
Elimination of HIV-1 Genomes by CRISPR/Cas9 Gene Editing - abetusk
http://www.nature.com/articles/srep22555
======
noname123
Reading the methods section, it looks the T-cells infected with HIV-1 that are
used for targeted for gene editing came from a cell line and incubated in a
cultured medium.

For people who are involved in dealing with CRISPR/Cas9, what is the effective
rate of gene editing done in vitro vs. in vivo?

Would the molecular machinery of CRISPR/Cas9 encounter any natural defense
mechanism of the human immune system when attempting to mutate target sites on
T-cells in say, lymph nodes of adult humans (contrasting to changing DNA in
the germ-lines of bacteria or even human embryo's)?

~~~
carbocation
> For people who are involved in dealing with CRISPR/Cas9, what is the
> effective rate of gene editing done in vitro vs. in vivo?

It will depend in part on the tissue of interest and the delivery vector. In
the mouse liver, the Broad group under Zhang has gotten efficiencies as high
as 40% using Staphylococcal Cas9 [1].

1 = [http://www.nature.com.sci-
hub.io/nature/journal/v520/n7546/f...](http://www.nature.com.sci-
hub.io/nature/journal/v520/n7546/full/nature14299.html)

~~~
itcmcgrath
I found this + the op's comments fascinating purely because I wouldn't have
been able to tell if both were real, or just fake technical talk of the ilk my
friends and I sometimes do with meaningless software/hardware jargon as a
joke, without Googling.

~~~
noname123
Well, for the record, I am a programmer working at the Broad, a genomics
center that is mired in the current CRISPR patent dispute (although I don't
work with CRISPR at all).

The answerer I believe is a MD/PhD at Mass General Hospital and before that a
research scientist at the Broad working on genomics (and so is the author of
the cited paper on mouse liver, small world here on HN). So I can be full of
BS, but I don't think the answerer is.

Tbh, I intentionally added a few more jargons more than necessary (I could
have just asked "Can gene editing be effective in live humans T-cells vs.
cells on a petri dish?") to field my question; but I intentionally did so to
filter out the pop-sci, speculative futurists to get answers from people in
life sciences research.

------
cryoshon
I was in HIV research for a time. I was unimpressed by this paper's title,
introduction, methods, etc, until the part where they discussed correcting
infected cells derived from human donors. As a general note, a running joke in
the field is that "anyone can cure HIV in vitro", so I practically tuned out
the sections where they discussed the work in T-cell cultures.

>Lentivirus (LV) mediated Cas9/gRNA delivery suppresses HIV-1 infection in
human T-cells.

That should be the actual headline of this paper. Yes, it's a big deal that
researchers were able to use a lentiviral vector (HIV without the disease
causing bits) to genetically alter infected CD4 T-cells. This is a shot at a
cure.

Simple infection of a person with the CRISPR-fortified lentiviral vector is a
new therapy idea which has the potential to trump the current antiretroviral
therapies because it can reduce or remove the HIV "viral reservoir"\-- the
places in the body where HIV can hang out and survive even when being
suppressed by antiretroviral therapy. I can imagine a therapy in which the
CRISPR-vector is injected into patient lymph nodes and other similar sites
with the intent of curing HIV for good. Won't be cheap for another decade,
though.

------
medymed
Seems like it could be effective to use CRISPR/Cas9 to insert the HIV
resistance gene mutation (CCR5-delta32, to replace the normal CCR5 gene) into
bone marrow stem cells and then inject those into the person after testing the
created cells to be HIV negative (or cutting the HIV out by means in this
paper). If HIV negative bone marrow cells available were sufficient to do this
it could be a 'cure' for the hematopoietic system. Or use HIV-negative induced
pluripotent stem (IPS) cell with hematopoietic potential derived from a tissue
not normally infected by HIV.

Such cells in principle would have a survival advantage over native cells and
would effectively perform a bone marrow transplant, maybe helped along by some
medicinal guidance. Pies in the sky, but using HIV-negative cells may avoid
the problem of removing divergent sequences of HIV in different people and
ineffective gDNAs.

------
benjismith
Awesome.

I've been avidly following the CRISPR/Cas9 developments over the past five
years or so, and the possibilities seem astonishing. I can't wait to see where
this all leads!

~~~
azinman2
Some cool stuff combined with very scary stuff, like eugenics and large scale
food chain modifications with unpredictable effects (already being planned
with mosquitos).

~~~
duaneb
> eugenics

Well, in this case CRISPR wouldn't make much sense for _negative_
eugenics—i.e. trimming the gene pool—vs _positive_ eugenics—purposely
introducing or manipulating existing genes.

While the latter is still scary, it's not exactly close to the mass
sterilization people think of.

~~~
azinman2
Negative eugenics is currently being planned for deployment to control which
mosquitos are able to reproduce.

[http://www.nature.com/nbt/journal/v34/n1/full/nbt.3439.html](http://www.nature.com/nbt/journal/v34/n1/full/nbt.3439.html)

[https://medium.com/mit-media-lab/kevin-esvelt-joins-the-
medi...](https://medium.com/mit-media-lab/kevin-esvelt-joins-the-media-
lab-3e33fe24f90#.y2qfqy39w)

~~~
afarrell
Which I applaud for the same reason I applaud some of the extinctions we have
caused.

~~~
ktRolster
Which extinction do you applaud?

~~~
spb
I can't help but feel like life is easier without sabertoothed tigers roaming
the streets

~~~
Dylan16807
There are non-extinct big scary cats. They don't cause much trouble. We'd be
doing fine without that extinction.

------
zymhan
Apparently this doesn't only diminish the infection, but can supposedly confer
resistance?

"When evaluating a therapeutic strategy based on CRISPR/Cas9, it is critical
to understand that not only will HIV-1 be eliminated from latently infected
cells, but the majority of uninfected cells will become resistant to HIV
infection. Thus, there is a high likelihood that rebounding viral infections
will be contained by the resistant cells."

~~~
L_Rahman
This confused me as well. Based on my skimming, the paper doesn't speculate on
why this appears to be true, but it's very interesting that the researchers
observed this phenomenon at all.

------
coryfklein
It has been common experience for me to check my internet news feed and see
"the cure" for HIV/AIDS/Cancer/Diabetes fairly regularly.

This article does not appear to claim to be "a cure", but does seem promising.
Can anybody comment on the significance of this and why the layman should care
about it?

~~~
azurelogic
Most HIV treatments center around suppressing the virus. For example, many
antivirals used in treatment interfere with viral reproduction. These people
are never cured, because part of what HIV does is it inserts itself into the
DNA of the infected individual's cells.

The technique proposed in this article would be revolutionary because it
suggests that we may eventually be able to use CRISPR to selectively cut that
HIV DNA out of infected cells, leading to a permanent cure, if 100%
eradication of HIV DNA can be achieved. Unfortunately, doing it in a petri
dish is much different than doing it in vivo, particularly with 100%
eradication.

~~~
x0x0
if we could create a reasonable number of healthy (uninfected) t-cells, would
an individual be left infectious but otherwise asymptomatic (because of the
retention of a working immune system)?

~~~
azurelogic
This would only make sense if you could make the uninfected t-cells totally
resistant to infection. What this paper suggests is that even this would be
unnecessary. The combination of antiretrovirals and CRISPR therapy would
prevent replication and excise the embedded viral sources. With enough of this
combined therapy, eradication may be obtainable, resulting in a properly cured
patient.

------
nonbel
Figure 1D is said to show "Detection of Cas9 protein by Western blot
analysis", so what does "bp" stand for in that figure (above the ladder)?
Usually it stands for base pair but that doesn't make sense when looking at
protein.

~~~
cryoshon
Base pairs. I don't know the exact math and haven't ever done Westerns, but
there's almost certainly a way of converting protein size (typically expressed
in kDa) to base pairs, since theoretically a large enough quantity of bp would
weigh as much as a protein of a given size.

The trick here is to understand that Western blots use gel electrophoresis to
detect protein size, meaning that heavier proteins will not traverse as far as
lighter proteins.

~~~
kyberias
Come on. Having actually done Westerns, there is absolutely zero chance that
the "bp"=base pair in that figure is on purpose. Western blot is a protein
blot and they are always measured by their molecular weight (kDa). Base pairs
would make no sense here. The numbers in the image are obviously kilo daltons.

How do we know the numbers are kilo daltons? Well, they say they're using
β-tubulin as a loading control. β-tubulin's molecular weight is about 50kDa
and that's what you can see in the image.

Also, look how the headers are not aligned properly in the image. As if the
image is a draft.

~~~
cybersiddhu
The bp==base pair stands correct. It refers to Fig 2C, that shows the display
of RT-PCR products(DNA made from RNA).

------
ogreveins
So it cuts the genomes out, does it keep the cells alive while doing so? I
heard that CRISPR/Cas9 doesn't rejoin the dna it cuts. I'm new to this and
would love to know from someone more knowledgeable about this.

~~~
jameskraus
Cells are always poised to repair DNA breaks. For example: over your lifetime,
your cells will have repaired thousands of DNA breaks due to gamma radiation.
The breaks due to CRISPR/Cas9 are similar to the breaks caused by gamma
radiation and likely to be repaired very rapidly. If the DNA break is not
resolved, there are other biochemical pathways to lead to apoptosis
(programmed cell suicide).

------
giarc
"...potentially serve as a novel and effective platform toward curing AIDS."

Researchers are usually quite conservative and the media are usually the ones
to sensationalize titles. Given the above quote, this is likely a major step
forward.

~~~
mikexstudios
No, that's standard boilerplate these days in papers. You have to "sell" the
science a bit to reviewers and connect your work to the broader picture. Those
in the field usually ignore statements like this.

~~~
fluxquanta
When I was a research assistant in college I was encouraged for one of my
presentations to emphasize safe hydrogen storage as a vehicle fuel source,
when really the research was all about how minuscule amounts of hydrogen
change the electrical/optical properties of thin metallic films (and
desorption was on the order of days). Selling it as a building block for safer
hydrogen storage was necessary to get people engaged.

~~~
refurb
Yup! I published a paper on organoselenium chemistry and was asked to add
commentary about potential "anti-cancer" properties.

Sure my work could be extended in that direction, but I had done zero work to
pursue it.

------
gravypod
How did they narrow down what section of DNA is removed? That sounds to me
like the really hard problem here.

I'm actually really interested in that kind of work.

~~~
kyberias
Knowing the exact location in the genome, they amplified the target sequence
from the DNA (using PCR) and sequenced it verifying the modification.

------
jcoffland
If this pans out I hope there won't be any nonsense where some pharma company
charges ~$80k for a full treatment like with the hep C cure.

~~~
roywiggins
$80k for an HIV cure is cheap compared to a lifetime of HIV treatment.

"The most recent published estimate of lifetime HIV treatment costs was
$367,134 (in 2009 dollars; $379,668 in 2010 dollars)."

[http://www.cdc.gov/hiv/programresources/guidance/costeffecti...](http://www.cdc.gov/hiv/programresources/guidance/costeffectiveness/index.html)

~~~
ascorbic
And so for that matter are the hep C treatments. They're that rare thing: an
effective cure for a serious, chronic disease that was previously only
treatable with long term medication with many nasty side effects.

------
atmosx
Thats the best news I've had in a long time. Great time to be a molecular
biologist.

------
jacobsimon
!!! Amazing

------
return0
If it does what it says on the box, it would be in nature, not scientific
reports.

