New Research Could Fine-Tune the Gene Scissors CRISPR

If you are a follower of this blog, you know I am following CRISPR research closely in regards to a potential cure for Kennedy's Disease (SBMA). If this process could reduce the number of CAG Repeats, we might have the cure. One issue that researchers are looking at is how to identify and minimize the potential side effects. Below is a link to the latest article on the fine tuning of the process.

New Research Could Fine-Tune the Gene Scissors CRISPR

GENETIC RESEARCH

When researchers and doctors use the tool CRISPR to correct genetic errors, it may have side effects on the human genome. Now, researchers from the University of Copenhagen have learned how the molecular machinery behind CRISPR works and thus expect to be able to fine-tune CRISPR and remove the undesired effects.

The introduction of the tool for gene editing, the so-called gene scissors CRISPR, in 2007 was a revolution within medical science and cell biology. But even though the perspectives are great, the launch of CRISPR has been followed by debate, especially focussing on ethical issues and the technology’s degree of accuracy and side effects.

However, in a new study published in the scientific journal Cell researchers from the Novo Nordisk Foundation Center for Protein Research have described how one of the CRISPR technologies, the so-called Cas12a, works – all the way down to the molecular level. This makes it possible to fine-tune the gene-editing process to only achieve the desired effects.

‘If we compare CRISPR to a car engine, what we have done is make a complete 3D map of the engine and thus gained an understanding of how it works. This knowledge will enable us to fine-tune the CRISPR engine and make it work in various ways – as a Formula 1 racer as well as an off-road truck’, says Professor Guillermo Montoya from the Novo Nordisk Foundation Center for Protein Research....

Molecular Film 

The researchers have used a so-called cryo-electron microscope to map the technology. The recently inaugurated cryoEM facility at the University of Copenhagen has established the state-of-the-art technology enabling the researchers to take photographs of the different shapes of the molecule when CRISPR-Cas12a cuts up the DNA strand.

They combined it with a fluorescent microscopy technique called ‘single molecule FRET’ that directly observes the motions of the molecules and the sequence of events for each individual protein.

Among other things, this sequence of events revealed to the researchers that three “pieces” of the CRISPR tools must change form for the DNA to be cut properly.

‘Our new study shows the precise series of events in the genome leading to gene editing. These three “pieces” that change, work like airport security checks. You have to complete all checks and in the right order to proceed’, says Associate Professor Nikos Hatzakis from the Department of Chemistry and the Nano-Science center.

[Click on the link above to read the entire article]

Is ASO a potential treatment for Kennedy’s Disease?

This is a follow up to my November 15, 2017, article, “MDAAnnounces SBMA Research Grant.” The research paper was a little over my head (nothing new for me), so I asked the KDA’s resident biology professor, Ed Meyertholen, to explain what Dr. Lieberman’s research was about. Below is Ed’s summary of the grant. For a short primer, I have included the link to a video on DNA-RNA.

"The grant the Andy Lieberman received was to continue the research on the use of Anti-Sense Oligonucleotides (ASO) as a treatment for Kennedy’s Disease (KD). To best understand how it works, it is important to remember the following:

1. KD is believed to be the result of a misfolded protein, specifically, the protein known as the Androgen Receptor (AR).

2. Proteins are built of specific sequences of amino acids, thus to make a protein, one must have amino acids and the sequence of the amino acids of the protein of interest.

3. The sequence of amino acids for any protein are hard coded into our genes - our DNA.  Thus to make a particular protein, the cell must find the gene that codes for the sequence for that protein and read the code to get the sequence.  The structure of the cell that makes the protein is the ribosome.

4. In KD, the misfolded protein is known as the Androgen Receptor (AR) and it misfolds because our DNA has an error in the sequence.  So, when our cells want to synthesize the AR, our instructions are faulty and when we make the resulting protein, it somehow causes cells to die albeit, slowly.

5. Protein synthesis requires two major steps, the first is the synthesis of an RNA copy (RNA is like DNA) of the gene (DNA) which codes for the protein of interest (this occurs in the nucleus).  The RNA synthesis is known as transcription.

6. The RNA copy (which contains the code for the protein) leaves the nucleus and goes to the ribosome.  Here the code is read and the protein is synthesized.  This actual making of the protein is known as translation.

7.  An ASO is a specially designed fragment of RNA that binds only to a specific RNA.  An ASO can be designed to bind specifically to any given RNA.  In this case, the ASO binds only to the RNA that is used to make the AR.  When the ASO binds to the RNA, the cell responds by destroying the RNA (that is what it does) - thus the RNA to make the AR is destroyed before the protein is made and thus no AR is synthesized and thus, it is hoped, no KD.

8.  Andy's grant is, as I understand it, will try to test this procedure on mice models of KD and involve investigating the best ways to deliver the ASO.  Let me also add, there have been several published studies that have shown that ASO's are effective in preventing KD in mice.  Other ASO's have been developed to treat other diseases and just recently, one was approved for use in a disease called Spinal Muscular Atrophy (this is not KD)."  

US scientists try 1st gene editing in the body

In recent years I have reported several times about CRISPR and gene editing. I am not a scientist, but, to a layman, this sounds like the best possibility for curing Kennedy's Disease. In the article referenced below, it discusses the potential benefits and possible dangers of gene editing.

There is much more research that needs to be done, but the possibility of a cure is now closer than ever before.

US scientists try 1st gene editing in the body

"...This time, the gene tinkering is happening in a precise way inside the body. It’s like sending a mini surgeon along to place the new gene in exactly the right location.

“We cut your DNA, open it up, insert a gene, stitch it back up. Invisible mending,” said Dr. Sandy Macrae, president of Sangamo Therapeutics, the California company testing this for two metabolic diseases and hemophilia. “It becomes part of your DNA and is there for the rest of your life.”

That also means there’s no going back, no way to erase any mistakes the editing might cause.

“You’re really toying with Mother Nature” and the risks can’t be fully known, but the studies should move forward because these are incurable diseases, said one independent expert, Dr. Eric Topol of the Scripps Translational Science Institute in San Diego.

Protections are in place to help ensure safety, and animal tests were very encouraging, said Dr. Howard Kaufman, a Boston scientist on the National Institutes of Health panel that approved the studies.

He said gene editing’s promise is too great to ignore. “So far there’s been no evidence that this is going to be dangerous,” he said. “Now is not the time to get scared.”..."

A short APNews animation video does a good job of explaining the process. 

(AP Animation/Marshall Ritzel)

Gene Editing Followup

A followup article in the New York Times by Pam Belluck dismisses some of the fears people have regarding Gene Editing. Since Huntington’s Disease is mentioned, that is a good sign for those of us with families living with Kennedy’s Disease. To read the entire article, follow the title link.

Gene Editing for ‘Designer Babies’? Highly Unlikely, Scientists Say

 “Now that science is a big step closer to being able to fiddle with the genes of a human embryo, is it time to panic? Could embryo editing spiral out of control, allowing parents to custom-order a baby with Lin-Manuel Miranda’s imagination or Usain Bolt’s speed?

News that an international team of scientists in Oregon had successfully modified the DNA of human embryos has renewed apprehensions that babies will one day be “designed.” But there are good reasons to think that these fears are closer to science fiction than they are to science.

Here is what the researchers did: repair a single gene mutation on a single gene, a defect known to cause — by its lonesome — a serious, sometimes fatal, heart disease. …”

“ … So are most physical diseases and psychiatric disorders. The genetic message is not carried in a 140-character tweet — it resembles a shelf full of books with chapters, subsections and footnotes.

So embryonic editing is unlikely to prevent most medical problems.

But about 10,000 medical conditions are linked to specific mutations, including Huntington’s disease, cancers caused by BRCA genes, Tay-Sachs disease, cystic fibrosis, sickle cell anemia, and some cases of early-onset Alzheimer’s. Repairing the responsible mutations in theory could eradicate these diseases from the so-called germline, the genetic material passed from one generation to the next. No future family members would inherit them.

But testing editing approaches on each mutation will require scientists to find the right genetic signpost, often an RNA molecule, to guide the gene-snipping tool.

In the study reported this week, it took 10 tries to find the right RNA, said Juan Carlos Izpisua Belmonte, a co-author and geneticist at the Salk Institute.

Dr. Greely noted that while scientists work to get human embryonic editing ready for clinical trials (currently illegal in the United States and many countries), alternate medical treatments for these diseases might be developed. They may be simpler and cheaper. …”

Photo:  https://rhapsodyinwords.com/2017/01/

The Last - I can only hope

A picture above my desk called, “The Last,” has a dual meaning for me. First off, I love the lithograph because it is so well done. To me it reflects the dying off of the American Indian culture. The second meaning is much more personal. I hope and pray I am the last male in my mother’s family with Kennedy’s Disease.

A NewYork Times article written by Pam Belluck reports on a recent study and potential milestone in genetic engineering. Nature,the International Weekly Journal of Science published the study this week. A portion of the article is shown below. Follow the links above to read the entire article and the study.

In Breakthrough, Scientists Edit a Dangerous Mutation From Genes in Human Embryos

“”Scientists for the first time have successfully edited genes in human embryos to repair a common and serious disease-causing mutation, producing apparently healthy embryos, according to a study published on Wednesday.

The research marks a major milestone and, while a long way from clinical use, it raises the prospect that gene editing may one day protect babies from a variety of hereditary conditions.

But the achievement is also an example of human genetic engineering, once feared and unthinkable, and is sure to renew ethical concerns that some might try to design babies with certain traits, like greater intelligence or athleticism.

Scientists have long feared the unforeseen medical consequences of making inherited changes to human DNA. The cultural implications may be just as disturbing: Some experts have warned that unregulated genetic engineering may lead to a new form of eugenics, in which people with means pay to have children with enhanced traits even as those with disabilities are devalued. …”

“… Scientists at Oregon Health and Science University, with colleagues in California, China and South Korea, reported that they repaired dozens of embryos, fixing a mutation that causes a common heart condition that can lead to sudden death later in life.

If embryos with the repaired mutation were allowed to develop into babies, they would not only be disease-free but also would not transmit the disease to descendants.

The researchers averted two important safety problems: They produced embryos in which all cells — not just some — were mutation-free, and they avoided creating unwanted extra mutations.

“It feels a bit like a ‘one small step for (hu)mans, one giant leap for (hu)mankind’ moment,” Jennifer Doudna, a biochemist who helped discover the gene-editing method used, called CRISPR-Cas9, said in an email. …”

"Desistance" is my current story I am in the final stages of editing. It is a Sci-Fi that takes place sixty years in the future. One of the storylines is the development of almost super-humans whose DNA has been edited to remove most of the known diseases. Perhaps it won’t be Sci-Fi after all.

Possible new therapy for motor neuron diseases

The University of Sheffield published the following news release yesterday. As always, additional research is required, but the premise is interesting.

New discovery in motor neurone disease and dementia could pave the way to novel treatments

"... When this series of nucleotides is expanded and repeated multiple times, neurodegenerative diseases can occur. The expansions of the gene forms genetic material called ‘R-loops’ which make the DNA vulnerable to breakages. They found that accumulation of R-loops and increased DNA breakage in neurons lead to neurodegenerative diseases.

Our cells have their own repair toolkits specially designed to fix breaks in DNA, however, the products of the expansion over-activate a process called autophagy – a process that gets rid of misfolded or “unwanted” proteins.

The new study, jointly directed by Professor Sherif El-Khamisy from the University of Sheffield’s Department of MBB and Professor Mimoun Azzouz from SITraN at the University of Sheffield, published today (17 July 2017) in Nature Neuroscience, shows that the expansion driven over-activation of this process can degrade some of the very precious DNA toolkits, meaning the cells will eventually die.

“We were able to shut down the out-of-control degradation process, which runs down the cell’s ability to fix genomic breaks, using genetic techniques,” said Professor El-Khamisy.

“Even though the DNA was still damaged, the cells were able to cope and did not die. Discovering this new mechanism and its consequence is a significant step towards developing new therapies for motor neurone disease and other neurodegenerative conditions. ..."

Click on the title to read the entire article.

SMA Drug Trial Goes So Well It Ends Early

This Los Angeles Times article on a new treatment for SMA Type 1. This is very promising news and I believe it will lead to additional breakthroughs for Type 2,3 and 4. SMA is different then SBMA (Kennedy's Disease), but antisense technology is being used in other progressive disorders and could be the bridge to a treatment.

Below is an excerpt from the Times article. You can read the entire article by following this link: Ionis shares leap 30% after a drug trial goes so well, it ends early

"The only option

Spinal muscular atrophy occurs in about 1 out of 6,000 to 10,000 births. It's caused by mutations in a gene that reduce production of a protein needed for survival of movement-controlling spinal neurons.

Nusinersen increases production of the protein from a closely related gene by altering how RNA made from the gene is translated into protein. This is done with antisense technology, a field pioneered by Ionis.

Drugs based on antisense technology are meant to block or change how targeted genetic instructions delivered through RNA affect the making of proteins, which are the building blocks of life. For instance, they could block certain mutations from resulting in protein synthesis that causes an unwanted medical condition.

Nusinersen has been granted orphan drug status in both the U.S. and the European Union — indicating it shows promise in treating a rare disease or condition — and it’s designated for fast-track review in the United States."

Image:  http://www.slideshare.net/pooranachithraflowry/antisense-therapy-48602109

Good Article on SBMA

JAMA Neurology published the following article in 2012. Another person living with Kennedy’s Disease recommended the article to me. The paper does a good job of explaining the condition to the layperson. 

MolecularPathophysiology and Disease-Modifying Therapies for Spinal and Bulbar MuscularAtrophy

Masahisa Katsuno, MD, PhD; Haruhiko Banno, MD, PhD; Keisuke Suzuki, MD, PhD; Hiroaki Adachi, MD, PhD; Fumiaki Tanaka, MD, PhD; Gen Sobue, MD, PhD

“Spinal and bulbar muscular atrophy (SBMA), or Kennedy disease, is an adult-onset lower motor neuron disease characterized by slowly progressive muscle weakness and atrophy. The disease is caused by the expansion of a trinucleotide CAG repeat encoding a polyglutamine tract within the first exon of the androgen receptor (AR) gene. During the 2 decades since the discovery of the AR gene mutation in SBMA, basic and clinical research have deepened our understanding of the disease phenotype and pathophysiology. Spinal and bulbar muscular atrophy exclusively affects men, whereas women homozygous for the AR mutation do not fully develop the disease. The ligand-dependent nuclear accumulation of pathogenic AR protein is central to the pathogenesis, although additional steps, eg, DNA binding and interdomain interactions of AR, are required for toxicity. Downstream molecular events, eg, transcriptional dysregulation, axonal transport disruption, and mitochondrial dysfunction, are implicated in the neurodegeneration in SBMA. Pathogenic AR-induced myopathy also contributes to the degeneration of motor neurons. Several potential therapies, including hormonal manipulation, have emerged from animal studies, some of which have been tested in clinical trials.”

Follow the title's link to the full article.

Kennedy's Disease Research; Future possibilities in

Last Saturday, Ed Meyertholen, the KDA's resident biology professor, was the KDA's Forum guest. As always, Ed provides information in a manner that is understandable to almost any audience.

Below are some of Ed Meyertholen’s comments and answers to specific questions concerning CRISPR and iRNA research. To read the entire forum discussion, follow this link: http://www.kennedysdisease.org/index.php/provide-support/chat-room-transcripts/2016-chat-room-transcripts/627-2016-february-06 .

“I am not a CRISPR expert, but the technique has been quickly (in the science view of quickly - several years) improving and it is expected that it will continue to do so. I do not think we will see this in humans for a while (years). 

In KD, I can imagine there may be issues on specificity - what cells to send CRISPR to, nerve cells or muscle (or both) and then one has to develop the procedures to specify the cells. The CRISPR work is very new and I do not know if anyone is working on it in Kennedy’s Disease. I suspect that someone will be soon. Nonetheless, I think that it has great possibilities - and I am usually not too optimistic. 

What happens is that they use a virus that normally attacks a specific cell type (like muscle), remove the DNA from the virus and replace it with the DNA needed to get CRISRP to work. The virus with the CRISPR DNA in injected into blood, goes to the muscle cells and instead of injecting viral (bad) DNA, injects the CRISPR DNA. The CRISPR DNA then lets the muscle cells make the CRISPR proteins which then should (and in the mouse, did) alter the DNA in those cells only. DNA in other than muscle cells is not affected. 

Kennedy’s Disease may give rise to other issues - the androgen receptor (the mutated protein) is made in many cells and may have effects (sexual issues, gynomatica (sp)), so we might want to alter the gene in other cells as well. It has great possibilities but it is not ready for prime time yet. This is the CRISPR paper:  http://www.ncbi.nlm.nih.gov/pubmed/26721684.

There was another paper from NIH (Kurt's lab) where they used iRNA to block the synthesis of the mutant androgen receptor and found that the mice got better. This is similar to work from LaSpada and from Lieberman that had been published a few years ago. These were done in conjunction with drug companies and I would not be surprised if there would be a clinical trial (I think that there is one starting using the same technique in Huntington's Disease) now. The other techniques with iRNA or ISO's are much closer to clinical use. If I remember correctly getting to nerve cells requires getting past the blood-brain barrier issue(s). Kurt's paper is here - http://www.ncbi.nlm.nih.gov/pubmed/26755334 .”

Photo:  http://www.science.ca/images/altman_rnase.jpg

CRISPR - Responses to Human Gene Editing

Earlier this month I posted an article on CRISPR and asked if it would be the answer we are waiting for in the Kennedy's Disease and other rare disease communities. The opportunity for human gene editing has generated a lot of interest and questions concerning its use. Below are two comments concerning this potential breakthrough.

______

May 18, 2015

National Academy of Sciences and National Academy of Medicine Announce Initiative on Human Gene Editing

Joint Statement by Ralph J. Cicerone and Victor J. Dzau

WASHINGTON -- The National Academy of Sciences and the National Academy of Medicine are launching a major initiative to guide decision making about controversial new research involving human gene editing.  Human gene-editing technologies, such as CRISPR-Cas9, may lead to promising new treatments for disease.  However, recent experiments to attempt to edit human genes also have raised important questions about the potential risks and ethical concerns of altering the human germline.  Future advances are likely to raise new questions.

Our initiative will include an international summit this fall to convene researchers and other experts to explore the scientific, ethical, and policy issues associated with human gene-editing research.  In addition, we will appoint a multidisciplinary, international committee to begin a comprehensive study of the scientific underpinnings and clinical, ethical, legal, and social implications of human gene editing.  The committee will consider and recommend standards, guidelines, and practices governing the use of gene-editing technologies in biomedical research and medicine.  An advisory group to steer the overall initiative will soon be announced.

We provided leadership in the past on emerging, controversial new areas of genetic research, such as human embryonic stem cell research, human cloning, and “gain-of-function” research.  In 1975, the Asilomar conference convened by the National Academy of Sciences led to guidelines for recombinant DNA research.  In keeping with these past efforts, we are prepared to work with the scientific and medical communities to achieve a comprehensive understanding of human gene editing and its implications in order to help guide researchers, clinicians, policy makers, and the public, here and around the world.

Ralph J. Cicerone is the president of the National Academy of Sciences, a private, nonprofit institution that provides science policy advice to the nation under an 1863 congressional charter. Victor J. Dzau is the president of the Institute of Medicine, which was founded as the health arm of the NAS in 1970.  Effective July 1, 2015, the IOM will become the National Academy of Medicine, and Dzau will be its first president.

Statement on NIH funding of research using gene-editing technologies in human embryos

April 29, 2015

Genomic editing is an area of research seeking to modify genes of living organisms to improve our understanding of gene function and advance potential therapeutic applications to correct genetic abnormalities. Researchers in China have recently described their experiments in a nonviable human embryo to modify the gene responsible for a potentially fatal blood disorder using a gene-editing technology called CRISPR/Cas9.

CRISPR-Cas9 is a customizable tool that lets scientists cut and insert small pieces of DNA at precise areas along a DNA strand. The tool is composed of two basic parts: the Cas9 protein, which acts like the wrench, and the specific RNA guides, CRISPRs, which act as the set of different socket heads. These guides direct the Cas9 protein to the correct gene, or area on the DNA strand, that controls a particular trait. This lets scientists study our genes in a specific, targeted way and in real-time.

Genomic editing is already widely studied in a variety of organisms. For example, CRISPR/Cas9 has greatly shortened the time it takes to produce knockout mouse models of disease, enabling researchers to study more easily the underlying genetic causes of those diseases. This technology is also being used to develop the next generation of antimicrobials, which can specifically target harmful strains of bacteria and viruses. In the first clinical application of genomic editing, a related genome editing technique (using a zinc finger nuclease) was used to create HIV-1 resistance in human immune cells, bringing HIV viral load down to undetectable levels in at least one individual. All of these examples of research using genomic editing technologies can and are being funded by NIH.

However, NIH will not fund any use of gene-editing technologies in human embryos. The concept of altering the human germline in embryos for clinical purposes has been debated over many years from many different perspectives, and has been viewed almost universally as a line that should not be crossed. Advances in technology have given us an elegant new way of carrying out genome editing, but the strong arguments against engaging in this activity remain. These include the serious and unquantifiable safety issues, ethical issues presented by altering the germline in a way that affects the next generation without their consent, and a current lack of compelling medical applications justifying the use of CRISPR/Cas9 in embryos. 

Practically, there are multiple existing legislative and regulatory prohibitions against this kind of work. The Dickey-Wicker amendment prohibits the use of appropriated funds for the creation of human embryos for research purposes or for research in which human embryos are destroyed (H.R. 2880, Sec. 128). Furthermore, the NIH Guidelines state that the Recombinant DNA Advisory Committee, “…will not at present entertain proposals for germ line alteration”. It is also important to note the role of the U.S. Food and Drug Administration (FDA) in this arena, which applies not only to federally funded research, but to any research in the U.S. The Public Health Service Act and the Federal Food, Drug, and Cosmetic Act give the FDA the authority to regulate cell and gene therapy products as biological products and/or drugs, which would include oversight of human germline modification. During development, biological products may be used in humans only if an investigational new drug application is in effect (21 CFR Part 312).

NIH will continue to support a wide range of innovations in biomedical research, but will do so in a fashion that reflects well-established scientific and ethical principles.

Francis S. Collins, M.D., Ph.D.
Director, National Institutes of Health