Using CRISPR to cut HIV out of infected cells

[Ygggdrasil]

A major impediment to curing patients with HIV is the fact that the virus integrates its DNA into the genome of the cells it infects. While antiretroviral drugs and therapies can prevent infection of new cells and kill cells that are actively producing virus, a "latent reservoir" remains that can cause an active infection to re-emerge after drug treatment has stopped. Therefore, current treatments for HIV-infected individuals requires them to take antiretroviral drugs for their entire lives.

One approach that various groups are exploring to attempt to rid the body of these latent reservoirs is to use https://www.physicsforums.com/insights/dont-fear-https://www.physicsforums.com/insights/dont-fear-crispr-new-gene-editing-technologies-wont-lead-designer-babies/-new-gene-editing-technologies-wont-lead-designer-babies/ to cut the virus out of infected cells. Recently, researchers published a study in which they performed a proof-of-principle experiment showing that they could use CRISPR to cut HIV out of infected mice (mice cannot normally be infected with HIV, a human virus, so the researchers used mice that had been engineered to carry humanized cells that are susceptible to HIV infection):

Yin et al. In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models. Mol Ther. 25 (2017). Published online 30 March 2017. doi:10.1016/j.ymthe.2017.03.012

See also the accompanying commentary article: http://www.sciencedirect.com/science/article/pii/S1525001617301703

The work builds off of previous studies by the same group showing that the virus could be cut out of human cells in the laboratory. The researchers also published a http://time.com/4340722/hiv-removed-using-smaller-scale demonstration in 2016. Their study engineers a virus (the normally non-pathogenic adeno-associated virus) to deliver CRISPR throughout the body. They are able to detect successful cutting of the virus at various sites in the infected mice's bodies.

Still, much more work needs to be done before this work could be applied to AIDS patients. Curing patients of HIV infection is a very difficult task as it requires eliminating all latent reservoirs. While the researchers show that they can detect successful cutting of the latent provirus in various cell types, they do not show evidence that their therapy removes all proviruses or that their delivery system can target all cells that may be harboring the latent HIV reservoirs. It will likely be very difficult for the researchers' method to eliminate 100% of the virus, and testing the ability of their delivery method to target all infected cells will be particularly difficult as mouse models of HIV may not harbor the same types of reservoirs present in humans. Still, the research represents an important step forward in developing CRISPR technology into a potential cure for HIV.

(Thanks to @mfb to pointing the study out to me)

 

[Drakkith]

It will likely be very difficult for the researchers' method to eliminate 100% of the virus

You can say that again. With a mouthful of peanut butter. "Very difficult" is likely a severe understatement.

 

[BillTre]

Pretty cool in many ways!

I'm not sure about how HIV works;
1) If you cut out all the genome inserted copies, will the cytoplasmic materials be able to maintain its infection in the cells?
2) Or will it get used up and the cellular infection reduce to nothing?
So, do you have to use both the drugs and the Crispr or can you just use the Crispr (over longer term)?

Does HIV really infect all the cells (or maybe cell types) in the body?

How many cell types does the virus infect?

 

[Drakkith]

Does HIV really infect all the cells (or maybe cell types) in the body?

How many cell types does the virus infect?

I believe HIV only infects certain types of immune cells.

From this wiki article:

HIV can infect a variety of cells such as CD4+ helper T-cells and macrophages that express the CD4 molecule on their surface. HIV-1 entry to macrophages and T helper cells is mediated not only through interaction of the virion envelope glycoproteins (gp120) with the CD4 molecule on the target cells but also with its chemokine coreceptors.

So it shouldn't be able to infect liver cells or skin cells and such.

 

[Andy Resnick]

The work builds off of previous studies by the same group showing that the virus could be cut out of human cells in the laboratory. The researchers also published a http://time.com/4340722/hiv-removed-using-smaller-scale demonstration in 2016.

Interesting application. Also interesting- they work with live HIV in the lab. I once had a tour of a BSL-3 lab (anthrax). No thanks...

 

[Ygggdrasil]

You can say that again. With a mouthful of peanut butter. "Very difficult" is likely a severe understatement.

Yes, that's the reason many are skeptical that such an approach could actually cure an individual of HIV.

I'm not sure about how HIV works;
1) If you cut out all the genome inserted copies, will the cytoplasmic materials be able to maintain its infection in the cells?
2) Or will it get used up and the cellular infection reduce to nothing?
So, do you have to use both the drugs and the Crispr or can you just use the Crispr (over longer term)?

As you suggest, the CRISPR approach would likely have to be combined with antiretroviral drugs. The drugs would deal with cells actively producing virus and prevent infection of new cells while the CRISPR approach would hopefully deal with latently infected cells.

Does HIV really infect all the cells (or maybe cell types) in the body?

How many cell types does the virus infect?

As @Drakkith mentioned, HIV mainly infects immune cells (specifically, HIV latches onto the CD4 protein to gain entry into the cell, so it only infects cells expressing CD4 and other required co-receptor proteins). However, immune cells reside in many locations in the body, so the CRISPR delivery method would need to access these cells all throughout the body.

 

[BillTre]

As @Drakkith mentioned, HIV mainly infects immune cells (specifically, HIV latches onto the CD4 protein to gain entry into the cell, so it only infects cells expressing CD4 and other required co-receptor proteins). However, immune cells reside in many locations in the body, so the CRISPR delivery method would need to access these cells all throughout the body.

That's what I thought.
I guess I was confused by one of the links saying:
"Khalili and his team engineered the animals to incorporate specific HIV genes into nearly every cell in their body, from the brain, heart, liver, kidney lungs and spleen to their blood cells.".
But that just the test animals.
HIV in many cell types would present greater problems for virus targeting and removal.

 

[Ygggdrasil]

That's what I thought.
I guess I was confused by one of the links saying:
"Khalili and his team engineered the animals to incorporate specific HIV genes into nearly every cell in their body, from the brain, heart, liver, kidney lungs and spleen to their blood cells.".
But that just the test animals.
HIV in many cell types would present greater problems for virus targeting and removal.

Yes, the mouse experiments use fairly artificial systems for studying HIV (because mice don't normally get infected by HIV and one would not want to do initial studies in humans or related primates). Most other studies employ lentiviruses to deliver the CRISPR system as HIV is also a lentivirus and would be expected to show similar tropism. Whether the AAV vectors they developed can recapitulate HIV's tropism is still unclear and would need to be addressed before this approach could be applied in more practical studies.

 

[BillTre]

They seemed vague about their success percentage-wise of removing HIV (guess that's the point of @Drakkith's comment).

Are those numbers known?

 

[Ygggdrasil]

They seemed vague about their success percentage-wise of removing HIV (guess that's the point of @Drakkith's comment).

Are those numbers known?

Not really. The authors mostly use qualitative assays to detect provirus cleavage, but don't do much to quantify the fraction of intact provirus remaining. Probably one of the reasons why this study was not published in a higher impact journal.

 

[DrDu]

Interesting application. Also interesting- they work with live HIV in the lab. I once had a tour of a BSL-3 lab (anthrax). No thanks...

HIV is level 2 and relatively harmless, as skin contact or inhalation won't usually lead to infection. I once worked in the development for test for HIV and Hepatitis and also had access to the labs. Working with Hepatitis B is much more risky.
Somewhere at home I have a book on how to set up a bacteriology lab for medical students from the beginning of last century.
It is recommended that the experiments with antrax are only to be performed by experienced students and that the wooden board on which experiments are to be performed are burnt afterwards :-)

 

[Andy Resnick]

HIV is level 2 and relatively harmless, as skin contact or inhalation won't usually lead to infection. I once worked in the development for test for HIV and Hepatitis and also had access to the labs. Working with Hepatitis B is much more risky.
Somewhere at home I have a book on how to set up a bacteriology lab for medical students from the beginning of last century.
It is recommended that the experiments with antrax are only to be performed by experienced students and that the wooden board on which experiments are to be performed are burnt afterwards :-)

Yeah, we also stopped mouth pipetting a while ago :)

One thing my students learn during their training in cell culture- the air we breathe is *filthy*. Full of bacteria, yeast, mycoplasma, mold spores... you name it, you breathe it in with every breath. One gains considerable experience in microbiology while doing cell culture.

 

[Mark Harder]

On the one hand, you want to be certain that HIV sequences are excised from 100% of genomes containing them. On the other hand, you need to prevent expressable DNA from even one cell from being excised if it's not an essential HIV gene. If one integrated HIV genome is ever expressed, a multitude of virions will be produced and infect other cells and a new infection will result. If one cell suffers a genetic disruption caused by mis-targeted CRISPR, mightn't a cancer be the result? That's the problem with biology. A single elementary event can have compounded consequences.

 

[mfb]

On the other hand, you need to prevent expressable DNA from even one cell from being excised if it's not an essential HIV gene.

A bit of collateral damage can be tolerable. It can lead to a slightly higher risk of cancer, but if you can cure HIV that way that is perfectly fine. An airplane flight gives you a slightly higher risk of cancer as well, and people use it just to get from A to B.

 

[Mark Harder]

A bit of collateral damage can be tolerable. It can lead to a slightly higher risk of cancer, but if you can cure HIV that way that is perfectly fine. An airplane flight gives you a slightly higher risk of cancer as well, and people use it just to get from A to B.

Quite true. A quantitative risk-benefit analysis must be done. That requires data which we don't have yet.

 

[Borg]

And now for the beginning of designer babies...
https://www.engadget.com/2018/11/26/chinese-https://www.physicsforums.com/insights/dont-fear-crispr-new-gene-editing-technologies-wont-lead-designer-babies/-edited-babies/.

A Chinese scientist claims to have created the world's first genetically-edited babies using the CRISPR/Cas9 tool. He Jiankui told the Associated Press that twin girls, Lulu and Nana, were born earlier this month following embryo-editing using CRISPR to disable the CCR5 gene, which allows the HIV virus to infect cells. An American scientist, Michael Deem, also reportedly assisted He on the project at the Southern University of Science and Technology of China.

Caveat...

Notably, He's work has not been published in a peer-reviewed scientific journal, meaning there's no independent confirmation of the research.

 

[Buzz Bloom]

https://www.engadget.com/2018/11/29/china-halts-gene-edited-baby-research/​

He's work has been met with severe backlash as scientists around the world have called it irresponsible and unethical while emphasizing that https://www.engadget.com/2018/01/18/first-human-https://www.physicsforums.com/insights/dont-fear-crispr-new-gene-editing-technologies-wont-lead-designer-babies/-study-in-the-us-could-begin-soon/ is not yet ready for human embryos since associated risks are not fully understood.

Other reports.

https://www.bbc.com/news/world-asia-china-46382662
https://www.nature.com/articles/d41586-018-07545-0​

 

[Laroxe]

Just to add to the complications.

Apparently its far more adaptable than being totally reliant on the CD4 receptor;

Many cell types share common epitopes with this protein, though CD4 lymphocytes play a crucial role. In macrophages and in some other cells lacking CD4 receptors, such as fibroblasts, an Fc receptor site or complement receptor site may be used instead for entry of HIV. Cells of the mononuclear phagocyte system, principally blood monocytes and tissue macrophages, T lymphocytes, B lymphocytes, natural killer (NK) lymphocytes, dendritic cells (Langerhans cells of epithelia and follicular dendritic cells in lymph nodes), hematopoietic stem cells, endothelial cells, microglial cells in brain, and gastrointestinal epithelial cells are the primary targets of HIV infection.

https://library.med.utah.edu/WebPath/TUTORIAL/AIDS/HIV.html

Then add, Human Arterial Smooth Muscle Cells
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2276423/

 

[Eagle9]

Hello guys

Although it is offtopic, but could you please tell me which genetic engineering technology is most suitable for transferring genes from one animal species to another (transgenic animals)? I thought that it was CRISPR/Cas9, but I was told that this technology is mainly used for gene editing. So, which technology is most suitable for transferring genes? Which book would you recommend me for this purpose?

 

[Ygggdrasil]

Here's a decently good starting point: https://blog.addgene.org/mouse-modeling-part-1-genetically-engineered-mice