HIV-1 genomeBy Gertrud U. Rey

Although antiretroviral therapy (ART) has been highly effective at controlling HIV-1 viral loads in the bloodstream of infected individuals, the virus remains latent in infected cells and starts replicating within a couple of weeks upon termination of therapy.

The discovery of CRISPR/Cas9 gene editing technology has provided renewed hope for alternative suppressive therapies and possible elimination of HIV-1 integrated proviral DNA from the genomes of infected individuals. However, even though this strategy has been somewhat successful in in vitro laboratory cell models, editing of HIV-1 DNA in cells and tissues that support viral reservoirs in vivo has been challenging. Accordingly, recent strategies have employed targeted vectors to deliver CRISPR/Cas9 complexes directly to infected subjects.

In a recent study at Temple University, the investigators sought to excise HIV-1 DNA from patient immune cell engrafts in mice by treating the mice with a CRISPR/Cas9 complex delivered in a lentiviral vector cocktail. The mice were immunodeficient animals that were “humanized” by injection with blood cells from healthy or HIV-1-positive human subjects. The cocktail (“therapeutic cocktail”) consisted of three separate lentiviruses encoding either

a) a CRISPR guide RNA targeting the 5’ HIV-1 long terminal repeat (LTR) motif;
b) a CRISPR guide RNA targeting the 3’ HIV-1 LTR motif; or
c) a Cas9 effector protein.

This cocktail would facilitate cleavage of integrated HIV-1 DNA on both 5’ and 3’ LTRs, resulting in the excision of a portion of each LTR and the entire HIV genome positioned between the two LTRs, corresponding to the gag-pol-env and accessory genes.

The experimental design, which aimed to assess the combined ability of the individual components in this therapeutic cocktail to edit the HIV-1 genome in vitro or in vivo, was as follows.

1. In vitro HIV-1 infection of healthy human blood cells, followed by in vitro treatment with therapeutic cocktail.

Blood cells isolated from healthy human subjects were infected with HIV-1 and treated with the therapeutic cocktail one week post-infection. The efficiency of excision of HIV-1 DNA was determined by sequencing the 5’ and 3’ LTR regions. There was a significant reduction (up to 65%) of HIV-1 DNA in treated cells versus non-treated control cells.

2. Engraftment of human in vitro infected HIV-1 positive cells into mice, followed by treatment with therapeutic cocktail in vivo.

Human HIV-1-positive blood cells infected in vitro were injected into immunodeficient mice to generate humanized mice. One week later, the mice were treated with the therapeutic cocktail. After another week, the mice were euthanized and the efficiency of excision of HIV-1 DNA was determined. In agreement with the results obtained in step 1, there was a drastic decline in HIV-1 DNA in engrafted treated animals versus engrafted non-treated control animals.

3. Ex vivo treatment of human blood cells derived from HIV-1-positive donors, with therapeutic cocktail.

Blood cells were isolated from three separate HIV-1-positive patients (patients 1-3), each of whom were on ART therapy and showed low or non-detectable virus levels. Their cells were treated with the therapeutic cocktail, and the efficiency of excision of HIV-1 DNA was determined. There was a significant decrease in the level of HIV-1 DNA (up to 68%) in treated cells versus non-treated control cells.

4. Engraftment of human blood cells from HIV-1-positive donors into mice, followed by treatment with therapeutic cocktail in vivo.

Blood cells from patients 1-3 were injected into immunodeficient mice to generate humanized mice. One week later, the mice were treated with the therapeutic cocktail. Two weeks later, the mice were euthanized and the efficiency of excision of HIV-1 DNA in the blood, spleens, and various other organs was determined. The efficiency of excision observed in the blood and spleens of mice engrafted with cells from patients 1 or 2 was as high as 96% compared to that observed in the blood and spleens of non-treated control mice. In contrast, the efficiency of excision was as low as 25% in mice engrafted with cells from patient 3. This difference in efficiency was attributed to variabilities observed in viral LTR sequences and guide RNA target sequences between different patient samples.

As a further assessment of the efficacy of this therapeutic strategy, the authors isolated cells from the blood and spleen of therapeutically treated mice that were engrafted with cells from patient 1 and co-cultured these cells with a cell line that is highly permissive for HIV-1 infection. They observed significantly reduced recovery of HIV-1 from cells taken from engrafted treated animals versus engrafted non-treated animals.

Although these results are promising, the system is still artificial, making it difficult to predict if it would work in humans. Furthermore, in vivo editing of the HIV-1 genome by CRISPR/Cas9 may not completely remove replication-competent virus from organs serving as reservoirs for viral latency, such as the spleen. As explained by John Coffin and Stephen Hughes on TWiV 510, the viral reservoir exists in a pool of infected cells that contain HIV integrated provirus. These cells are capable of clonally expanding, and surprisingly, not all offspring of a clone exhibit identical levels of viral expression. Developing effective strategies to identify and eliminate such pools of cells is a prevailing challenge in this field. Likely, complete elimination of HIV-1 by CRISPR may only be accomplished in combination with other therapies, such as ART, and may require personalized CRISPR/Cas9 complexes that are perfectly matched to a particular viral genome.

A PhD Lab Coat Ceremony

The Graduate School of Biomedical Sciences at the Icahn School of Medicine at Mt. Sinai recently conducted the first PhD Lab Coat Ceremony in New York City. I was honored to be the Keynote Speaker at this event. My address follows.

Forty-three years ago this month I was in your shoes. I had just joined the PhD program in biomedical sciences here at Mt. Sinai, and while we didn’t have a white coat, it was exciting times nonetheless.

Forty-three years is a long time, but looking back, I now realize that I was completely clueless. I didn’t know very much about scientific research, I didn’t know what I would be doing one day, but it didn’t matter – I was curious and enthusiastic and in the end it seemed to have all worked out. But you might ask, how did that happen if you were clueless? My answer is: Mentors. I found people who I trusted and I listened to what they told me.

I’ll give you an example from very early in my career. I had just graduated from Cornell in 1974 but I didn’t know what to do with my life. I knew that I liked science so I got a job as a microbiology lab technician. I loved the work but I soon realized that I had to return to school because I didn’t know anything about microbiology. Then I read a book called ‘Fever’ about the first identification of Lassa virus in Africa. I fell in love with viruses.

Remember that I’m living at home, I have no support network, no advisors, so I decided to apply to masters degree programs in the area. Then one night in August 1975 I went to dinner at Ed Kilbourne’s home. Ed and I were undergraduates at Cornell and had become friends; his parents lived nearby.

Some of you in this room surely recognize that name: Ed’s father, Edwin, was for many years chair of Microbiology here at Mt. Sinai. After dinner Dr. Kilbourne asked me what I was going to do with my life. I told him I wanted to work on viruses – which must have pleased him, as a virologist – and I told him I was applying to masters’ programs. He told me to forget the masters, just come to Mt. Sinai and get a PhD. The next week I interviewed with the program, was accepted, and moved into a dorm on 96th and 1st.

After three lab rotations, I entered Peter Palese’s laboratory to do my dissertation research. Again, someone I trusted advised me that it would be a good choice. When I was ready for postdoctoral work, I asked Peter what lab to go to, and he said: only the best, David Baltimore.

At each stage of my career I found mentors who I trusted and then listened to their advice. So my advice to you is: find a mentor you trust, and listen to them! It’s of no use if you do not listen.

To this day, I seek out mentors and listen to them. And to this day, when either Peter or David ask me to jump, I say, how high?

You are just beginning what I think is the best career you can have: a career in science. Notice that I didn’t call it a job, because it’s not. Nor are you entitled to a career in science: it is a privilege, not a right. You have to earn that right, and to do so won’t be easy. You’ll work harder than most of your peers, and for less money. But a career in science is not about money: it’s about changing the world.

Many humans live long and healthy lives because of science. Not all of you will make seminal discoveries, like reverse transcriptase or CRISPR-Cas. Not all of you will even go on to a career in basic research. But you will all play some role in advancing science, whether at the bench, in business, or in communication.

43 years in science have taught me many other important lessons. One worth telling you is that science is not about you. It is not about building a big lab, scoring many research grants, publishing papers in prominent journals, or even winning a Nobel Prize. It is about discovery. It is about understanding how life works. Everything else is second to that goal.

Sometimes your discoveries will improve people’s lives, but that won’t always be your goal. In the 43 years behind me, a new phrase has crept into the science lexicon: translational research. This means nothing more than doing research that has a direct use, like making a vaccine or curing a disease.

The problem is that the public has come to believe that translational research is all there is, or all that there should be. They don’t understand that translational research stems from discoveries that were not intended to cure anything, but were the fruits of a scientist’s curiosity. Abraham Flexner recognized this problem in his 1939 essay, ‘The usefulness of useless knowledge’, from which I quote:

I am not for a moment suggesting that everything that goes on in laboratories will ultimately turn to some unexpected practical use or that an ultimate practical use is its actual justification. Much more am I pleading for the abolition of the word ‘Use’ and for the freeing of the human spirit. To be sure, we shall thus free some harmless cranks. To be sure, we shall thus waste some precious dollars. But what is infinitely more important is that we shall be striking the shackles off the human mind and setting it free for the adventures which in our own day have taken Hale and Rutherford and Einstein and their peers millions upon millions of miles into the uttermost realms of space and, on the other, loosed the boundless energy imprisoned in the atom. What they have done out of sheer curiosity has transformed human life.

The key is to have the ‘right’ balance between what I’ll call curiosity driven, fundamental research, and translational research. I’m not sure that most people realize this, and I’ll bet that members of Congress – which of course appropriates much of the biomedical research dollars in this country – don’t either. It’s a problem you will have to deal with in your careers.

I want to tell you about a few other problems with science – they WILL affect your careers, and perhaps your generation will solve them.

The first concerns the publication of scientific findings– the very means by which our careers are made. You need to publish your work in scientific journals because that is how you will be judged at every step of your career, whether it be finding a new position, securing grant support, obtaining academic tenure, and much more.

We would not have a problem if you were judged solely by the contents of your papers. Unfortunately, you are often judged – sometimes in addition to, sometimes only by – which journal the work is published in.

This issue was less of a problem when I was a PhD student, but has grown substantially in the past 43 years. Over that time publishers learned that they could make billions of dollars in profit by publishing our work. The elite journals – like Science, Cell and Nature and their derivatives, maintain an exclusive cachet by limiting the number of papers they review and publish. This practice inflates the ‘journal impact factor’ – a supposed measure of the impact of work in the field.

The problem is that not all good science is published in these luxury journals, and not all of what they publish is good! Yet, when it comes to hiring, promotion, and funding, the committees involved make decisions based on the journal and not the contents of the paper.

In other words, the professional editors of luxury journals are playing an enormous role in crucial decisions about our careers. I hope you’ll agree with me that this practice is entirely unacceptable. I prefer that my scientific colleagues make the decisions about my career.

There is another problem in scientific publishing which revolves around the fact that the research we do is supported by tax dollars, but many journals keep scientific papers behind a paywall – so the public can’t see what they are paying for. This practice is also unacceptable, but it drives profits for journals.

Open access has been the solution to this problem – but not for all journals. The Public Library of Science, and many society journals like those of the American Society for Microbiology and The Microbiology Society are open access. But the luxury journals won’t make all their journals open access – to do so would be to forfeit their enormous library subscription revenue. But why do these journals have to make so much money? Do we really need printed journals? And how expensive is it to have a process to review papers, and simply post them online? It turns out not very much, but the problem is that the luxury journals also spend a good deal of money on the commentaries that fill up the front of each issue. Why should the cost of these writings be carried by scientists?

Solving these two publishing problems – the impact factor and open access – will not be easy. We are partly responsible for them and we are now addicted. But I think there is a solution, and I call it biorXiv with peer review.

You’ve probably heard of biorXiv – essentially a server run by Cold Spring Harbor to which you can submit preprints of your scientific papers. That way everyone can see the results of taxpayer-supported research. Many of these papers get submitted to scientific journals and end up behind a paywall with an inflated impact factor. But I propose to cut out the journals. Let’s add a peer-reviewing system to biorXiv and keep it open access. The costs to maintain it should be far less than for the luxury journals, and could be supported by page charges and advertising.

The ideal science publishing platform is one where the site of publication is irrelevant. What matters is the science. I envision a world where we hear no more ‘congratulations on your Cell paper’ but rather ‘congratulations on your paper identifying the cell receptor for poliovirus’.

Another science problem that also impacts all of you concerns science and the public. If you’re like me, you must bristle at the idea that there are ‘alternative facts’. We have a President who disdains science: he does not believe in climate change and vaccines and took 19 months into his administration to appoint a science advisor. Much of the public is apathetic to science, and wants to know nothing about it, despite the fact that it makes their lives better. I’m not sure of the source of this disdain, but one possibility is a poor science education starting in elementary school.

I think scientists must take part of the blame for a science-disinterested public. Few of us take the time to tell the public what we are doing; we leave that to science writers. Some of these writers do a good job, but most do not and they certainly do not have the passion that we do for the subject. And as you surely know, passion for the subject is the key to teaching.

When I was a student, communicating science to the public was hard. The internet and social media has changed all that.  It’s now easy to engage the public with blogs, videos, podcasts, Facebook, Twitter, Instagram and more. I’ve been doing this for 12 years and I can tell you that the public wants to hear about what you do! If scientists engaged the public as a single voice, we could easily drown out those who do not believe in vaccines or climate change. To touch on a problem I mentioned earlier, these venues could be used to tell your story of how curiosity drove what turned out to be a fundamental discovery. Some of my favorite such stories include restriction enzymes, PCR, and CRISPR-Cas. You should keep a few of them in the back of your mind for instant use in public situations.

As you move forward in your science career, there will be many demands on your time and it will be easy for you to ignore communication. But I urge you to start now and establish good habits. There are many ways for you to communicate through writing and multimedia; or you could simply have a presence on Twitter or Facebook where much of the public visits.

And if you don’t want to do any of that – here’s a simple way to communicate your science. The next time someone asks you ‘what do you do?’, tell them: I work for you. The conversation will definitely continue!

A career in science is an amazing journey. You get to solve problems, talk with smart and clever people on a daily basis, and help make the world a better place (only science does that – not business, or law, or government – they are just facilitators). I wish you all a fulfilling career in this wonderful field, and don’t forget to always be curious, passionate, and kind.

By David Tuller, DrPH

When I first began examining the PACE trial in detail, I turned to clinical trial experts to vet my concerns. One of them was biostatistician Bruce Levin, a professor at Columbia University’s Mailman School of Public Health, to whom I was referred by a mutual colleague. After he reviewed the trial, he pronounced it to be a mess—a conclusion that fueled my determination to investigate the matter.

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By David Tuller, DrPH

I’ll be in Bristol later this week for the CFS/ME Research Collaborative’s annual conference. I was not welcome last year, since I was at that point engaged in harshly criticizing the organization for its unwillingness to acknowledge that its deputy chair had falsely accused me of libel. This year, things have changed and both the chair and the new deputy chair have graciously welcomed me.

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Ned Landau joins the TWiV team to discuss restriction of HIV replication by SAMHD1, and a viral antagonist that can be used to produce a dendritic cell vaccine.

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Gray Mouse LemurLemurs are primates found only on the island of Madagascar and a few small neighboring islands. Some of these animals have endogenous lentiviruses in their genomes. How did these viruses infect the isolated lemurs?

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Vincent and Dickson travel to the 44th Retrovirus meeting at Cold Spring Harbor Laboratories, where they speak with John Coffin, Stephen Hughes, Ya-Chi Ho, and Matt Takata about the meeting and their work on HIV-1.

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Poliovirus by Jason Roberts

Poliovirus by Jason Roberts

The global withdrawal of the Sabin type 2 poliovirus vaccine is a test of the feasibility of the plan, declared by the World Health Assembly in 1988, to eradicate all polioviruses.

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By David Tuller, DrPH

Cochrane–formerly called the Cochrane Collaboration–is respected worldwide for its systematic reviews of medical treatments. These reviews are often cited as the definitive source of information about treatment efficacy and safety. In taking on the thankless task of assessing the data on commonly used interventions, Cochrane performs an invaluable public health service and has advanced the cause of evidence-based decision-making in medicine.

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The TWiV team considers whether those who can do, can’t teach, and newly discovered viruses of planarians and Aplysia with the largest RNA genomes.

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