New way to more efficiently deliver CRISPR/Cas9 therapeutic to mice with Tyrosinemia type I

University of Massachusetts Medical School researchers have found a way to more efficiently delivery a CRISPR/Cas9 therapeutic to adult mice with the metabolic disease Tyrosinemia type I that may also prove to be safer for use in humans. A study published in Nature Biotechnology shows that administering the treatment by combining two delivery mechanisms already in clinical trials for other diseases led to correction of the mutated gene that causes the rare liver disorder in 6 percent of liver cells -- enough to effectively cure the disease in mice.

"This is the first study to provide proof that the CRISPR/Cas9 gene editing system can be administered in a therapeutically applicable formulation to repair genes in live, adult animals," said Wen Xue, PhD, assistant professor of molecular medicine and a member of the RNA Therapeutics Institute at UMass Medical School. "Until now it's not been possible to deliver CRISPR/Cas9 in a way that was suitable for clinical trials. By using an RNA guide and DNA repair template delivered via viral vector followed by a Cas9 in a lipid nanoparticle, we've take a huge step forward to overcoming this hurdle."

"This finding really excites us because it makes us think that this is a gene repair system that could be used to treat a range of diseases -- not just Tyrosinemia but others as well," said senior author Daniel G. Anderson, PhD, associate professor of chemical engineering at the Massachusetts Institute of Technology and a member of the Koch Institute for Integrative Cancer Research and the Institute for Medical Engineering and Science.

CRISPR/Cas9 has become a powerful gene editing tool that is revolutionizing biomedical research by making it easier to inactivate or activate genes in a cell line for study. An adaptive immune system used by bacteria to defend itself against bacteriophage and other types of foreign genetic material, the system consists of two components: a molecular scalpel (Cas9) that cuts DNA and an RNA guide complex that unlocks the scalpel when a matching genetic sequence, defining the exact spot to cut, is found.

Scientists can reprogram the CRISPR/Cas9 system with artificial guide RNAs to cleave specific sequences within mammalian genomes. The cell's natural DNA repair processes glue the genome back together, often excising a small portion. If a corrected copy of the disease mutation is also delivered when the cut is made, the cell can stitch the genome back tighter with the corrected gene, leading to permanent repair of the genome and correction of the disease gene.

In order to use this technology effectively all three of these elements, including the DNA repair template, must be efficiently and safely delivered to the nuclei of target cells. "Our previous research (published in 2014) showed that it was possible to correct the genetic mutation that causes Tyrosinemia in mice using CRISPR/Cas9 delivered through high-pressured injection," said Dr. Xue. "This approach isn't suitable for clinical applications, though, because it can cause damage to the liver and we'd have to deliver so much of it that we'd be doubling the blood volume."

Type 1 Tyrosinemia, also known as hepatorenal tyrosinemia, is caused by the inability to metabolize the amino acid tyrosine. It is caused by a mutation in the FAH gene which codes for the enzyme fumarylacetoacetate hydrolase. This leads to a toxic build-up of metabolites in the blood and urine, causing severe damage to the liver and kidneys. Diagnosed in infants, treatment for the disease includes restriction of tyrosine in the diet, the drug nitisinone and in some cases liver transplant.

The challenge for Xue and colleagues was to develop a CRISPR/Cas9 delivery system that was more efficient than the 1 in 250 cells that were repaired via high-pressured injection in the previous study while also being potentially safer for human application. To achieve this, they turned to two genetic delivery systems already in clinical trials--an adeno associated virus (AAV) vector and a lipid nanoparticle.

They loaded a CRISPR guide RNA and the genetic repair template into a reprogrammed AAV vector and injected it into mice. Because these genetic materials are being delivered with a viral vector, they can be expressed over a prolonged period of time, alleviating the need to deliver them simultaneously with Cas9. Without the Cas9 messenger RNA to cut the genome, the CRISPR guide and repair template remain inactive in the cells.

A week later, after the liver cells have had time to begin producing the RNA guide strand and the DNA template, a lipid nanoparticle is used to deliver the Cas9 messenger RNA. This is a better delivery vehicle for Cas9 RNA because it is typically too big to fit inside an AAV. Additionally, when Cas9 is delivered with a viral vector into the cell, it will continue expressing long after the damaged DNA has been repaired. This increases the likelihood of an off-target edit that could potentially damage the genome. The Cas9 messenger RNA begins to degrade relatively quickly (within days) after delivery and allows for short-term expression of the Cas9. This greatly reduces the risk for potential off target cutting by CRISPR/Cas9 system.

When these elements were delivered to adult mice with Tyrosinemia type I, the animals experienced a reduction of weight loss, alleviation of liver damage and generation of liver cells with the corrected FAH gene. Deep sequencing of the treated liver cells revealed that 6 percent of them harbored the corrected FAH gene--about 1 in 16 or a 15-fold improvement over the previous study. It also reduced off-target cutting of the genome by seven-fold.

"We combined the clinical suitable non-viral and viral delivery systems to allow efficiently gene repair in vivo, and to minimize the side effects," said lead author Hao Yin, PhD, research scientist at MIT.

Because AAV vectors are already in clinical trials for other human disorders and Dr. Anderson has similar lipid nanoparticle in clinical development, the researchers are optimistic that this CRISPR delivery method could be used in humans, although more studies are needed.

"The hope is that because we've used two delivery methods already in clinical development for patients, it will expedite a path to clinical trials for a CRISPR treatment of Tyrosinemia type I," said Xue. "What's more, the platform we've devised is modular, so it can potentially be tailored to treat other diseases, especially in the liver."