The ability to precisely adjust the expression of genes inside the human body — ramping up protective ones and tamping down harmful ones — holds enormous potential for treating and preventing disease. But the standard molecular tools used to tune genes, such as the CRISPR system, are too bulky to package and deliver into the body's cells.
When delivered by viral vectors, for example, which are packed with the DNA instruction manual for cells to build CRISPR themselves, the lengthy instructions for CRISPR hardly fit inside a single virus. For therapies that may require adjusting multiple genes, delivering multiple CRISPR components separately becomes complicated and inefficient.
Now, Stanford Medicine scientists have developed a travel-sized solution — an ultracompact gene activation tool, called TIGRa (pronounced "tiger A"), small enough that its short DNA instructions can be packed inside viral vectors with room to spare.
Similar to CRISPR-based gene activation tools, once inside the cell, TIGRa finds the target gene and recruits the cell's transcriptional machinery to ramp up expression of that sequence. But compared with its CRISPR counterparts, TIGRa is more versatile regarding which genes it can target and more efficient at activating multiple genes at once.
In a mouse model, the researchers used TIGRa to turn up two protective genes in retinal ganglion cells, the nerve cells in the eye that are damaged in glaucoma and other retinal degenerative conditions. When the retinal ganglion cells were injured, the mice that received the treatment retained partial vision; those that did not became nearly blind.
"This is a tool that can be used to activate genes that otherwise lie dormant in our bodies," said Yang Sun , MD, professor of ophthalmology and senior author of the study published Aug. 10 in Cell Stem Cell. Sun's lab studies the cellular biology of glaucoma and other eye disorders. The eye is an ideal testing ground for gene activation therapies — a relatively accessible organ with clear signals. "It's easier to measure whether the eye is able to see than whether the liver is working," he said. "In the visual system, you can tell right away."
Ultracompact system
TIGRa is based on a system of gene-targeting enzymes known as TIGR-Tas discovered only last year by researchers at the Broad Institute of the Massachusetts Institute of Technology and Harvard University. Similar to the CRISPR-Cas system (often referred to simply as CRISPR), TIGR-Tas was discovered in microbes: CRISPR evolved in bacteria as an immune system that snipped and stored pieces of DNA from infectious viruses; TIGR-Tas evolved in parasitic bacteria and viruses, perhaps as a competitive genetic weapon, though its function is not yet clear. Both are RNA-guided, DNA-targeting systems, meaning they use a snippet of guide RNA like a mug shot to find and cut a matching piece of DNA. By customizing the guide RNA, scientists can direct these systems to different parts of the genome.
In the nearly 15 years since CRISPR's discovery, researchers have re-engineered this find-and-cut system into a suite of CRISPR tools, including those that edit a cell's DNA sequence and others that only activate or inhibit gene expression. They're now commonplace research tools in the lab, but their corpulence has limited their therapeutic use inside the body.
"The existing CRISPR activation tools are very powerful, but their size is too large. They're not suitable for the classical adeno-associated virus delivery," said Zhiquan Liu , PhD, a postdoctoral scholar in ophthalmology and lead author of the study.
TIGRa is the first gene activation tool based on the much more compact TIGR-Tas system. It's less than half the size (as measured by the number of amino acids) of comparable CRISPR gene activators.
Small but efficient
When Liu set out to develop a smaller gene activation tool, he cast a wide net, testing more than a dozen molecular prototypes based on different gene-targeting systems. He was surprised when TIGRa, one of the smallest, emerged as the most efficient gene activator.
"Usually when the size is smaller, the efficiency will be lower, but TIGRa had very high activation efficiency compared with the others, even though it's also very, very small," Liu said. "When we first saw the results, we were all very excited."
The researchers then designed several variants of their TIGRa prototype, making further space-saving modifications. TIGRa-Pro and TIGRa-Ultra are slightly smaller but more powerful gene activators, and TIGRa-Mini is an extra-small version with about 70% the efficiency of the original.
In cells in the lab, the team showed that TIGRa could activate up to 12 different genes simultaneously. As a demonstration, they used TIGRa to reprogram adult fibroblasts into an embryonic state (known as induced pluripotent stem cells), a feat that requires activating seven different genes at once.
TIGRa also matched or outperformed CRISPR gene activators in activating diverse genes of therapeutic interest, including those that could counter retinal degeneration, congenital muscular dystrophy and obesity.
"We tested nine different genes, and in six of them the TIGRa system shows better activation efficiency," Liu said.
Preserving vision
To test therapeutic delivery, the researchers packed TIGRa into viral vectors that were then injected into the eyes of mice. The TIGRa was customized to activate two genes, CaMKIIa and CaMKIIb, known to have protective effects on retinal ganglion cells. Two weeks later, when the mice were given a retinal injury that causes blindness, those that had received the TIGRa treatment retained about a third of their vision.
"We demonstrated that with the TIGRa injection, the retinal ganglion cells' survival rate improved twofold," Liu said. "We saw significant retinal structure preservation and visual protection."
The treated mice still showed preserved vision four months later.
The results are a promising proof of concept that may still be several years away from treating retinal degeneration in humans. "In our mouse study, we saw efficient protective effects with these two genes, but it's not enough," Liu said. "In the future, because these neurodegenerative diseases are very complex, we need to regulate more genes and in different combinations to get a better effect."
TIGRa is likely to find wider applications. The researchers have filed a patent for the tool. "Because it's very small and versatile, it could be used for many different diseases throughout the body, including heart conditions, liver conditions, skin conditions, cancer, neurodegeneration, stroke and any number of things," Sun said. "I'm optimistic about the application of this for humans. If you target the right genes in the appropriate diseases, it could happen faster than other gene therapies."
The study received funding from the National Institutes of Health (grants R01-EY025295 and R01-EY032159), the U.S. Department of Veterans Affairs, a VA Merit Award, a Children's Health Research Institute Award, a Research for Prevention of Blindness grant, the International Retinal Research Foundation and a National Eye Institute P30 Vision Research Core Grant.
