New CRISPR discovery - may be more useful

[mcmich]

https://futurism.com/the-new-cas9-scientists-discover-casx-and-casy-enxymes-in-bacteria/

[Renegadow]

On 12/29/2016 at 3:12 AM, mcmich said:

https://futurism.com/the-new-cas9-scientists-discover-casx-and-casy-enxymes-in-bacteria/

thanks for remind us the CRISPR potential

[Evaluate]

Thanks for sharing.  I think it's too early to say if it will affect HSV research at all.  The issues that CRISPR/Cas 9 must overcome in the near future are to do with the vector, which newly discovered CasX and Y would still use.

[mcmich]

True...but since the size of CasX and CasY are much smaller then it may be able to reach many more places. Also, I remember reading about a way to trigger HSV to replicate, thus making the CAS able to target the replicating cells and eliminating the virus.

I think the way to make people non-infectious would be a triple therapy approach (similar to HIV). Drugs, vaccine, and CRISPR combined to make it virtually impossible to infect someone. Also, there are broad spectrum antivirals in works (currently not being in drug trials for HSV - such as CMX-001) that would work with the current drugs reducing shedding further. I had been looking forward to CMX-001 to pass phase 3 and then try to get it off label.

http://aac.asm.org/content/55/10/4728.full

[Evaluate]

42 minutes ago, mcmich said:

Also, I remember reading about a way to trigger HSV to replicate, thus making the CAS able to target the replicating cells and eliminating the virus.

There are a few ways to force HSV out of latency, one of them being Protesome-26 inhibitors.  The issue with that is the risk of neuron death.  There might be ways to reduce the damage such as use with antivirals, effective vaccines etc, but damage may result still.

[Renegadow]

 

Sort of update/informative post:

 

 

 

 

COMMERCIALIZING CRISPR CURES

Biotech firms and partnerships have proliferated since the discovery of the gene-editing system.

Company	Selected conditions	Delivery strategy
Casebia Therapeutics	Severe combined immunodeficiency	Ex vivo, no details disclosed
	Hemophilia A, ear and eye disorders, congenital heart disease	In vivo, no details disclosed
CRISPR Therapeutics	Glycogen storage disease Ia, Duchenne muscular dystrophy, Hurler syndrome (MPS-1)	In vivo, no details disclosed
CRISPR Therapeutics and Vertex Pharmaceuticals	β-thalassemia, sickle cell disease	Ex vivo, no details disclosed
	Cystic fibrosis	In vivo, no details disclosed
Editas Medicine	Leber congenital amaurosis 10 (LCA10)	AAV injection into eye
	β-thalassemia, sickle cell disease	Ex vivo electroporation with ribonucleoproteins
	Duchenne muscular dystrophy, cystic fibrosis, α-I antitrypsin deficiency	AAV or lipid nanoparticles
Editas Medicine and Juno Therapeutics	CAR-T cells for cancer	Ex vivo electroporation with ribonucleoproteins
GenEdit	None disclosed	Gold nanoparticles, lipid and polymer nanoparticles with ribonucleoprotein
Intellia Therapeutics and Regeneron Pharmaceuticals	Transthyretin amyloidosis	Lipid nanoparticle with Cas9 mRNA
Intellia Therapeutics and Novartis	Hematopoietic stem cell (HPSC) transplant, CAR-T cells for cancer	Ex vivo electroporation
Locus Biosciences	Carbapenem resistant Enterobacteriaceae	Guide RNAs delivered in bacteriophage, Cas proteins already in bacteria

AAV = adeno-associated virus. CAR = chimeric antigen receptor. Sources: Company websites, presentations, and interviews

---

HOW TO DELIVER CRISPR

Researchers are focused primarily on three ways to make a therapeutic agent out of CRISPR, in which a guide RNA directs the Cas9 enzyme to a specific location in DNA for precise editing.

	DNA	RNA	Protein-RNA complex
Delivery method in brief	Deliver both Cas9 and guide RNA in a DNA plasmid	Deliver Cas9 as mRNA alongside guide RNA	Deliver Cas9 protein alongside guide RNA
Most common packaging	AAV: Adeno-associated virus	Various kinds of lipid and polymer nanoparticles	Ribonucleoprotein (Cas9 and guide RNA)
Amount of Cas9 protein delivered to cells	High, indefinite production of Cas9	Medium, short-lived production of Cas9	Precise dose of Cas9 delivered
Advantages	Established gene delivery system and potential for delivery to specific tissues	Improved release of mRNA inside cells and potential for repeat dosing if needed	Lower risk of off-target gene edits and potential for easier manufacturing
Liabilities	Higher risk of off-target cutting	Potential for unexplained toxicities	Difficulty delivering in vivo and potential immune response, especially if repeat dosing is needed
Tissues targeted (as reported in animals)	Localized injection into most tissues (including brain and muscle) and systemic injection to liver	Systemic injection primarily to liver and spleen	Primarily blood, immune, and stem cells removed from the body; localized electroporation to skin

Sources: Company websites, presentations, and interviews

Protein power

 

Lipid nanoparticle innovation may be blossoming, and CRISPR developers are confident they can reach the clinic more quickly and safely than with RNAi, but that delivery vessel is by no means foolproof.

Rodger Novak, chief executive officer of CRISPR Therapeutics, whose founders include another of CRISPR’s co-inventors, Emmanuelle Charpentier, points out that “CRISPR has an advantage” over RNAi, which turns down protein production only temporarily and needs to be readministered periodically. Those repeat injections can cause liver toxicity, a side effect that has slowed down the initially rapid progress of RNAi companies. Although the technology is maturing, there are no approved RNAi drugs.

“The whole lipid nanoparticle field is a little bit weird,” Ross Wilson of UC Berkeley says. The literature is full of “success stories that are never followed up on; they just fizzle.”

Wilson is one of several researchers working on delivering Cas9 as a protein rather than as mRNA in a lipid nanoparticle or as DNA in a virus. Researchers call this form of CRISPR a ribonucleoprotein, which is the active form of the guide RNA hooked up to the Cas9 enzyme in a single, ready-to-go complex.

David Liu of Harvard University says delivering CRISPR in a virus gives “the least amount of control” because it manufactures the Cas9 protein indefinitely. If there are too many Cas9 enzymes in a cell, there is a greater chance that one of them may accidentally cut DNA in the wrong place. Directly delivering the protein gives “the most control” because lower levels of Cas9 in each cell means a lower risk of potentially dangerous off-target cutting, Liu says. His group developed cationic lipid nanoparticles for CRISPR ribonucleoprotein delivery (Nat. Biotechnol. 2015, DOI: 10.1038/nbt.3081).

Liu, along with Qiaobing Xu of Tufts University, also created lipid nanoparticles that are biodegradable inside cells. The system binds negatively charged CRISPR ribonucleoproteins initially, but releases them upon entering the chemically reducing environment of the cell (Proc. Natl. Acad. Sci. USA 2016, DOI: 10.1073/pnas.1520244113).

Wilson is looking to find a way to deliver CRISPR ribonucleoproteins without the hassle of lipid nanoparticles. To do that, he needs to make the ribonucleoprotein complex stable in the bloodstream, able to escape the cell’s endosome, and even able to home in on a particular tissue type. But there is a downside. “The immunogenicity of Cas9 could be a real issue,” Wilson says.

Other scientists are crafting even more exotic delivery systems for CRISPR, including a yarn ball-like structure called a DNA nanoclew developed by Chase Beisel and Zhen Gu of North Carolina State University. Their nanoclew uses repeated stretches of DNA complementary to the guide RNA wrapped up in a ball to deliver Cas9 protein to cells. (Angew. Chem. Int. Ed. 2015, DOI: 10.1002/anie.201506030).

Remaining challenges

 

Even as the field works out the delivery kinks, therapies are expected to soon reach people. Clinical trials using ex vivo gene editing in humans with CRISPR is anticipated to start in the U.S. this year, with in vivo gene editing likely in 2018 and 2019.

Casebia Therapeutics, a joint venture between Bayer and CRISPR Therapeutics, is making its commitment to the delivery challenge clear, with plans to hire a head of delivery. James Burns, CEO and president of Casebia, says, “There are some approaches that we can take now, but to really harness or achieve CRISPR’s full potential, we are going to have to invest in new delivery technologies.” Currently, most CRISPR-based companies are taking an agnostic, “whatever works” approach, testing both AAV and lipid nanoparticles for their first rounds of treatment.

Berkeley’s Corn points out another problem with CRISPR that many people conveniently gloss over. “We are really good at breaking sequences and not really good at fixing them,” he says. Some conditions can be cured using Cas9 to cut out a mutation or turn a gene off. But there are many more conditions where faulty DNA needs actual correcting. That requires a third component: a DNA template strand to tell the cell’s repair machinery how to fill in a cut made by Cas9.

“Delivering all three has been really challenging and it has not been demonstrated in in vivo systems with any lipid nanoparticles yet,” says Kunwoo Lee, who is now CEO of the start-up GenEdit that he founded in February 2016 shortly before finishing his Ph.D. in Murthy’s lab at Berkeley. GenEdit focuses on applications of CRISPR that will require a DNA template for repair.

Lee and his colleagues at GenEdit already have a few scientific studies under review, including one that uses gold nanoparticles as a core material to load the three components of the CRISPR system. They are also working on lipid and polymer nanoparticle systems, all designed to deliver CRISPR ribonucleoproteins. Although that strategy is promising for minimizing off-target cutting, it may also be the furthest away from being an injectable treatment in the clinic.

“There is simply no way that any particular delivery modality is going to provide the means to address all of those targets, so it really needs to be an all-of-the-above approach,” says Erik Sontheimer of the RNA Therapeutics Institute at the University of Massachusetts Medical School. And from the looks of it, the CRISPR companies are approaching it as such.

“I’ve heard many people say buckle up because there will be a trough of disillusionment that has to be traversed before it can become a clinical reality,” Sontheimer says. “But the potential payoff is so clear, that there will be enough staying power if and when that comes.” 

https://cen.acs.org/articles/95/i7/CRISPRs-breakthrough-problem.html

 

Edited February 16, 2017 by Renegadow

[Malcolm]

1 hour ago, jolando123 said:

No one would just activate HSV out of latency unless you had something there to stop replication.  You need a book end.  You force it to replicate and then stop it from doing so.  It's then a dead-end and the virus dies off.  This is largely how the hepatitis C antivirals work (though there is no latent reservoir).  In theory you would use those stimulators + therapeutic vaccine + valtrex + pritelivir.

Can you find any evidence that suggest the latent "master copy" of the genome is able to reactivate and exit the nerve cell? As far as I know, the latent genome stays inside the nerve cell's nucleus and reactivation involves producing copies. I hope you're right, and I'm wrong.

Edited February 17, 2017 by Malcolm

[Malcolm]

The problem with Cas9 is not even delivery but the fact that it is too big and clunky to access the latent genome. Cas9 is really only useful for cheap prototyping and finding the role of certain genes cost effectively. It's a programmable protein which comes with overheads in terms of it's size and it's effectiveness. For an actual end-user treatment, you are better off using Zinc-finger Nucleases or Homing Endonucleases, both of which have proven to better access latent genomes which are tightly wrapped around histones. I wanted to pursue ZFNs until I found out a lab in the US has just recently looked at using them (I can't find the name of the lab, it's not Cullen).

Edited February 17, 2017 by Malcolm

[Sanguine108]

yeaaahh, I don't think there will ever be a cure for hsv in one's lifetime.   It's gonna be one of those things where ya got it for the rest of this life.

[Joseph_Esp]

7 minutes ago, Sanguine108 said:

yeaaahh, I don't think there will ever be a cure for hsv in one's lifetime.   It's gonna be one of those things where ya got it for the rest of this life.

Haha really useful comment! 

[Unrequited]

19 hours ago, Malcolm said:

The problem with Cas9 is not even delivery but the fact that it is too big and clunky to access the latent genome. Cas9 is really only useful for cheap prototyping and finding the role of certain genes cost effectively. It's a programmable protein which comes with overheads in terms of it's size and it's effectiveness. For an actual end-user treatment, you are better off using Zinc-finger Nucleases or Homing Endonucleases, both of which have proven to better access latent genomes which are tightly wrapped around histones. I wanted to pursue ZFNs until I found out a lab in the US has just recently looked at using them (I can't find the name of the lab, it's not Cullen).

Fred Hutch - Jerome Lab

http://research.fhcrc.org/jerome/en/members.html

[Unrequited]

Just now, Unrequited said:

Fred Hutch - Jerome Lab

http://research.fhcrc.org/jerome/en/members.html

They are hitting about 5-10% removal of latent HSV virus in vitro - When they break it, they'll make it... boom! 

 

[Unrequited]

On 31/12/2016 at 9:08 PM, mcmich said:

True...but since the size of CasX and CasY are much smaller then it may be able to reach many more places. Also, I remember reading about a way to trigger HSV to replicate, thus making the CAS able to target the replicating cells and eliminating the virus.

I think the way to make people non-infectious would be a triple therapy approach (similar to HIV). Drugs, vaccine, and CRISPR combined to make it virtually impossible to infect someone. Also, there are broad spectrum antivirals in works (currently not being in drug trials for HSV - such as CMX-001) that would work with the current drugs reducing shedding further. I had been looking forward to CMX-001 to pass phase 3 and then try to get it off label.

http://aac.asm.org/content/55/10/4728.full

http://aac.asm.org/content/55/10/4728/T2.expansion.html

Pity it talks about increased efficacy but doesn't say what that is in terms of percentage..  Although, the data based on

ACV, 30; CMX 2.5 mg/kg

was 4/15 in terms of mortality. However, as you will see CMX by itself did not perform very well at all.

 

[Cas9]

20 hours ago, Malcolm said:

The problem with Cas9 is not even delivery but the fact that it is too big and clunky to access the latent genome. Cas9 is really only useful for cheap prototyping and finding the role of certain genes cost effectively.

I beg your pardon! 

[Renegadow]

Non HSV

but  CRISPR delivery news

 

AsianScientist (Feb. 22, 2017) -

South Korean researchers have used CRISPR-Cas9 gene editing to treat symptoms of age-related macular degeneration (AMD) in mice. Their findings have been published in Genome Research. It is estimated that almost one in every ten people over 65 has some signs of AMD, and its prevalence is likely to increase as a consequence of the aging population. AMD is a form of blindness which causes distorted vision and blind spots. AMD in older adults and retinopathy of prematurity in newborns are the leading cause of blindness in those respective age groups. In both diseases, abnormally high levels of vascular endothelial growth factor (VEGF) are secreted.

In AMD, VEGF causes the formation of new blood vessels in the eyes but also leads to leakages of blood and fluid into the eye, damaging an area at the center of the retina called macula. Injections of anti-VEGF drugs are the most common treatment for AMD, but at least seven injections per year are required because VEGF is continuously overexpressed by the cells of the diseased retinal pigment epithelium. Instead of such invasive treatments, scientists at the Institute for Basic Science (IBS) believe that gene therapy with the third generation gene editing tool CRISPR-Cas9 could improve the situation. “The injections tackle the effects, but not the main cause of the problem.

By editing the VEGF gene, we can achieve a longer-term cure,” explained Professor Kim Jin-Soo, Director of the Center for Genome Engineering at IBS. CRISPR-Cas9 can precisely cut and correct DNA at the desired site in the genome. The CRISPR-Cas9 system works by cutting DNA at a target site, in this case, inside the VEGF gene. Two year ago, IBS scientists proved that a pre-assembled version of CRISPR-Cas9 called Cas9 ribonucleoprotein (RNP) can be delivered to cells and stem cells to modify target genes. The pre-assembled complex works rapidly and degrades before the body has time to build up an immune response against it. Despite these advantages and previous successes, the difficulty in delivering pre-assembled CRISPR-Cas9 has limited its use in therapeutic applications.

 

In this study, the research team successfully injected CRISPR-Cas9 into the eyes of a mouse model with wet AMD and locally modified the VEGF gene. Initially, they found that the delivery of the pre-assembled CRISPR-Cas9 complex is more efficient that the delivery of the same components in a plasmid form. Secondly, the complex disappeared after just 72 hours. Scientists assessed the whole genome of the animals and found the CRISPR-Cas9 complex modified only the VEGF gene and did not affect other genes. The progression of the eye disease was monitored by looking at choroidal neovascularization (CNV), the creation of new blood vessels between the retina and the sclera, a common problem of ‘wet’ macular degeneration.

The researchers found that the CRISPR-Cas9 complex reduced the CNV area by 58 percent. Moreover, cone dysfunction, a likely side effect that takes only that days to show in mice, did not occur a week after the treatment. “We have developed a treatment to suppress CNV by inactivating the VEGF gene, one of the causes of AMD. We envision that, in the future, surgeons will be able to cut and paste disease-causing genetic elements in patients,” explained Kim. While CRISPR-Cas9 is conventionally used to correct mutations causing hereditary diseases or cancer, this study suggests a new therapy for non-hereditary degenerative disease.

“We believe that this is a new therapeutic modality for the treatment of non-hereditary degenerative diseases,” said study co-author Professor Kim Jeong Hun from Seoul National University. “We confirmed the effect on the animal models of the disease and now we wish to continue with preclinical trials.”

The article can be found at: Kim et al. (2017) Genome Surgery Using Cas9 Ribonucleoproteins for the Treatment of Age-related Macular Degeneration. ——— Source: Institute for Basic Science. Disclaimer: This article does not necessarily reflect the views of AsianScientist or its staff. Read more from Asian Scientist Magazine at:

https://www.asianscientist.com/2017/02/in-the-lab/crispr-age-related-macular-degeneration/ 

Edited February 23, 2017 by Renegadow