Gene Editing Takes the Stage at Discovery on Target 2016
By Dana Barberio
October 5, 2016 | The CRISPR-Cas9 system is revolutionizing genomic engineering with its unprecedented ability to precisely modify the DNA of essentially any organism—sparking new research, patent battles, and some ethical concerns. In the Advances in Gene Editing, Gene Silencing, and Gene Therapy Tracks at the 2016 Discovery on Target conference*, we heard from researchers on their exciting new advancements in CRISPR-CAS9 and beyond. The implications for cancer, anti-viral therapies, genetic diseases, and organ transplantation are vast.
One of the key issues we heard in panel discussions and throughout the conference was with delivery - how best to get the gene editing or gene silencing machinery into cells.
Nanoparticles offer a unique solution to these delivery problems, as discussed in a talk by Daniel Anderson, Professor of Chemical Engineering, Harvard-MIT Health Sciences and Technology. “There are natural destinations in our body for nanoparticles, such as the spleen, bone marrow, and the kidney,” said Anderson. Their group encapsulates RNA in a tiny lipid-like shell—a 50-100 nanometer nanoparticle. “The vision is to make small interfering RNA (siRNA) molecules bound to nanoparticles, and get them inside cells, where they can engage RNA interference machinery for specific destruction of target RNA, blocking protein production.” The team has learned that sequence selection is important, and chemical modification and nanoparticles can be used for delivery. “We are confident in the use of nanoparticles to deliver to hepatocytes, but it’s not limited to hepatocytes,” said Anderson. Endothelium can also be accessed with siRNA nanoparticles.
Anderson’s group also used nanoparticle delivery with the CRISPR-CAS9 system in a mouse model, resulting in 60% indel formation. Their studies have included humans with tyrosinemia, who have mutations in a gene coding for the enzyme fumarylacetoacetate hydrolase. They were able to use CRISPR-CAS9 to correct the mutation.
At the University of Iowa, researchers are tackling one family’s inherited eye disease with gene editing. Alexander Bassuk, Professor and Chair in Pediatric Neurology at the University of Iowa, discussed two brothers with RPGR mutation- X-linked retinitis pigmentosa, an inherited condition that causes progressive vision loss in boys and young men, and blindness later in life. Their mother had the same disease in one eye only.
The Iowa team generated induced pluripotent stem cells from patient cells, and then used the CRISPR-CAS9 system to target the faulty gene. They got precise corrections of the targeted nucleotide in the gene but also some non-homologous end joining, as is often the case with CRISPR-CAS9. “Our efficiency was low; less than 13% of the cell population was corrected, and we still have work to do,” said Bassuk. As far as they know, they were the first to go from skin biopsy to induced pluripotent stem cells and then to correct a gene associated with eye disease. “This was a proof of concept, and really a labor of love, as the members of this family were friends of mine from childhood, and the work was done on a $50,000 donation,” Bassuk said. This was a great first step, but researchers still need to increase the efficiency of repair and improve off-target effects to make it clinically therapeutic, and to correct the disease in the patients. Bassuk’s lab is now working with a mouse model with the human version of the gene. Down the road, “we can use this approach for all retinal diseases. The retina is attractive because (during treatment) we can see the retina from the outside, and we can directly inject. Skin and blood are also obvious places to go. In terms of the nervous system, the eye is going to be the first place we get CRISPR correction,” predicts Bassuk.
Researchers are also exploring gene editing to fight chronic viral Infections that can be controlled with current therapies but have no cure, such as HIV, Hepatitis B (HBV) and Herpes Simplex Virus (HSV).
At the Fred Hutchinson Cancer Research Center at the University of Washington, Keith Jerome is working with a class of gene editing tools called homing endonucleases, which have high specificity for the viruses they target and limited off-target effects.
In order to inhibit the ability of the virus to replicate, they start with a non-pathogenic, replication-defective AAV (adeno-associated virus) to deliver HSV-specific homing endonucleases. After infecting mice with HSV, they introduce the AAV delivery vector under the skin. They think the AAV is entering in through nerve endings, traveling down the axon to access the trigeminal ganglia and deliver the gene-editing system.
Their results are promising: they’re able to detect insertions and deletions in 2-4% of the viral genome using Next-Gen sequencing. The targeted endonuclease is highly specific, with no evidence of off target mutagenesis in the mouse genome. “One of the major challenges we’re dealing with now, not only with HSV, but also with HBV and HIV therapy, is really moving this delivery efficiency up,” said Jerome. For now, research indicates that targeted mutagenesis of chronic viruses such as HSV, HBV and HIV is possible, and that mutagenesis of the virus leads to a reduction in viral replicative capacity. “The ultimate success of viral disruption as a therapeutic approach is critically dependent on efficient delivery systems,” Jerome told the audience.
Gene Therapy Genesis
In the sci-fi like “Genesis Project”, Walter Low, Professor and Associate Head of Research, Neurosurgery at the University of Minnesota Medical School, is experimenting with growing human organs in pigs. Since pigs have organs similar in size to that in humans, pigs can be used as incubators to grow human organs from human donor stem cells. Using gene therapy, the researchers knock out genes important for organ development in the pigs, and create a niche where the human donor stem cells can occupy that niche to create an organ system, said Low. This involves knocking out the transcription factors that are key to organ and cell development.
The basic concept is to use gene therapy technology to create deficient pig blastocysts that cannot develop organs, then to take human stem cells and introduce them into the deficient blastocyst. The blastocyst is then transferred into a surrogate mother pig, whose offspring would develop a human organ made from stem cells. The organ would then be transplanted into an individual who needs a liver, kidney, or heart.
As first steps, their group showed that specific gene knockouts of transcription factors involved in organ development resulted in an absence of targeted organ development. They achieved this for a variety of organs and immune cells: pancreas, lung, kidney, liver, thymus, T cells, and B cells. Their next step is to generate human organs in the pigs, and they’ve already done some proof-of- concept with other animal models.
An important ethical issue was raised by the audience surrounding the implications of off-target effects generated through introduction of human stem cells into pigs, including the possibility of accidently generating a humanized brain in the pigs. Yes, you heard correctly. They will need to work closely with regulators on this one.Finally, in a wrap up of the gene editing track, we heard some thoughts on the big picture of gene editing in a panel discussion.
“What’s really important… is the careful selection of disease targets. I would suggest targets in which there is substantial unmet need,” said Bryan Cullen, Professor of Molecular Genetics and Microbiology at Duke University. It’s also key that when you edit or insert a gene through gene editing, that “you see something meaningful, such as a clinical biomarker, so that there is no dispute that it worked, that you got the construct in and it was targeted.”
Anderson added: “I would say the greatest therapeutic potential is in delivery of immune cells that are going to attack the tumor, rather than delivery to the tumors themselves. Potentially some of that can be done ex-vivo.”
* Discovery on Target hosted by Cambridge Healthtech Institute, September 19-22, Boston, Mass.