00 8:58 pm
Msg: 15472 of 15538 Enzo’s mission statement (as I’ll call it from now on) can be boiled down to: "The objective is to produce modified CD4+ T-cells which are resistant to the HIV-1 virus. There also must be *enough of these modified CD4+ T-cells (>200+/ml) that the patient does not have symptoms of AIDS."

The procedure that Enzo is using consists of a number of steps:
1) Removal of CD34+ stem cells from the patient via leukopheresis. Leukopheresis increases the concentration of stem cell, making the delivery of DNA (step 2) more efficient.
2) Delivery of genetic material conferring HIV-1 resistance to the stem cells. This is where Enzo’s REAL strength is. Their delivery system has produced results (transfection efficiencies) that are unheard of.
3) Reintroduction of the modified stem cells into the patient. Again, Enzo made a big leap with the 18 hr transfection time. This substantially reduces the risk that the cells will become more specialized while outside of the patient’s body. If no stem cells are reintroduced, genetically modified macrophages & monocytes won’t survive more that 2-3 months. If Enzo has documentation of modified monocytes surviving for >4 months, this is a STRONG indication that viable, modified stem cells were reintroduced to the patient.
4) Waiting while the modified stem cells produce modified descendants, including CD4+ T-cells. The Enzo paper in Virology provides in vitro evidence that these cells are resistant to HIV-1; there is little reason to think that they shouldn’t be resistant in the patient as well.
5) Allowing the level of modified cells to reach a high enough level to avoid the syptoms of AIDS. This is the final step; if Enzo reaches >200+ modified (resistant) cells/ml, game over. Thanks for coming, AIDS, but your time is up.

HIV-1 trial (part IV)
by: icposse2000 (27/M/Bethesda, MD) 2/2/00 8:58 pm
Msg: 15473 of 15538 Now before everyone uncorks the champagne, let me talk about some of the potential pitfalls in Enzo’s strategy. There are a number of hurdles which still need to be overcome before the strategy can be considered successful:

1) No modified stem cells: If no modified stem cells are returned to the patient, it is impossible to expect resistant CD4+ T-cells to emerge in high enough levels to prevent AIDS. A lack of modified stem cells could be a result of poor transfection efficiency, specialization of stem cells while outside of the patient (so that they are no longer stem cells when they are returned), or poor expression of antisense HIV RNA. All indications from the abstract and press release suggest that these hurdles have been overcome. The transfection efficiency has been extraordinarily high. The short incubation time increases the possibility that the cells will remain stem cells outside of the patient . As I noted above, the presence of modified monocytes >4 months old is a strong indication of viable modified stem cells. The abstract also documents antisense RNA levels of >1000 copies/cell. Looking good so far...
2) Complications with gene therapy: The recent death of a patient at U Penn undergoing gene therapy trials has put a damper of the enthusiasm surrounding clinical trials involving gene therapy. The patient at U Penn is different from the patients in the Enzo HIV-1 trial b/c the virus was directly injected into the patient and the patient’s particular condition made him susceptible to side effects of viral injection. (The patient had severe liver failure; introduction of virus into his hepatic artery resulted in massive cytokine production which led to disseminated intravascular coagulation and multi-system organ failure. The chances ofsimilar complications in the HIV-1 trial is virtually nil.) The main danger in the HIV-1 trial is that the HGTV43 vector integrates into the chromosome. This poses a theoretical r isk that the patient could develop cancers of the immune system (leukemia or lymphoma), b/c HGTV43 might integrate into and knock-out tumor suppressor genes (which play a role in limiting cancer; think of this possibility as a loss of brakes). HGTV43 could also integrate near an oncogene (which plays a role in promoting cancer); if this happened, it could potentially cause a lack of regulation of the oncogene (think of this as a stuck accelerator). However, as I noted in my post yesterday, there is no indication of any safety concerns with the HGTV43 vector. Nevertheless, this is always a potential risk, and the recent death of the patient at U Penn may slow Enzo’s trial even though it is essentially unrelated. Better safe than sorry…

HIV trial (part V)
by: icposse2000 (27/M/Bethesda, MD) 2/2/00 8:59 pm
Msg: 15475 of 15539 3) No resistance of modified cells to HIV: If the modified cells do not have high enough levels of antisense RNA, they will not be resistant to HIV. However, the in vitro data from the Virology paper indicates that transfected cells ARE resistant to HIV-1. While the extrapolation of in vitro results to the patient is not always certain, there is currently no reason to think that the modified cells in the patient shouldn’t be resistant.
4) Mutation of the HIV-1 virus so that it is no longer inhibited by the antisense RNA. This is a classic weakness of traditional anti-HIV pharmaceuticals (reverse transcriptase inhibitors, protease inhibitors, even the recently announced integrase and adhesion inhibitors). The HIV-1 virus mutates very rapidly. Given the immense numbers of different HIV-1 virus particles in a patient, it is possible (even likely) that at least one virus particle will be resistant to the drug. Given the laws of natural selection, this one viral particle will soon be the dominant strain and the virus will no longer be inhibited by the pharmacologic agent. This is where the true beauty of Enzo’s system comes into play. First, Enzo’s HGTV43 vector encodes for 3 separate antisense RNA sequences; in order for a viral particle to be resistant to the antisense RNA, it must have simultaneous mutations in all 3 regions which are targets for the antisense. This GREATLY decreases the odds of developing of the virus developing resistance to HGTV43. Second, and even more important, is the fact that the virus will select for CD4+ T-cells which ARE resistant. Just as the virus will mutate so that it can survive in an environment containing an anti-HIV drug, the CD4+ cells which are resistant to the virus will live (and produce more CD4+ T-cells via clonal selection), while those that are susceptible to the virus will die. Again, via the principles of natural selection, this favors the emergence of resistant CD4+ T-cells. The equivalent of this in traditional HIV pharmacology would be something like a protease inhibitor that mutated to avoid the viral mutations. (Note: since the virus mutates faster than the T-cells, it is still favored, but to a much lesser degree than in traditional HIV pharmacology).

HIV-1 trial (part VI)
by: icposse2000 (27/M/Bethesda, MD) 2/2/00 9:00 pm
Msg: 15477 of 15539 5) Levels of modified CD4+ cells too low (<200/ml) to prevent AIDS. This is the concern that was raised on yesterday’s posting…what if the levels of resistant cells are 1 in 10,000 or less? As was pointed out yesterday, the survival advantage of resistant cells over susceptible cells will rapidly reduce this ratio. Furthermore, the proliferation of resistant CD4+ and CD8+ cells which have encountered antigen (clonal selection of T-cells) will further boost the number of resistant cells. By my calculations, the CD34+ stem cells will need to produce greater than 1 million resistant CD4+ T-cells in order to keep CD4+ T-cell levels above 200 cells/ml. (200 cells/ml x 1000ml/L x 5 L of blood). One additional danger to the resistant T-cells is that they may still be killed via syncytia formation; antisense RNA will not make them less susceptible to his fate.

All in all, I am bullish on Enzo’s chances. There are clearly some hurdles left, but Enzo’s strategy allows the immune system to do much of the work for it. Even with the concerns I’ve outlined above, Enzo is in the best position of any company I’ve heard about. And that’s without factoring in the ultimate trump card:
There is a possibility (even likelihood?) that one or more of the CD4+ T-cells will be more than just resistant to HIV-1…it may be specific FOR HIV-1. This could result in the development of a specific immune reaction against the HIV-1 virus and virally infected cells by a resistant population of T-cells. The development of this kind of a response would make an HIV-1 infect ion similar to the flu…your immune system would take care of it on its own after a week or two. This is the Holy Grail of AIDS immunotherapy, but I think that Enzo’s strategy uses the laws of natural selection to drastically increase the chances of reaching that milestone. Go Enzo. Thanks for reading, all who made it!!
-icposse2000

HIV-1 (supp 1)
by: icposse2000 (27/M/Bethesda, MD) 2/2/00 9:00 pm
Msg: 15478 of 15539 Before we dive too deep into the science, I’m going to talk a little about the hematopoietic system and how the HIV-1 virus works. I think this will help make the discussion on Enzo’s HIV-1 trial more understandable.
The "hematopoietic system" is a fancy name for all of the cells present in the blood and immune system. The CD34+ stem cell is the grand-daddy of the hematopoietic system…all other blood and immune cells are derived from these cells. CD34+ stem cells are continually dividing; some of these ‘daughter’ cells remain CD34+ stem cells (so you don’t run out of them), while others move down one of several paths to more specialized cells.
In the simplest terms, CD34+ cells can specialize into one of 3 types of cells: red blood cells (which are the oxygen carrying cells in the blood), platelets (which are involved in helping the blood clot when you cut yourself), and immune cells (which help you fight off infection). For the purposes of what Enzo is doing, only the immune cells are important. In fact, since both red blood cells and platelets do not have a nucleus, Enzo’s "stealth vector" is not even present in these cells.
The immune cells are a complex group of cells which all play a role in fighting infection. One arm of the immune system is the "innate" immune system, which consists of relatively primitive cells which look for classic bacterial and viral markers. These cells are the ‘first on the scene’ of an infection and provide the your body’s first line of defense against pathogens (second, if you want to be technical; the skin and mucous membranes are really the first).

HIV-1 trial (supp 2)
by: icposse2000 (27/M/Bethesda, MD) 2/2/00 9:01 pm
Msg: 15479 of 15539 While the innate immune system is holding back the pathogen (bacteria, virus, etc.), the second arm of the immune system (called the ‘adaptive’ response) is developing. The adaptive immune response is a much more powerful weapon against pathogens, especially viruses. There are basically 3 cells involved in the adaptive immune response:
1) Antigen presenting cells (APCs), which pick up little pieces of virus and present them to T-cells. B-cells, which are responsible for producing antibody, are a type of APC.
2) CD4+ ("helper") T-cells, which are responsible for ‘double-checking’ the material being present by the APCs to make sure that it is foreign (part of the invading organism). If the CD4+ T-cells determine that the APC is presenting it with foreign material, it can stimulate the B-cells to produce antibody and CD8+ ("cytotoxic") T-cells to check for pieces of the virus inside cells.
3) CD8+ T-cells have a sophisticated system of reading MHC I molecules (think of these as cellular ID tags). When a virus infects a cell, it often changes the ID tag on the cell, and the CD8+ T-cell can recognize that the cell has been infected. Infected cells are killed by the CD8+ T-cells, helping to prevent the virus from spreading.

There are 2 critical points to keep in mind for the discussion of Enzo’s technology:
1) immense number of T-cells (both CD4+ and CD8+) exist in the body; this is necessary because the chance of an individual T-cell being able to recognize that the material presented by an APC is relatively slim. (Another way of saying this is that only 1 T-cell in 100,000 or more is able to positively identify the material as foreign; the others aren’t sure and can’t start an immune response).
2) When that rare T-cell that can identify the ‘foreign’ material is found, it divides many times so that many clones of the original T-cell are present. Thus, after the initial phase of recognition, many virus-specific CD4+ and CD8+ T-cells are present. This is called T-cell ‘clonal expansion’; remember the term.

HIV-1 trial (supp III)
by: icposse2000 (27/M/Bethesda, MD) 2/2/00 9:01 pm
Msg: 15480 of 15539 HIV-1 is a virus that tends to infect CD4+ ("helper") T cells. The virus uses the CD4 molecule (and at least one co-receptor molecule) to gain access into these CD4+ T-cells. A battle then ensues between the virus, which is infecting and killing these CD4+ T-cells and the bone marrow stem cells, which are producers of the CD4+ T-cells. After a pronounced interval (usually between 5 and 10 years), the virus begins to win this battle, and the patients CD4+ T-cell levels drop.
Uninfected people tend to have CD4+counts of between 500-1000 cells /ml. Once a patient’s CD4+ T-cell count drops below 200 cells/ml, constitutional symptoms begin to appear; at levels below 100 cells/ml, the patient is severely immunocompromised and can be killed by bacteria and viruses that wouldn’t hurt healthy people. At levels below 200 cells/ml, the patient is said to have AIDS (acquired immunodeficiency syndrome, the disease caused by HIV-1 infection). The patient is immunocompromised because the CD4+ T-cells orchestrate the immune response against infection; although other immune cells are present in higher (even normal) numbers, the lack of CD4+ T-cells means that the patient can’t coordinate an attack to get rid of the invading organisms.
The HIV-1 virus has two main ways of killing CD4+ T-cells. First, the virus can directly infect cells and kill them. Second, the virus can promote the formation of syncytia (clumping of cells) which will trap and eventually kill uninfected CD4+ T-cells. Other immune cells besides CD4+ T-cells are also susceptible to HIV-1 (especially macrophages); however it is the decline of CD4+ T-cells which is responsible for the onset of AIDS and the eventual death of the patient.

*****

Viral loads (I)

by: icposse2000 (27/M/Bethesda, MD)

2/7/00 4:50 pm

Msg: 16443 of 16483

Hi to all!!

Sorry I haven’t posted in a while…I tried to have as computer-free a weekend as I could tolerate…Before I start with today’s post, I realized that I forgot to answer the question of how I heard about Enzo during my last Q&A session. I was informed of Enzo by the poster ‘spanky23’ on this board. He is a good friend, extremely knowledgeable about biological science and medical implications of research, and also recommended affymetrix to me when it was $17 a share (but of course I didn’t listen…), for what that’s worth.

I have noticed some questions recently regarding viral loads and their use in monitoring of AIDS therapeutic agents. I thought I would use this post to try to clear up the issues surrounding viral loads and how they do (and don’t) apply in Enzo’s case.

As I mentioned in the review on HIV biology, there is a continual battle between the HIV-1 virus, which infects and destroys CD4+ T-cells, and the bone marrow stem-cells, which are the predeccesors and producers of these CD4+ T-cells. Since HIV has a very long latent period (5-10 yrs, on average) between infection and the eventual development of AIDS, doctors utilize a number of prognostic tests to learn more about who is winning the battle between the virus and the stem cells.

Viral loads (II)

by: icposse2000 (27/M/Bethesda, MD)

2/7/00 4:51 pm

Msg: 16444 of 16484

There are two prognostic tests which are widely used in monitoring the patient with HIV:

1) CD4+ T-cell count. As I mentioned in the review on HIV biology, normal people have CD4+ T-cell levels of 500-1000 cells/ml of blood or greater. During the latent phase of HIV infection (when the patient has the virus but has not yet displayed signs of AIDS), the CD4+ T-cell count is usually between 200-500 cells/ml. Once the CD4+ T-cell count drops below 200 cells/ml, the patient does not have enough of these CD4+ T-cells around to orchestrate an immune response against usually benign bacteria and viruses. This immunodeficiency brought on by the drop in CD4+ T-cells is the disorder known as AIDS. Monitoring of the absolute CD4+ T-cell level gives doctors information about the strength of the patients immune system; monitoring changes in the CD4+ T-cell levels provides information on whether the immune system is becoming stronger or weaker.

2) HIV viral loads. The HIV virus infects CD4+ T-cells (and certain other cells of the immune system), replicates in them (often producing 1000 or more viral particles for each cell infected by a single viral particle), and then leaves the cell and enters the bloodstream, looking for more cells to infect. The HIV viral load is a measure of how much virus is present in the blood, and therefore is a indicator of how much the virus has recently been produced by infected cells. The HIV viral load is usually measured by a technique called reverse transcriptase-polymerase chain reaction (RT-PCR). This is basically a way of converting RNA (the genetic material of HIV is RNA) into DNA and then amplifying the information to measurable levels. One of the key advantages of measuring HIV viral loads compared to CD4+ T-cell levels is that they change much more rapidly and provide more timely feedback as to whether a therapeutic is working or failing…

Viral loads (III)

by: icposse2000 (27/M/Bethesda, MD)

2/7/00 4:51 pm

Msg: 16446 of 16484

When a patient initiates traditional anti-retroviral therapy (consisting of reverse-transcriptase inhibitors, nucleoside analogs, and protease inhibitors), doctors can see how well the patient is responding to the treatment by measuring the viral load. If the drugs are effective at preventing HIV replication, the patients viral load will decrease over time (usually several weeks to several months). With the HIV drug ‘cocktails’, the viral load is often reduced to unmeasurable levels, meaning that the virus has essentially disappeared from the blood. This does NOT mean that the virus is gone…only that it has not had much success replicating inside infected cells. Therefore, a drop in the viral load is a strong indication that a traditional anti-HIV pharmacologic has been effective in inhibiting viral replication.

Just as a drop in viral load can be an indication that a therapeutic is working against HIV, a rise in viral load can indicate that a drug is becoming less effective against the virus. For instance, the HIV drug ‘cocktail’ was greeted with enthusiasm by the medical community because the viral load dropped off dramatically in many patients soon after starting the therapy. However, after several years, many patients’ viral loads began to creep back up.

This suggests that a mutated strain of the virus (one that is not as inhibited by the drugs) had emerged and had begun replicating in cells and spreading virus into the blood. At this point, the only choice is to try to introduce one or more new drugs to try to stop the virus…but it is almost always a losing battle.

As you can see, measurements of the viral load are of great importance in monitoring how well a patient is responding to traditional anti-HIV pharmacologics. But Enzo’s HIV-1 strategy is anything BUT traditional…

Viral loads (IV)

by: icposse2000 (27/M/Bethesda, MD)

2/7/00 4:52 pm

Msg: 16447 of 16484

The importance of viral load measurements breaks down in Enzo’s case (especially at this

stage) for several reasons…

1) Little is known at this time about the patient population of the stage I clinical trial. Are the patients on a standard HIV drug cocktail? Are they people with HIV strains that have mutated to evade the cocktail? These questions are of great importance because they can influence (even determine) the viral loads in these patients. For instance, patients who are responding to the drug cocktail will have undetectable viral loads…regardless of whether Enzo’s approach is effective or not. Likewise, patients that are not responding to the drug cocktail will probably have high viral loads…even if Enzo’s approach IS working (see #2).

2) Enzo’s strategy is not to inhibit HIV replication…it is to repopulate the immune system with cells that are resistant to the replication of the virus. The trouble with using viral loads to monitor the efficiency of Enzo’s approach is that these patients will have two types of CD4+ T-cells…the unmodified CD4+ T-cells which are SUSCEPTIBLE to viral infection, and the HGTV43-modified CD4+ T-cells (descendants of the transfected CD34+ stem cells) which should be RESISTANT to viral replication. As a result, the virus may be replicating away in the susceptible cells (leading to detectable viral loads) even if the resistant cells are working perfectly and no viral replication is occurring within that population of cells.

Therefore, the viral load is NOT a useful measurement of whether Enzo’s strategy is working. The viral load will only become a useful measurement when the patient has only one population of CD4+ T-cells (either all sensitive to HIV infection…as in normal individuals…or all resistant to HIV infection…as Enzo hopes). As long as two populations of cells exist, it will be difficult to apply viral load measurements to monitor the treatment’s effectiveness.

Viral loads (V)

by: icposse2000 (27/M/Bethesda, MD)

2/7/00 4:52 pm

Msg: 16448 of 16484

With antisense treatment of HIV, Enzo has entered uncharted waters. Traditional indicators of a therapeutics efficacy are no longer valid…I think that the REAL questions that need to be asked to determine whether Enzo is making progress are:

1) Is there a subpopulation of resistant CD4+ T-cells? The press release last week suggested that a subpopulation exists, but no data was yet released on whether those cells were resistant to HIV. This question needs to be answered.

2) What is the proportion of modified CD4+ T-cells to normal T-cells, and how is that ratio changing? As I mentioned in the posts commenting on Enzo’s HIV-1 study, Enzo needs to achieve levels of 200+ modified CD4+ T-cells/ml in order to have successfully repopulated the immune system with resistant cells. This correlates to a ratio of approximately 1 resistant cell in 2-5 cells in normal individuals. It would be useful to know what this ratio is at the present time. It is important to remember, though, that it is likely that there would be a change in the ratio (in favor of the resistant cells) over time as the virus kills off non-resistant cells.

I would suspect that these are the kinds of questions that Enzo will be focusing on, rather than worrying about viral loads. However, given that Enzo is in stage I trials, I doubt that they are looking too intently at these (efficacy) endpoints. Just my 2 cents…

I hope that this helped reduce some of the confusion surrounding viral loads and Enzo’s strategy…as always, if there are questions/comments/corrections/etc. I’d be happy to try to address them. Thanks to all who made it through…

-icposse2000