Using CRISPR to edit the globin gene in blood stem cells has resulted in the highest level of gene correction achieved to date in preclinical models of sickle cell disease (SCD). To be sure, "highest" is still not particularly high. The study, which was published in the Oct. 12, 2016, issue of Science Translational Medicine, showed successful gene correction in 2 percent to 5 percent of blood stem cells.

But that rate may be sufficient to result in a clinical benefit.

"In SCD, the sickle cells are basically deformed, and they are unstable, and they don't last very long in the circulation," David Martin, who is one of the paper's corresponding authors, told BioWorld Today.

And because the human body naturally has a "tremendous capacity" for generating blood cells, "you may have to make more red cells, but the normal ones will end up dominating in the circulation. . . . They just outlast the sickle cells."

Sickle cell anemia results from a point mutation in the gene for hemoglobin. To suffer from outright sickle cell disease, individuals need two copies of the mutation. Having only one copy of the mutation, though, is partially protective against malaria, and so the net result is that "sickle cell disease is one of the world's most common and most severe genetic diseases," said Martin, who is chair of the Center for Genetics and deputy director of the Children's Hospital Oakland Research Institute.

In the U.S., according to the Clarivate Analytics Incidence & Prevalence Database, the number of outright SCD cases is between 70,000 and 100,000, with an additional 3.5 million heterozygous carriers.

The only curative treatment for sickle cell disease is a bone marrow transplant. But because bone marrow transplant itself is a risky procedure, it is not often used, though the disease itself can be fatal and shortens life expectancy.

Because SCD results from a point mutation in blood stem cells, it is a good candidate for gene repair therapy.

In 2015, researchers from Sangamo Biosciences Inc. reported using zinc finger nucleases to correct the beta-globin gene. They were able to achieve a correction rate of roughly 1 percent in preclinical experiments.

Martin said he was "not prepared to comment in detail on the zinc finger work," but said that it was unlikely that CRISPR is inherently better at gene repair.

"The difference between what zinc fingers do and what CRISPR does at the level of repair is minimal," he said. In both cases, "you're trying to change to a specific sequence" by inducing DNA breaks in the genome at a specific location and repairing them with a template that is provided by the experimenter.

For both CRISPR and zinc finger nucleases, "once the cleavage has occurred, what happens afterward depends on cellular mechanisms" that are the same in each case."

Zinc finger nucleases consist of proteins, and their amino acid sequence is not obvious just from knowing the DNA sequence that is being targeted. In the CRISPR system, though, the genome sequence is targeted with an RNA molecule whose sequence can more or less bet determined by anyone who knows Watson-Crick base pairing rules.

In the work now published in Science Translational Medicine, the team described using a ribonucleoprotein complex "comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor, to enable efficient replacement of the SCD mutation" in blood stem cells.

That system "is vastly cheaper and more flexible in the stage of developing the proper reagent," Martin said.

As a result of that greater simplicity and flexibility, his team, with those of co-corresponding authors Jacob Corn and Dana Carroll, "were able to test a lot of different combinations of guide RNAs and donor template molecules, and then to take the optimal ones and apply them in human stem cells, and we were able to do that really, really fast."

"And that," he added, "is probably why our results are better."

In their paper, the team demonstrated a 2 percent to 5 percent correction rate in blood stem cells that engrafted into the bone marrow. The overall repair rate was higher, but most of those cells were not true stem cells, but progenitor cells, which can make all types of blood cells but do not do so long-term.

Martin, Corn, Carroll and their colleagues hope to take their approach into the clinic. "The goal in the near-term is a pre-IND meeting with the FDA that would lead to a clinical trial," Martin said.

While the path to the clinic may include some work on increasing repair efficiency, "probably the major issue here is the safety, because all of the gene editing technologies are new, but particularly CRISPR," Martin acknowledged.

The team is now taking a detailed look at any possible off-target effects of their system, particularly in sites like tumor suppressors that could spell trouble. So far, Martin said, they have "seen evidence for some off-target cleavage, but not anything that would cause anyone to worry."

But he also acknowledged that "a single cell, if it receives a mutation in a critical gene, can become a clone that seeds a malignancy."