Wave of the future – cutting and pasting genes

Israeli scientists are being introduced to a new technique to cure some genetic diseases by a Tel Aviv University biologist and geneticist.

By JUDY SIEGEL-ITZKOVICH
DNA structure [Illustrative] (photo credit: INIMAGE)
DNA structure [Illustrative]
(photo credit: INIMAGE)
Anyone familiar with a word-processing program has much experience cutting and pasting for editing text and even images.
Although “gene editing” for eventually treating and even curing human diseases is almost unknown to the general public at large, geneticists in the US and elsewhere are very excited about this new and promising field. An Israeli scientist who spent several years conducting research at Stanford University in California who is responsible for advances in gene editing returned last summer to Israel to launch a startup that could become a pioneer in implementing the technology.
Dr. Adi Barzel, who earned master’s and doctoral degrees in biology and genetics at Tel Aviv University, will soon join its faculty as well as head a startup R&D he cofounded in California called LogicBio Therapeutics that is moving to the Ness Ziona Science Park. He will lecture on gene editing and gene therapy at the upcoming Biomed Conference of Israeli Advanced Technology Industries (IATI BioMed), which is due to be held in Tel Aviv between May 24 and 26. The event, to include an exhibition of companies and startups, is considered one of the world’s leading biomed events.
In an interview with The Jerusalem Post, Barzel said he was born at Kibbutz Kfar Hahoresh in the Lower Galilee to a family of academics, but not scientists. After his army service, he was accepted to a program of excellence at TAU, going directly to master’s of science studies instead of beginning with a bachelor’s degree, and he focused on the study of yeasts. With his wife, Dr.
Bat Zion Shachar (then a doctor at Assaf Harofe Medical Center in Tzrifin and now a gynecologist at Sheba Medical Center at Tel Hashomer) and their first two children (a third child, a son, was born at Stanford), Barzel went for post-doctoral studies in Stanford. There, he worked in the lab of Prof. Mark Kay, the Dennis Farrey Family Professor in Pediatrics and Professor of Genetics, who specializes in human gene therapy and viral vectors, and who has received numerous awards in his specialty.
Kay participated in the establishment of Barzel’s startup company whose headquarters will remain in the US, while the R&D Center will be in Ness Ziona. The startup is now focused on a new way of genome editing that could cure hemophilia (uncontrolled bleeding) in mice and is reportedly safer than older methods. Barzel, Kay and colleagues recently published a scientific paper in the prestigious journal Nature reporting on a genome editing technique that, without nucleases, could ameliorate hemophilia in the rodents. The technique developed by the Stanford researchers could provide a safer, longer-lasting method of introducing working copies of genes that provide the missing function.
LogicBio Therapeutics, which already licensed four patents owned by TAU, is so new, said Barzel, that it has only four employees, including the Israeli co-founder, and hasn’t had time to establish its own website.
“We make viral vectors,” said Barzel, including a DNA molecule contained in a protein capsid. It is the protein capsid that allows the DNA to reach its target tissue, cells and even intra-cellular targets.
Manipulating DNA by cutting and pasting began in the 1970s, but it was limited to bacteria and did not involve complex genes in mice or humans, he said. Then, in the 1980s, a new family of nucleases that are much more specific because they cleave only at specific unique sequences was developed. These allow manipulation in large genomes. If you were to use a nuclease with non-unique cleavage sites, then this nuclease would just shear the genome to little pieces.
These are enzymes that can split bonds between organic molecules called nucleotides, which are the building blocks of DNA and RNA. It was discovered in France, but this was long before gene therapy applications were envisioned. The first nuclease was in yeast, but it took a decade before it was used by a US researcher in mammalian cells and another 10 years before it was tried on animals in the context of gene therapy and a third decade before it was utilized last year as gene therapy for human patients. The enzymes discovered in the 80s and first used in human cells in the ‘90s were “homing endonucleases,” and the nucleases being used in the clinical trials are called Zinc Finger Nucleases (ZFNs) and TALENs (transcription activator-like effector nucleases). The newest technique, CRISPR (clustered regularly interspaced short palindromic repeats), should reach clinics soon. Another important leap in that decade was the ability to perform specific manipulations in fertilized eggs and later in embryonic stem cells that earned scientists a Nobel Prize. Genes could then be inserted into the mouse genome in an effort to make disease models.
“In the late 1990s, scientists discovered how to use discoveries for genome editing.
There was a great leap at the time – even though today it looks very old. I don’t think their initial aim in the ‘90s was to repair defective genes; it was still only a scientific tool. A combination of techniques was used with nucleases to induce higher rates. That meant the development of new animal models and much-more-diverse applications.”
THE FIVE-YEAR-OLD gene editing technique called TALEN uses, in effect, a bacterial gene combination that serves as a biological editing tool, or tiny scissors that can sever a defective gene in a cell and replace it with an artificially made healthy one. It has been used in the lab on animals and on human cells that cured cancer in children.
It could eventually help cure a variety of single-gene diseases in people. Focusing on a single location in one gene and creating a TALEN is relatively difficult, but CRISPR/ Cas9 is much simpler and cheaper to use, said Barzel. He noted that the biggest advantage is ease of use.
For those who might worry that CRISPR could get into the hands of terrorists, Barzel stressed: “There is no reason for a terrorist to use CRISPR. Like the other gene editing platforms, CRISPR is designed for specificity.
Terrorists don’t care about specificity.
The public should indeed be concerned about the use of nucleases in gene therapy and in the future also in embryo engineering, because mistakes can and will happen – but this is not terrorism, and in any case the pros outweigh the cons.”
Thus, while there are some risks and ethical problems in gene editing, conceded Barzel – and there should be supervision – gene editing is not the most important thing to be supervised by governments or other agencies. The possibility of terrorists creating pathogenic bacteria or viruses is much more worrisome.”
He and his TAU colleagues hope to be involved in work on cancer immunotherapy, fighting pathogenic infections and gene targeting of broadly neutralizing antibodies; elucidating the mechanism of gene targeting using viral vectors; and devising methods to increase the rate of gene targeting by homologous recombination.
Barzel has gone even further, developing a gene-editing method that is independent of nucleases that could pose risks in gene therapy.
“Rare cutting nucleases are all based on microbial proteins. If implemented in humans, it could result in adverse immune responses. Also, nucleases are never 100 percent specific, so it’s best to avoid them.”
The ability to insert a working copy of a gene into a patient’s genome is a tantalizing goal for many clinicians treating genetic diseases.
Now, researchers at the Stanford University School of Medicine have devised a new way to carry out this genetic sleight of hand.
His technique is different because one need not deliver an endonuclease enzyme to cut the DNA at specific location or genetic switches called promoters to activate the new gene’s expression, Barzel said.
His new and unique technology, which is being used in the Ness Ziona startup, is called “GeneRide.” It uses adeno-associated viral vectors (AAV) that used to be viruses, but were engineered so they are not dangerous.
“Viruses can replicate, but viral vectors cannot replicate in our cells. Therefore, viruses are often harmful while viral vectors can be used in gene therapy. I insert a correct gene at an unrelated but desired location.”
AAVs do not cause disease.
“The DNA of our choice is inserted instead into the capsule. It can be very exact and prevent the appearance of undesirable phenomena.
We are unique for using AAV for genome editing, so we have an edge on other companies.”
It can take 10 to 20 years for the proposed drug to go through all the pre-clinical and then clinical trials until the drug is approved, but once a drug is approved by the regulatory authorities, it can reach the market very fast. Thus biomed companies may thus remain startups for a long time.
“My company already has received $4 million in investments and we expect to get many times more,” he added.
“In two or three years, we hope to start clinical trials for treating hemophilia.
As it is an orphan [rare and serious] disease, potential treatments get priority in the FDA, and companies get grants and permission to speed up development. Such diseases should best be treated early on in patients. The more terrible the disease is, the faster a company will receive FDA approval for drugs that have been proven safe.
“Hemophilia treatments are hard on patients. Clotting factors have to be infused three times a week. Our technique would be a one-time thing, and it could become a proof-of-concept for many other diseases.
It could be infused even by a nurse or technician, with a doctor’s guidance, and not only by a physician.”
Delivery of the healthy gene for hemophilia involves targeting the liver when injected into a vein, or eventually even just into the muscle, thus serving as a factory to produce a product such as clotting factor.
“One can target just one out of 1,000 cells in the liver to make up for systemic deficiency or clearing toxic substances from the plasma,” he said.
ASKED TO predict what he expects in his field in 10 years, Barzel said that “the prospects are great. Gene therapy will move far beyond genetic diseases.”
Already, using a combination of techniques, there are children being cured of acute lymphoblastic leukemia, using engineered immune cells that fight diseases and not healthy cells. We are advancing.
The life expectancies of cancer patients is rising. Diseases that weren’t treatable in the past are treatable today.”
Babies suffering from severe combined immunodeficiency (SCID), who were confined to “bubbles” and called “bubble babies” because they totally lacked an immune system, have been freed from their plastic prisons after being completely vulnerable to infections.
“Bubble babies were treated by the integration of special retroviral vectors to reconstitute their immune systems,” said the biologist/geneticist. Of the first 20 babies to be treated, however, five contracted leukemia, because the vector was integrated next to an oncogene. But in general, the procedure is still a success. Today, the relevant cells are taken out of the body, treated and then reinfused into the body.”
Initially, “gene editing will be very expensive, and it has to be regulated. But I’m sure that it will eventually be included in the basket of health services, because it will be a one-time treatment and not a lifelong treatment for a chronic disease and thus, much less expensive.”