Genome editing is a field of science that’s been around for awhile, but has experienced an explosion of activity and interest in recent years. Chances are that even your grandmother has heard about the recent story where for the first time, gene editing saved a one-year-old girl from dying of leukemia.

Microsoft word versus genome editing

To give you an idea of what this technique involves, think back to the last time you had to write a report. You let all your ideas flow out onto the page, but then realize that certain sentences or paragraphs need to be rearranged, removed, or added. So you copy, paste, and move stuff around with your mouse and keyboard until you’re satisfied.

Tools for editing the genome (which contain all of our genes) work a similar way, but they cut and paste DNA sequences in the human genome instead of words on a page. Scientists have figured out how to use these “genetic scissors” to delete genes (so they no longer have function) and to correct disease-causing mutations (by pasting in the normal DNA sequence of a gene to restore function). Both these abilities make genome editing a highly valuable tool for scientists to model diseases and to develop therapies to treat them.

There are multiple tools that researchers are currently using to modify the human genome. The main ones are fancifully named ZFNs, TALENs, and CRISPRs. All three use engineered proteins called nucleases to cut strands of DNA at specific locations in the genome. A cell’s DNA repair machinery will then either glue the DNA strands back together (this typically results in the loss of DNA and gene function), or repair the break by copying and pasting in the missing sequence of DNA from a template (you can correct disease-causing mutations this way by providing a donor template). We don’t have time to get into more details about how these tools work, but you can learn more by reading this fact sheet from Science Media Centre.

Some cells are more stubborn than others

While genome editing technologies offer many advantages for modifying human genes, it’s not a perfect science. There are still many limitations and roadblocks that need to be addressed to make sure that these tools can be safely and effectively used as therapies in humans.

Besides the obvious worry about “off-target effects” (when the genetic scissors cut random sections of DNA, which can cause big problems), another issue with genome editing tools is that some types of cells are harder to genetically modify than others.

Such is the case with blood stem cells, also known as hematopoietic stem and progenitor cells (HSPCs), that live in our bone marrow and make all the different blood cells in our body. Initial studies reported difficulty in delivering genome editing tools into human HSPCs, which is a problem if you want to use these tools to help cure patients suffering from genetic blood or immune diseases.

Have no fear, blood-stem cell editing is here

We are happy to inform you that a CIRM-funded study published today in Nature Biotechnology has developed a solution to the problem of hard-to-edit blood stem cells. Scientists from the USC Keck School of Medicine and from Sangamo BioSciences developed a new delivery method that allows for efficient genome editing of human HSPCs using zinc finger nucleases (ZFNs).

They used a viral delivery system to deliver ZFNs to distinct locations in the genome of HSPCs and successfully inserted a gene sequence that made the cells turn green under a fluorescent microscope. The virus they used was a harmless form of an adeno-associated virus (AAV), which can enter certain cells and delivery the researcher’s DNA cargo with a very low chance of altering or inserting its own DNA into the HSPC genome.

Using an AAV that was exceptionally good at entering HPSCs, they virally delivered ZFNs to specific gene locations in HSPCs that had been isolated from human blood and from fetal liver tissue. They found that delivering the ZFNs as mRNA molecules allowed the protein versions they turned into to be temporarily expressed in HSPCs. This produced a high rate of gene insertion (ranging from 15-40% of cells treated), while keeping off-target effects and cell death low. Even the most hard-to-edit HSPCs, called the primitive HSPCs, were modified. This result was really exciting because no other study has reported gene editing with this level of efficiency in this primitive population of blood stem cells.

The tools work but what about the cells?

After proving that they were able to successfully edit the genomes of HSPCs with high efficiency, they next asked whether the modified cells could grow in culture and create new blood cells when transplanted into mice.

While their method to deliver ZFNs into the HSPCs did cause some of the cells to die (around 20%), the majority that survived were able to multiply in a dish and specialize into various blood cells when grown in cultures. When the modified HSPCs were taken a step further and transplanted into immune-deficient mice (meaning their immune system is compromised and won’t attack transplanted cells), they not only survived, but they also specialized into many different types of blood cells while still retaining their genomic modifications.

Now here is where I want to give the researchers a high five. They decided that once wasn’t enough, and challenged their modified HSPCs to a second round of transplantation. They collected the bone marrow from mice that received the first transplant of modified HSPCs, and transferred it into another immune-deficient mouse. Five months later, they found that the modified cells were still there and had generated other blood cell types. Because these modified HSPCs lasted for so long and through two rounds of transplants, the authors concluded that they had successfully edited the primitive, long-term repopulating HSPCs.

Next stop, the clinic?

In summary, this study offers a new and improved method to genetically modify blood stem cells in all their forms.

So what’s next? The obvious hope is the clinic.

It’s a likely future as the study was conducted in collaboration with Sangamo BioSciences. They specialize in ZFN-mediated gene therapy and have a number of preclinical therapeutic programs, many of which focus on genetic diseases that affect the blood and immune system, as well as ongoing clinical trials using ZFNs to treat patients with HIV/AIDs. (One of these trials is funded by CIRM, read more here).

In a USC press release, Dr. Michael Holmes, VP of Research at Sangamo and co-senior author on the paper hinted at future clinical applications:

Michael Holmes, Sangamo BioSciences

 

Our results provide a strategy for broadening the application of gene editing technologies in HSPCs. This significantly advances our progress towards applying gene editing to the treatment of human diseases of the blood and immune systems.

 

 

Co-senior author and USC Professor Dr. Paula Cannon echoed Dr. Holmes:

Gene therapy using HSPCs has enormous potential for treating HIV and other diseases of the blood and immune systems.

One last question

A question that I had after reading this exciting study was whether other genome editing tools such as CRISPR could produce better results in blood stem cells using a similar viral delivery method.

CRISPR is described as a faster, cheaper, and easier gene editing technology compared to ZFNs and TALENS (for a comparison, check out this fun article by The Jackson Laboratory). And many scientists, both in academia and industry, are pushing CRISPR gene editing towards clinical applications.

When I asked Paula Cannon about which gene editing technology, ZFNs or CRISPRs, is better for therapeutic development, she said:

Paula Cannon, USC Professor

In terms of advantages, CRISPRs are easier to work with initially, and this makes them a great lab research tool. But when it comes to developing something for a clinical trial, its much more of a long game, so that initial advantage disappears. The ZFNs I work with have been previously optimized and are well characterized, and the CCR5 ZFNs are already in the clinic so they have a big advantage in that regard when you are trying to develop something for the next clinical application.

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Related Links:

• USC Press release 
• CIRM-funded clinical trial for HIV