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!)

 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….

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.

 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…

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).

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!!

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).

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 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.