Zinc Finger Proteins Put Personalized HIV Therapy Within Reach

[geobee]

I went in yesterday to Quest to get screened for the Sangamo trial.   As I mentioned before, they are using a laser to blast the wholes in the cells to let the ZFN get in there and do the work of modifying the cell.  So the process is apherisis, modify the cells, put them back in.  Because there are not using a virus as a vector, this should work better as one's body won't be producing ABs to the viral vector.

They have to do a tropism test.  30% of those come back as "no result" b/c there is not enough virus in the blood to obtain a sample.  If I come back as CCR5 only, then I'll be eligible for the trial, which won't be until October/Nov.

Other news:  The monoclonal AB trial is going well.  Most getting UD as long as they are taking it (weekly injection).  Also, CalImmune second arm should be starting soon.  The conditioning is going to be necessary to get the modified stem cells into the marrow.

[Jmarksto]

Thanks for the update geobee, and wishing you the best on the test and trial - thank you.

Also, do the monoclonal AB's penetrate the reservoirs?

[geobee]

I don't know if the AB's penetrate the reservoirs or not.  I'm guessing that they don't.  From what I read before, the virus comes back after you stop the AB treatment.

What a person at Quest did say as that the participants in the study had a favorable view of the injections which allowed them to not take daily meds.  For me, I'd rather take meds than inject myself.

[Jmarksto]

Given that the ABs go directly to the blood stream I would expect fewer side effects (even minor ones).  I think I would prefer the injections - even if it was just to eliminate or adjust my lunch schedule a little more.  It would be nice to have the option anyway.

Thanks again for posting, interesting stuff!

[freewillie99]

Another article from today's SFGate talking about the Sangamo and Calimmune stem cell trials and their potential to effect a functional cure.

http://www.sfgate.com/health/article/Stem-cell-therapy-could-lead-to-HIV-cure-5640362.php

Stem cell therapy could lead to HIV cure
Erin Allday
Published 5:02 am, Wednesday, July 23, 2014

Two teams of scientists with strong ties to the Bay Area are racing to develop a stem cell therapy that would provide a practical cure for people living with HIV infection, leaving them with an immune system capable of keeping them healthy without daily medication even as some virus remains circulating in their bloodstream.

Both groups of researchers are trying to capitalize on the DNA of so-called elite controllers - people who are naturally resistant to HIV due to a genetic mutation that prevents the virus from latching on to their immune cells. It was an elite controller who donated bone marrow to Timothy Brown, the "Berlin patient," who was the first in the world to be cured of HIV. Doctors attribute Brown's rebuilt HIV-resistant immune system to the genetic mutation in the bone marrow.

Bone marrow transplants are not an effective cure for HIV for the general population because they're risky and expensive. But stem cells, drawn from a patient's own bone marrow and altered to be HIV-resistant, may be able to do the job using the same premise.

"If you could make a person's immune system mutated in a way that HIV could not infect it, then you may be able to cure the HIV," said Dr. John Zaia, a virologist with the Beckman Research Institute near Los Angeles, who's working with Sangamo Biosciences in Richmond on a technique to engineer and transplant stem cells. "That's the premise anyway," he said. "And it's based on that one case in the Berlin, that one transplant."

The teams at the forefront of stem cell HIV therapy are led by Sangamo and Calimmune, a San Diego company that is testing its treatment in patients in Los Angeles and San Francisco.

Calimmune was the first to start human clinical trials, in July 2013, and last month reported that the first group of patients was doing well enough that they were ready to begin treating a second group. Sangamo expects to start clinical trials as early as this fall.

Both groups are being funded in part by the California Institute for Regenerative Medicine, the state's stem cell agency.

The research is based on the discovery in the mid-1990s of a specific genetic mutation that blocks a protein called CCR5. The protein is found on the surface of some cells where it acts as a receptor, allowing HIV to attach and ultimately fuse with the cell. Without CCR5, it's much more difficult, although not impossible, for the virus to infect a cell.

Genetic mutation

In elite controllers, the CCR5 receptor is mutated in such a way that HIV cannot latch onto it. Scientists believe that only about 1 percent of people worldwide have the CCR5 genetic mutation.

Nearly 10 years ago, when Timothy Brown needed a bone marrow transplant to treat his newly diagnosed leukemia, his doctor in Berlin decided to attempt an experiment, and chose a donor who was known to be an elite controller.

Bone marrow contains stem cells that stay active during a person's lifetime, constantly replenishing the cells that make up their blood and immune system. In a bone marrow transplant, stem cells from the donor replace the patient's own stem cells and build a new immune system.

Brown had been HIV-positive since about 1995, and his doctor wondered if replacing his immune system with cells from a donor who was naturally resistant would kill the virus and cure the leukemia at the same time. It worked.

But bone marrow transplants are risky procedures - patients can die from rejection of the donor cells or from infection while their immune system is rebuilt from scratch. They're also expensive and require lengthy recovery. And it's impractical to collect donor tissue only from the rare individuals who are elite controllers.

So bone marrow transplants aren't considered a worthwhile treatment for everyone who has HIV, especially since antiretroviral drugs are so effective.

Instead, scientists believe that they can draw stem cells from the bone marrow of patients infected with HIV, and genetically engineer those cells so that they have the mutated CCR5 receptor. Then, the same cells can be transplanted back into patients, where they will supply the immune system with cells that are HIV-resistant.

'Warrior cells'

"By actually treating the patient's own cells and giving them back, you're essentially helping to engineer an immune system that becomes long-term protective against HIV," said Dr. Louis Breton, chief executive of Calimmune. "The stem cells give the patient a group of highly protected warrior cells to do the job that we want our immune system to do, which is to kill off any disease and virus."

In theory, once the stem cells are implanted in the patients again, they'll be able to indefinitely replenish the immune system, and no further transplants or other treatments would be necessary.

Because scientists don't plan to destroy the patients' old immune system before transplanting the engineered stem cells, patients would continue to make cells that are vulnerable to HIV.

But the hope is that enough of the HIV-resistant cells will be produced that the body will be able to mount an immune response to fight the virus and keep it in check - providing what's called a "functional" cure, in which the virus remains but doesn't cause damage.

"Obviously eradication of all viral strains in the system would be extraordinary," Breton said. "This step we're taking is essentially a one-time treatment instead of lifetime."

The same premise could someday help scientists build a vaccine against HIV. But for now, the two teams of scientists are focused on a specific group of patients - those who aren't doing well on antiretroviral therapy and are at risk of their infection developing into AIDS.

The Sangamo and Calimmune teams have different strategies for engineering their stem cells and transplanting them back into patients. But the basic premise is the same, and the teams are nearly neck-and-neck in their research.

Calimmune was the first to put the stem cells into patients. Sangamo hasn't begun clinical trials with stem cells yet, but earlier this year it reported some success from a study involving transplanted T-cells - a type of immune cell that is destroyed by HIV. The T-cells were genetically engineered to resist HIV, using a technique that Sangamo will apply to stem cells in upcoming trials.

Scientists familiar with the research said they're encouraged both by the premise of the stem cell work and the results that have been produced so far. But even if both teams are successful, the treatment won't be practical on a global scale until it's made much simpler, said Dr. Warner Greene, director of the Gladstone Institute of Virology and Immunology in San Francisco.

Ideal therapy

Ideally, doctors would like to see a therapy in which patients are given a single injection that causes their stem cells to be re-engineered in the body, without having to be removed and treated in a lab. If the ongoing research proves successful in causing a functional cure, Greene said, the next step would be developing a procedure that's much more efficient.

"A lot of the stem cell work involves transplants. As proof of principle, that's great, but when it comes to treating tens of millions of people, it's just not going to work," Greene said. "We always have to have in mind a scalable solution. We must avoid therapies that are only useful to 100 rich people in the world."

San Francisco resident Michael Petrelis said he understands the need to remain wary of any promise of a cure - HIV is complicated, and there may never be a single treatment or therapy that works for everyone to wipe out the disease. But he said hope is something that helps keep him going.

"In addition to my AIDS cocktail, one of the things that's helped keep me alive is my hope for a cure," said Petrelis, 55, who has lived with HIV for more than a decade. "I'm optimistic that I can survive long enough to take advantage of stem cell research or something else.

"A lot of times, the researchers and even people with AIDS say we shouldn't use that 'C' word," he said. "And I'm of the thinking that yes, we should. We have to."

[greenbean]

One patient remains off therapy with control viral load for over a year
and another one for 34 weeks.

I think this approach will eventually work. It is just a matter how they can get enough resistant CD4 cells (20-30% is pretty good. the more the better of course).

Hope for the best.

[dico]

In previous studies, mathematical models have shown that we need at least 50% of our peripheral blood to be changed in order to have a long term remission.

They can change at best 20 to 30% of the PBMC so it won't lead to a cure.

[geobee]

Quest let me know that I've passed the first round of qualifications for the trial.  One day I may find out for myself whether this works or not!  (Once I start the trial, I'm embargoed from telling the results until it's over).

[greenbean]

In previous studies, mathematical models have shown that we need at least 50% of our peripheral blood to be changed in order to have a long term remission.

They can change at best 20 to 30% of the PBMC so it won't lead to a cure.

Which study shows that at least 50% is needed? If the study doesn't consider ccr5 mutation even 100% is useless like the Boston patients. Btw both two sangamo patients have experienced longer remission despite having only around 20% cells modified. my feeling is that modifying stem cell is the ultimate cure

Also. With the ability to retreat. Mathematically they can achieve over 50% modification with multiple treatments

[greenbean]

Quest let me know that I've passed the first round of qualifications for the trial.  One day I may find out for myself whether this works or not!  (Once I start the trial, I'm embargoed from telling the results until it's over).

Glad to hear that!!! I pray that you will get benefits from it. Hopefully one day I can be in the trial too. Especially the stem cell one

[freewillie99]

And then there's this from the New England Journal of Medicine:

http://www.nejm.org/doi/full/10.1056/NEJMc1405805

Shift of HIV Tropism in Stem-Cell Transplantation with CCR5 Delta32 Mutation

N Engl J Med 2014; 371:880-882August 28, 2014DOI: 10.1056/NEJMc1405805
 
Article

To the Editor:

Infection with the human immunodeficiency virus (HIV) requires entry into target cells by binding of the viral envelope to the CD4 receptor and to either the chemokine (C-C motif) receptor 5 (CCR5) or the chemokine (C-X-C motif) receptor 4 (CXCR4). Homozygosity for a 32-bp deletion in the CCR5 allele (CCR5 delta32) prevents cellular entry of CCR5-tropic (R5-tropic) HIV type 1 (HIV-1) strains. In 2009, there was a report1 about an HIV-1–infected patient with acute myeloid leukemia in whom the viral load remained undetectable after allogeneic stem-cell transplantation from a donor who was homozygous for the CCR5 delta32 mutation and after the discontinuation of antiretroviral therapy. This case gave rise to hope for new strategies for eradicating HIV-1 infection. However, this case has remained unique. Furthermore, in HIV-1–infected patients undergoing allogeneic stem-cell transplantation from donors with nonmutated CCR5, viral rebound has been reported.2

Here, we present the case of a 27-year-old patient with HIV-1 infection and anaplastic large-cell lymphoma. Because of a poor prognosis after progression of the T-cell lymphoma, stem-cell transplantation was planned, and a donor who was homozygous for the CCR5 delta32 mutation was identified. We determined the viral tropism of HIV-1 by genotyping the V3 amino acid sequence and applying geno2pheno bioinformatic software to predict viral coreceptor use,3 which indicates the probability of classifying an R5-tropic virus falsely as a CXCR4-tropic (X4-tropic) virus (false positive rate of <5%, X4-tropic; false positive rate of 5 to 10%, intermediate; false positive rate of >15%, R5-tropic). Before the patient underwent transplantation, the tropism from viral RNA was predicted to be either R5-tropic (false positive rate, 24.7%) or intermediate (false positive rate, 8.2%), whereas the V3 sequence from proviral DNA was classified as intermediate (false positive rate, 6.6%) or X4-tropic (false positive rate, 4.4%).

The patient discontinued antiretroviral therapy before the initiation of myeloablative treatment but resumed therapy 3 weeks after transplantation because of a rebound of 93,390 copies of HIV RNA per milliliter (Figure 1Figure 1 Human Immunodeficiency Virus (HIV) Levels, HIV Tropism, and Effect of Antiretroviral Therapy in a Patient with Anaplastic Large-Cell Lymphoma.
). The V3 sequence was related to the previous genotypes from this patient, as indicated by the presence of identical mutations in all V3 sequences (see the table in the Supplementary Appendix); it also carried several specific mutations resulting in the prediction of an X4-tropic virus (false positive rate, 0.4%). Antiretroviral therapy effectively suppressed viral replication until the patient had a relapse of the T-cell lymphoma, when antiretroviral therapy was again stopped. Two weeks before the patient died, the HIV-1 RNA level was 7,582,496 copies per milliliter.

The genotypic analyses of HIV-1 variants in this patient showed a shift from a dominantly R5-tropic HIV before stem-cell transplantation toward an X4-tropic HIV after transplantation. This shift of tropism was probably driven by transplantation with stem cells homozygous for the CCR5 delta32 mutation. This case highlights the fact that viral escape mechanisms might jeopardize CCR5-knockout strategies to control HIV infection.4

Lambros Kordelas, M.D., Ph.D.
Jens Verheyen, M.D.
Stefan Esser, M.D.
University of Duisburg–Essen, Essen, Germany
lambros.kordelas@uk-essen.de

for the Essen HIV AlloSCT Group

[tryingtostay]

What does all that ^  mumbo jumbo say? 

[buginme2]

What does all that ^  mumbo jumbo say?

Guy had ccr5 tropic virus, he got a stem cell transplant with stem cells from a ccr5 delta 32 deleted donor (just like the Berlin patient did) then his virus rebounded with cxcr4 tropic virus, then the patient died. 

[Ptrk3]

I think that the "mumbo jumbo" points out that HIV is a wily bastard that can adjust its specific genotype population to specifically-tailored genotype eradication attempts (one genotype is attacked so if the patient has multiple genotypes of HIV the genotype not eradicated will thrive and take over):  http://en.wikipedia.org/wiki/Host_tropism

At the very least, the letter points out that one type of treatment won't be a universal cure. 

I'm starting to understand that I'm going to be on Atripla for the rest of my life

Is this the "take away" other people on the forum take from this letter?

[buginme2]

At the very least, the letter points out that one type of treatment won't be a universal cure. 

I'm starting to understand that I'm going to be on Atripla for the rest of my life

Is this the "take away" other people on the forum take from this letter?

Dude you won't be on Atripla for the rest of your life.  My god Atripla is an older and nasty ass med.  But, your gonna be on treatment for the rest of your life.  Move to a new med with less side effects and get comfortable because it's going to be a long ride.

Oh, I didn't take that away from this report.  This report is interesting but it's one patient who sadly appeared to be very sick with advanced cancer and HIV and died.  Poor guy.

[Ptrk3]

Yeah, but I was hoping that I wouldn't need to be on any treatment for the rest of my life and was thinking that there might be a cure soon.

Thanks, though, for your realistic perspective.  You have adjusted and accepted your status well.  I wish I can do the same.

I frequently get seriously depressed because of my status.  Do you think it could be the Atripla (the sustiva) that contributes to my depression?  What other med should I seek?  I was diagnosed about 13 months ago and have been on Atripla ever since (my VL was undetectable in about 6 weeks).  I am still so sad and sometimes can't even leave the house.

[Jeff G]

I frequently get seriously depressed because of my status.  Do you think it could be the Atripla

If you are having bouts of depression that has gotten worse or began after Atripla then you probably need to make a change . I personally feel that if depression is a factor a person should never be put on this drug to begin with .

[Ptrk3]

Thanks.  I see my ID the week after next.  I'll bring the matter up. 

[buginme2]

Yeah, but I was hoping that I wouldn't need to be on any treatment for the rest of my life and was thinking that there might be a cure soon.

What's your definition of "soon"?  Everyone wants a cure for HIV and it will probably happen soon, if your definition of soon is measured in decades rather than months or years.

[Ptrk3]

Good question.  I was thinking of "soon" as within 10 years, but that does not seem likely after all, not at this point.  I'm in middle age, so decades could well be the rest of my life.

[tryingtostay]

I was thinking of "soon" as within 10 years.

I believe 10 yrs or within is possible with the study at Temple. 

[Ptrk3]

Thanks.  Within 10 years, that would be nice, indeed!

[tryingtostay]

Thanks.  Within 10 years, that would be nice, indeed!

I believe it is the best way to go above all else.  What better way to remove HIV-1 from the body than to remove it from the DNA?  The problem is the delivery method last I gathered.  I believe this is the best hope above all other research.  I know there was a geneticist in here doubting the study with his knowledge and research with HIV-1 but he pointed out that it's only because gene therapy is in it's infancy.  It's coming and what I think is other cures will come along with it!  And that's fucking fantastic for all mankind

[hotguyinTX]

Interesting. Actually there is one recent study showing that people with Delta32 mutation is losing their "resistance" to HIV because so many HIV+ people have cxcr4 strains while people with two genes called rs9264942 still maintain viral control which is likely due to the way their immune system handle the infection rather than the mutation that prevent HIV infection or slows replication. 

http://www.pubfacts.com/detail/21860345/Rising-HIV-1-viral-load-set-point-at-a-population-level-coincides-with-a-fading-impact-of-host-genet

But on the other hand, it clearly shows that HIV virus do need some receptors to replicate - what happens if we engineer cells to lack cxcr4 and delta32? Or are there other "doors" for them to open?

[geobee]

I'm still in the running for the Sangamo trial.  As I mentioned above, one of the things they have to do a tropism test to see if you have CCR5 virus.   Since they have so little virus to test (if you're UD), they often can't determine this.    Also, it takes months (apparently) to complete.

[buginme2]

I'm still in the running for the Sangamo trial.  As I mentioned above, one of the things they have to do a tropism test to see if you have CCR5 virus.   Since they have so little virus to test (if you're UD), they often can't determine this.    Also, it takes months (apparently) to complete.

Ya but the Sangamo trial isn't a clinical trial to cure HIV at this point anyway.  It's pretty much already been determined not to cure HIV (treating only cd4 cells) but may improve cd4 recovery and may possibly offer a clinical improvement. Possibly, maybe.

Sangamo and City of Hope are now looking to see if treating and transplanting hematopoietic stem cells could cure the infection http://defeathiv.org/institutions/
And
http://defeathiv.org/clinical-studies/

But they are ONLY doing clinical trials on patients with cancer who are getting a stem cell transplant anyway.


Even so....this research is still decades from actual clinical significance.

[freewillie99]

But they are ONLY doing clinical trials on patients with cancer who are getting a stem cell transplant anyway.

Your other comments can be insightful, but that is just blatantly incorrect when it comes to Sangamo's upcoming HSC trial.  Assume you're only referring to the COH trial.

Even so....this research is still decades from actual clinical significance.

Really?  Better let CIRM know they're wasting millions of dollars of taxpayer money on a hoax.

[buginme2]

Show me where I said it was a hoax?  I just saw a presentation from Paula Canon who pretty much said the said the same thing

[freewillie99]

Paula Cannon said we were "decades away" from modified HSC efficacy in the clinic?  Given the certainty with which you proclaim your hyperbolic assertions I'm surprised you didn't post a link.

[buginme2]

I think you misunderstood the point.

The current Sangamo and Calimune trials that are treating cd4 cells and infusing them back into the patient are not expected to yield results that will in effect cure the patient.   They may offer a clinical benefit by 1. Restoring cd4 counts in patients who's cd4's don't recover after HAART and restoring cd4/8 ratio to normal.

However, the FDA will only approve this as a treatment IF this a. turns out to be true and b. actually shows that this affects outcomes meaning does raising a cd4 count and restoring the cd4/8 ratio have clinical meaning?   The only way to determine that is with a phase 3 trial that measure outcomes.  Meaning...a very long clinical trial and we aren't anywhere near starting that (so at least a decade here). 

2.  Can the current treatment lower the viral load to say <200 without HAART?  Not expecting a cure but maintaining a low viral load without treatment?  The obstacles to this are enormous and currently not expected.  Even so, let's say it does maintain a low level viral load.  If it's not low enough the virus will mutate and your whole concept is shit.  The only way the FDA will approve such a treatment when there are currently effective and well tolerated treatment is if it can show clinical benefit over a long period of time.  Again, we are no where near a phase 3 trial and the duration of that trial would need to be significant.   Especially when just last week it was shown that over time it's quite possible your modified immune system that's resistant to ccr5 tropic virus won't matter since it's mutating to become cxcr4 tropic. 

3.  There is the idea that completely destroying ones immune system with chemotherapy and radiation and rebuilding it from scratch using modified stem cells using sangamo's system could lead to eradication (cure).   The NIH has funded this idea with >$20 million grant.  Sangamo and City of Hope and Fred Hutch (who invented the stem cell transplant) are studying this.  Clinical trials at this point are ONLY open to hiv positive patients with cancer.  I posted the links above.  Just last week there was a conference about this work with many speakers including Francois Barre-Synoise, Paula Cannon, Hans Peter Kiem, Keith Jerome among others.  I can tell you NOT ONE OF THEM is expecting a "cure" any time soon.  Especially using this technology.  It's too soon  and currently too dangerous. 

Read the links I posted above. 

PS. Everyone is free to hope.  If you want to believe this will cure you, knock yourself out. 

Personally I'd rather take a Stribild or Tivicay/Truvada then be irritated and exposed to high dose chemo and a stem cell transplant.....no thank you.

>>>Does anyone really think the FDA is going to approve any of this when we currently have effective and well tolerated treatments that basically restore life expectancy to near normal?  This needs to hit a fucking grand slam to get approved and so far, I don't see it.

[Ptrk3]

Thanks, to all the folks in this thread for their spirited and intelligent discussion of this topic.  I'm learning much from reading the links and back-and-forth.  I appreciate the time folks are spending in expressing complicated thoughts in easy-to-understand language for the rest of us.

[buginme2]

Thanks, to all the folks in this thread for their spirited and intelligent discussion of this topic.  I'm learning much from reading the links and back-and-forth.  I appreciate the time folks are spending in expressing complicated thoughts in easy-to-understand language for the rest of us.

Don't forget none of us are scientist so everything we say (me included) is really..well..shit.  So feel free to jump in

[Hoyland]

I think I should point out that the Calimmune trial is not only treating CD4 cells but also Hematopoietic Stem/Progenitor Cells. Because Cal-1 is DNA based and treats HSPC's, which will divide and differentiate into immune system cells, more and more HIV resistant cells will be produced over time.

I should also point out that Cal-1 includes a fusion inhibitor which is there to attempt to block any non-CCR5 cell entries.

[geobee]

I agree that the Sangamo trial isn't a cure attempt, probably.  But... and it's a big but... it's better than the earlier trials.  In the old trial, you could only get one infusion b/c your body would create an antibody to the delivery vector.  In the new trial, you can get more than one infusion b/c the cells are being modified differently.   I don't think this will make a difference, but it might.

[dico]

I agree that the Sangamo trial isn't a cure attempt, probably.  But... and it's a big but... it's better than the earlier trials.  In the old trial, you could only get one infusion b/c your body would create an antibody to the delivery vector.  In the new trial, you can get more than one infusion b/c the cells are being modified differently.   I don't think this will make a difference, but it might.

It will make a difference but the problem is that HIV can mutate. So the CCR5 knockdown will only temporarily work. There are no preclinical studies showing monkeys or rabbits have been cured using gene therapy.

[JakaTingkir]

It will make a difference but the problem is that HIV can mutate. So the CCR5 knockdown will only temporarily work. There are no preclinical studies showing monkeys or rabbits have been cured using gene therapy.

g
why do you need rabbits n monkeys?...there is human got cure about this method aka berlin patient...

the key point is how to make the body produce NEW modified cd4 cells. That’s my thought.

[bmancanfly]

Anyone who wants to understand how slow research progresses,  just look at how long this thread has been going on.

And this treatment is nowhere close to being ready for prime time.  Let alone a "cure".

[buginme2]

Anyone who wants to understand how slow research progresses,  just look at how long this thread has been going on.

And this treatment is nowhere close to being ready for prime time.  Let alone a "cure".

Hahaha for real this thread started six years ago and sure they have moved forward a little bit but. ....

Not to mention when/if this doesn't work... me thinks there's going to be a lot of pissed off poz forum members. 


I don't think I've seen such investment in one particular area of research.

[tryingtostay]

So the big hurdle with alot of these trials is to sustain the immune cells?  Then what?  I'm no expert but there will be problems with HIV still in the blood when and after they find a way to make our immune cells well, immune to HIV. 

[Matts]

A case study from á german university:

a patient got a stem-cell transplantation with CCR5-Delta 32 mutation. The receptor switched from CCR5 to CXCR4 and the Patient died at the end with a viral load of > 7 Million copies. Maybe it is illusive to believe in  a cure with a CCr5 knockout-strategy.

Timothy R. Brown just had luck and is  a special single case. Correct me if I am wrong.

http://www.nejm.org/doi/full/10.1056/NEJMc1405805

http://cid.oxfordjournals.org/content/59/4/596.long

"Shift of HIV Tropism in Stem-Cell Transplantation with CCR5 Delta32 Mutation

Infection with the human immunodeficiency virus (HIV) requires entry into target cells by binding of the viral envelope to the CD4 receptor and to either the chemokine (C-C motif) receptor 5 (CCR5) or the chemokine (C-X-C motif) receptor 4 (CXCR4). Homozygosity for a 32-bp deletion in the CCR5 allele (CCR5 delta32) prevents cellular entry of CCR5-tropic (R5-tropic) HIV type 1 (HIV-1) strains. In 2009, there was a report1 about an HIV-1–infected patient with acute myeloid leukemia in whom the viral load remained undetectable after allogeneic stem-cell transplantation from a donor who was homozygous for the CCR5 delta32 mutation and after the discontinuation of antiretroviral therapy. This case gave rise to hope for new strategies for eradicating HIV-1 infection. However, this case has remained unique. Furthermore, in HIV-1–infected patients undergoing allogeneic stem-cell transplantation from donors with nonmutated CCR5, viral rebound has been reported.2

Here, we present the case of a 27-year-old patient with HIV-1 infection and anaplastic large-cell lymphoma. Because of a poor prognosis after progression of the T-cell lymphoma, stem-cell transplantation was planned, and a donor who was homozygous for the CCR5 delta32 mutation was identified. We determined the viral tropism of HIV-1 by genotyping the V3 amino acid sequence and applying geno2pheno bioinformatic software to predict viral coreceptor use,3 which indicates the probability of classifying an R5-tropic virus falsely as a CXCR4-tropic (X4-tropic) virus (false positive rate of <5%, X4-tropic; false positive rate of 5 to 10%, intermediate; false positive rate of >15%, R5-tropic). Before the patient underwent transplantation, the tropism from viral RNA was predicted to be either R5-tropic (false positive rate, 24.7%) or intermediate (false positive rate, 8.2%), whereas the V3 sequence from proviral DNA was classified as intermediate (false positive rate, 6.6%) or X4-tropic (false positive rate, 4.4%).

The patient discontinued antiretroviral therapy before the initiation of myeloablative treatment but resumed therapy 3 weeks after transplantation because of a rebound of 93,390 copies of HIV RNA per milliliter (Figure 1Figure 1Human Immunodeficiency Virus (HIV) Levels, HIV Tropism, and Effect of Antiretroviral Therapy in a Patient with Anaplastic Large-Cell Lymphoma.). The V3 sequence was related to the previous genotypes from this patient, as indicated by the presence of identical mutations in all V3 sequences (see the table in the Supplementary Appendix); it also carried several specific mutations resulting in the prediction of an X4-tropic virus (false positive rate, 0.4%). Antiretroviral therapy effectively suppressed viral replication until the patient had a relapse of the T-cell lymphoma, when antiretroviral therapy was again stopped. Two weeks before the patient died, the HIV-1 RNA level was 7,582,496 copies per milliliter.

The genotypic analyses of HIV-1 variants in this patient showed a shift from a dominantly R5-tropic HIV before stem-cell transplantation toward an X4-tropic HIV after transplantation. This shift of tropism was probably driven by transplantation with stem cells homozygous for the CCR5 delta32 mutation. This case highlights the fact that viral escape mechanisms might jeopardize CCR5-knockout strategies to control HIV infection.4 "




Lambros Kordelas, M.D., Ph.D.
Jens Verheyen, M.D.
Stefan Esser, M.D.
University of Duisburg–Essen, Essen, Germany
lambros.kordelas@uk-essen.de
---------------------------

About the Berlin Patient:

"Dependence on the CCR5 Coreceptor for Viral Replication Explains the Lack of Rebound of CXCR4-Predicted HIV Variants in the Berlin Patient

Abstract

The “Berlin patient” is the first patient cured of HIV-1 infection after allogeneic transplantation with nonfunctional CCR5 coreceptor stem cells. We demonstrate that CXCR4-predicted minority viruses present prior to transplantation were unable to rebound after transplantation due to their dependence on CCR5 for replication and high genetic barrier toward CXCR4 usage.

The “Berlin patient” is the first individual in whom human immunodeficiency virus (HIV) type 1 (HIV-1) infection has been cured following allogeneic transplantation to treat acute myeloid leukemia [1, 2]. The stem cells used were homozygous for the Δ32 frameshift mutation (CCR5Δ32), resulting in absence of the CCR5 receptor at the cellular surface [3]. Heterozygosis for CCR5Δ32 is associated with slower disease progression, and individuals homozygous for CCR5Δ32 are naturally resistant to CCR5-tropic HIV [3]. Binding of the viral envelope glycoprotein gp120 to a coreceptor (CCR5 or CXCR4) is essential for HIV entry into CD4+ host cells. HIV coreceptor tropism is mainly determined by the third hypervariable loop of the viral envelope (gp120-V3) [4].

The Berlin patient had a plasma viral load of 6.9 × 106 copies/mL during an episode of treatment interruption prior to stem cell transplantation (SCT). The viral population was predicted to be CCR5-tropic based on standard genotypic tropism testing (Geno2Pheno[coreceptor] false-positive rate [FPR] 24.2%) [1]. Detailed analysis using ultradeep sequencing detected a 2.9% minority viral population predicted to be CXCR4-tropic (Geno2Pheno[coreceptor] FPR range, 2.7%–9.3%). Two months after SCT, successful reconstitution of CD4+ T cells with the homozygous CCR5Δ32 phenotype was observed [1]. These donor derived T cells displayed normal levels of CXCR4 coreceptor surface expression. Given the detection of CXCR4-predicted viral variants prior to SCT, one would expect these variants to appear after SCT, especially as antiretroviral therapy was discontinued during the transplant procedure. However, no viral rebound was observed, and the patient remained free of HIV infection for >5 years post-SCT [5].

Previously, we demonstrated that some viruses capable of using CXCR4 have a clear CCR5 coreceptor preference in vivo [6]. Based on these findings, we postulate that a rebound of the CXCR4-predicted variants in the Berlin patient did not occur due to dependence on the CCR5 coreceptor for viral replication.
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DISCUSSION

The Berlin patient is the first patient with a cured HIV infection. Prior to SCT, the viral population was genotyped and a minority population was predicted to use the alternative CXCR4 coreceptor in a Web-based algorithm (Geno2Pheno[coreceptor]) [1]. Considering the normal levels of CXCR4 coreceptor expression on the donor-derived cells and susceptibility of these cells for CXCR4-tropic virus, it was remarkable that HIV did not rebound post-SCT in the absence of combination antiretroviral therapy (cART) [2]. Our study demonstrates that CXCR4-predicted minority viruses present prior to transplant were unable to rebound after transplant due to their dependence on CCR5 for replication and a high genetic barrier toward CXCR4 usage.

The main determinant of coreceptor usage is the gp120-V3 loop. Genotypic prediction algorithms, such as Geno2Pheno[coreceptor], use this region to predict coreceptor tropism. The result of the Geno2Pheno[coreceptor] interpretation is given as a quantitative value, the FPR, which defines the probability of classifying a CCR5-predicted virus falsely as CXCR4-predicted variant. Varying the threshold value for FPR classification changes the sensitivity and specificity for CXCR4 tropism prediction. In clinical practice, conservative cutoff FPR values that show good correlations with virological outcome during CCR5 inhibitor–based therapy are generally applied to prevent underestimation of the presence of CXCR4-tropic viruses (FPR cutoffs 3.5%–10%) [11, 15]. The lowest FPR of the minority variants in the Berlin patient prior to SCT was 2.7%. Although this is below the lowest cutoff FPR suggested for tropism prediction based on deep sequencing (3.5%) and therefore genotypically predicted to be CXCR4-tropic, the variant was dependent on CCR5 for viral entry in phenotypic assays. This single case study shows that predicted genotypic coreceptor tropism may not always reflect biological behavior and suggests that larger studies are needed to explore the use a lower cutoff FPR for maraviroc eligibility in clinical practice. In case of a CCR5Δ32-SCT procedure, a more lenient FPR and/or phenotypic testing should be considered.

Envelope regions outside the gp120-V3 sequence can modulate coreceptor affinity [16], and commercial phenotypic coreceptor usage assays are based on (near) full-length envelope sequences representing the plasma population virus [11]. Unfortunately, prior to SCT, full envelope sequences were not generated from the Berlin patient, and no additional samples were stored to enable full envelope sequencing. Therefore, we were inherently limited in assessment of the coreceptor phenotype of the minority variants and were restricted to the V3 loop sequences generated by ultradeep sequencing prior to SCT. In absence of patient-derived full envelope sequences, we decided to clone the gp120-V3 loop sequences of the Berlin patient in the background of a CXCR4-tropic HIV-1 laboratory strain to limit bias for CCR5 usage.

In the Berlin patient, CCR5-tropic permissive cells could still be detected for at least 5.5 months in the colon, and proviral DNA was observed 2 months after SCT [1, 2]. More than 5 years post-SCT, extremely low levels of HIV DNA and RNA were intermittently detected using very sensitive assays [5]. Given the volatile combination of long-lived CCR5-expressing cells and a potential CCR5-tropic reservoir, HIV replication could potentially have continued. Residual replication of CCR5-tropic viruses in the setting of increasing numbers of CCR5−CXCR4+ cells may then result in evolution toward CXCR4 usage. We tested the evolution potential for the most CXCR4-predicted patient-derived viruses and did not observe viral evolution toward CXCR4 usage, suggesting a relatively high barrier for coreceptor switch. Moreover, it has also been hypothesized that the number of residual CCR5-expressing CD4+ cells after SCT was too low to support replication of the CCR5-tropic variants and therefore evolution toward CXCR4 usage [17].

A recent study in which 2 HIV-infected heterozygote CCR5Δ32 patients with a small viral reservoir received a CCR5WT/WT transplant in the presence of cART unfortunately demonstrated viral rebound after interruption of cART [18, 19]. In the Berlin patient, aside from absence of CXCR4-tropic virus, the lack of a rebound of CCR5-tropic virus immediately post-SCT in the absence of cART indicates that transplantation of CCR5Δ32/Δ32 stem cells was pivotal to the apparent cure of HIV. These results provide a rationale for CCR5-based SCT and gene therapy studies in which in-depth analysis of HIV coreceptor usage is essential.
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[greenbean]

I still believe gene therapy is a promising approach to cure HIV. Again, the problem is delivery and it won't be a cure for everyone. (e.g. people with dual tropism)

Page 14 of this presentation shows a hetero controlling viral load for over a year

http://files.shareholder.com/downloads/SGMO/3465274679x0x780854/8f05992e-1a46-41b2-904d-040803103d0a/MS%20NYC%20091014%20FINAL.pdf

Page 15 showing two patients without CCR5 mutation controlling viral load without HART.

looks like it is heading to the right direction

[freewillie99]

Timothy R. Brown just had luck and is  a special single case. Correct me if I am wrong.

Or maybe this guy just had bad luck and is a special single case?  We have no way of knowing of course, but unprovable doom and gloom assertions delivered as fact provide nothing.

Btw, Tim Brown was reported to be dual tropic pre-stem cell transplant.

[leatherman]

Timothy R. Brown just had luck and is  a special single case. Correct me if I am wrong.

interesting links you provided - but they said nothing about what the man himself went through. While all that biological/scientific stuff is certainly intriguing, Tim Brown himself went through HIV, acute myeloid leukemia, chemotherapy, antiretrovirals, radiation, Stem cell transplant, relapse of acute myeloid leukemia, a second Stem cell transplant, leukoencephalopathy, colonoscopies, liver biopsies, major side effects, physical therapy, extended recovery time.... while the man is lucky to be alive because just one of any of those issues had a huge potential of causing his death, if I was him I don't know that I would be feeling all that "lucky".

http://onlinedigeditions.com/display_article.php?id=1280628

[dico]

Hi Geobee,

Before you are being embargoed tell us what are the news about the Sangamo trial ? What dose of cyclo you will take (1 mg or 1,5 mg ?)

Thanks

[geobee]

Hi dico --

I'm not on the trial yet.  They said it might take a while.  They have to do a tropism study to see if I'm CCR5 or X4.  Because I'm undetectable it takes a long time for them to get enough sample to do it.   They said it would be a few months -- and it has.  I'll give them a call and see what's up.

[dico]

Thank u for ur swift reply.

[freewillie99]

Looks like Sangamo was granted a US patent today, 10/28/14, to modify / knock out CXCR4 for "treatment and prevention of CXCR4-tropic HIV infection" using their Zinc Finger tech.  Normally I'd paste the body of the article to this post, but it's so long I'm just going to give a link.

http://www.investorvillage.com/smbd.asp?mb=1933&mn=68693&pt=msg&mid=14313758

[Hoyland]

http://www.youtube.com/watch?v=plvv07vd5iI

A talk by Paula Cannon about HIV cure with gene therapy.

[Cosmicdancer]

It's good to see that Paula Cannon is hoping to get approval to start a clinical trial with USC/City of Hope/Sangamo using hematopoietic stem cells in January, 2015. 

[freewillie99]

And here....we....go...

Giving HIV the [zinc] finger: using targeted nucleases to create an HIV-resistant Immune system.

Dr. Paula Cannon's presentation 1/27/15

http://mediacast.ic.utoronto.ca/20150127-OSCI/index.htm#

Sounds like the long awaited HSC trial will begin "any day now".