Labs the world over are jumping onto the gene editing bandwagon (and into the inevitable patent arguments). And it’s hard to blame them. As these technologies have evolved over the last two decades starting with the zinc finger nucleases (ZFNs), followed by transcription activator-like effector nucleases (TALENs) and CRISPR—they’ve become ever more powerful and easier to use.
But one question keeps coming up: How precise are these systems? After all, a method that selectively mutates, deletes or swaps specific gene sequences (and now can even turn genes on) is only as good as its accuracy.
Algorithms can predict the likely “off-target” edits based on the target’s DNA sequence, but they’re based on limited data. “The algorithms are getting better,” says Richard Frock, PhD, a fellow in the laboratory of Frederick Alt, PhD, at Boston Children’s Hospital. “But you still worry about the one rare off-target effect that’s not predicted but falls in a coding region and totally debilitates a gene.”
Frock, Alt (who leads Boston Children’s Program in Cellular and Molecular Medicine, or PCMM), fellow Jiazhi Hu, PhD, and their collaborators recently turned a method first developed in Alt’s lab for studying broken chromosomes into a quality assurance tool for genome editing. As a bonus, the method—called high-throughput genome translocation sequencing (HTGTS)—also reveals the “collateral damage” gene editing methods might create in a cell’s genome, information that could help researchers make better choices when designing gene editing experiments. …
CRISPR—a gene editing technology that lets researchers make precise mutations, deletions and even replacements in genomic DNA—is all the rage among genomic researchers right now. First discovered as a kind of genomic immune memory in bacteria, labs around the world are trying to leverage the technology for diseases ranging from malaria to sickle cell disease to Duchenne muscular dystrophy.
In a paper published yesterday in Cell Stem Cell, a team led by Derrick Rossi, PhD, of Boston Children’s Hospital, and Chad Cowan, PhD, of Massachusetts General Hospital, report a first for CRISPR: efficiently and precisely editing clinically relevant genes out of cells collected directly from people. Specifically, they applied CRISPR to human hematopoietic stem and progenitor cells (HSPCs) and T-cells.
“CRISPR has been used a lot for almost two years, and report after report note high efficacy in various cell lines. Nobody had yet reported on the efficacy or utility of CRISPR in primary blood stem cells,” says Rossi, whose lab is in the hospital’s Program in Cellular and Molecular Medicine. “But most researchers would agree that blood will be the first tissue targeted for gene editing-based therapies. You can take blood or stem cells out of a patient, edit them and transplant them back.”
The study also gave the team an opportunity to see just how accurate CRISPR’s cuts are. Their conclusion: It may be closer to being clinic-ready than we thought. …
We all remember Dolly the sheep, the first mammal to be born through a cloning technique called somatic cell nuclear transfer (SCNT). As with the thousands of other SCNT-cloned animals ranging from mice to mules, researchers created Dolly by using the nucleus from a grown animal’s cell to replace the nucleus of an egg cell from the same species.
The idea behind SCNT is that the egg’s cellular environment kicks the transferred nucleus’s genome into an embryonic state, giving rise to an animal genetically identical to the nucleus donor. SCNT is also a technique for generating embryonic stem cells for research purposes.
While researchers have accomplished SCNT in many animal species, it could work better than it does now. It took the scientists who cloned Dolly 277 tries before they got it right. To this day, SCNT efficiency—that is, the percent of nuclear transfers it takes generate a living animal—still hovers around 1 to 2 percent for mice, 5 to 20 percent in cows and 1 to 5 percent in other species. By comparison, the success rate in mice of in vitro fertilization (IVF) is around 50 percent.
My first car was my grandfather’s 1980 Chevrolet Malibu. For about two years before my family gave it to me, it sat unused in Grandpa’s garage—just enough time for all of the belts and hoses to rot and the battery to trickle down to nothing.
Why am I telling this story? Because it’s much like what happens to the DNA in our blood-forming stem cells as we age.
Hematopoietic stem cells (HSCs) spend very little of their lives in an active, cycling state. Much of the time they’re quiescent or dormant, keeping their molecular and metabolic processes dialed down. These quiet periods allow the cells to conserve resources, but also give time an opportunity to wear away at their genes.
“DNA damage doesn’t just arise from mistakes during replication,” explains Derrick Rossi, PhD, a stem cell biology researcher with Boston Children’s Hospital’s Program in Cellular and Molecular Medicine. “There are many ways for damage to occur during periods of inactivity, such as reactions with byproducts of our oxidative metabolism.”
The canonical view has been that HSCs always keep one eye open for DNA damage and repair it, even when dormant. But in a study recently published in Cell Stem Cell, Rossi and his team found evidence to the contrary—which might tell us something about age-related blood cancers and blood disorders. …
If you follow cancer biology, then you’ve probably heard of ubiquitin before. Ubiquitin tags a cell’s damaged or used proteins and guides them to a cellular machine called the proteasome, which breaks them down and recycles their amino acids. Proteasome-blocking drugs like Velcade® that go after that recycling pathway in cancer cells have been very successful at treating two blood cancers—multiple myeloma and mantle cell lymphoma—and may hold promise for other cancers as well.
Less well known, however, is the fact that ubiquitin helps normal, healthy cells raise an alarm when viruses attack. Ubiquitin works with a protein called RIG-I, part of a complex signaling pathway that detects viral RNA and triggers an innate antiviral immune response.
Your immune system’s B cells can produce antibodies against an amazing number of pathogens—viruses, bacteria, etc.—without ever having encountered them. That’s because, as they develop, your B cells reshuffle their antibody-producing genes into an amazing number of possible combinations—more than 100 million—to produce what’s called your primary pre-immune B cell repertoire.
It’s long been thought that in people and in mice this reshuffling process—called V(D)J recombination, after the B cells’ antibody-coding V, D and J gene segments—takes place in two places: the bone marrow and the spleen. But new research from a team led by Frederick Alt, PhD, and Duane Wesemann, MD, PhD, suggests that there may be one more place B cells go to undergo recombination: the gut. What’s more, that reshuffling in the gut may be influenced by the microbes that live there.
If you are a scientist and you want to turn off a gene, one option that’s been gaining traction is RNA interference (or RNAi). In this molecular process—first discovered in plants and only 12 years ago detected in mammals—bits of RNA called small interfering RNAs (siRNAs) cancel out a gene’s messenger RNA, effectively muffling that gene.
Labs can order custom-made, chemically synthesized siRNAs for just about any DNA sequence they want to silence. The tricky part is deciding what the right sequence is—especially when that gene is part of a virus, where genes can mutate pretty quickly.
However, a biotechnology approach to producing siRNAs could make it relatively easy for just about any lab that can master recombinant DNA technologies to make a number of siRNAs against multiple sequences within the same target gene: a potential bonus for companies seeking to make drugs that rely on RNAi. …
Our immune system has immense powers of observation. It needs to in order to fend off the millions of bacteria, viruses, fungi, you name it, that we get exposed to every day.
I’m not talking about antibodies and T cells—parts of the immune system’s adaptive arm, which is fine-tuned to recognize a specific virus or bacterium. Rather, I’m talking about pattern recognition proteins—biological sensors capable of recognizing features and structures that only bacteria or viruses have. These make up the immune system’s innate arm, which essentially primes the body to attack anything that looks remotely like it doesn’t belong.
For instance, our cells carry sensors that can detect double-stranded RNA (dsRNA), which certain kinds of viruses use to encode their genome—like the rotavirus, which causes severe diarrhea in infants and small children. Our genome, by contrast, is encoded in DNA, and the RNA we make is single-stranded; if there’s dsRNA present, it means there’s a virus around.
The malaria parasite (or parasites: four species of Plasmodium can cause malaria in people) has a really complex life cycle. That complexity has allowed this mosquito-borne parasite from bringing untold misery to the human race for millennia. The World Health Organization thinks it causes 216 million cases of disease every year, while the U.S. Centers for Disease Control and Prevention estimates that some 3.3 billion people live at risk of malaria infection around the globe. Even in the United States, where malaria was officially eradicated 60 years ago, there are still about 1,500 cases every year.
All these numbers add up to one fact: we need a vaccine, badly. This is where malaria’s complexity becomes a problem. …
It used to be that there were two kinds of lymphoma, a cancer of the white blood cells: Hodgkin’s lymphoma, and everything else (aka non-Hodgkin’s lymphoma). Now doctors recognize more than 20 different types of non-Hodgkin’s lymphoma, based on cell type, genetic/genomic features, what the cells look like under a microscope, where the tumors form, etc.
With greater knowledge of what makes a lymphoma a lymphoma has also come the recognition that each type, subtype and sub-subtype responds to the same treatment differently—or not at all.
That’s led to a more targeted approach to discovering and developing anti-lymphoma drugs, based on the unique molecular features of a particular subtype. A team of researchers including Hao Wu, PhD, of the Program in Cellular and Molecular Medicine at Boston Children’s Hospital, is getting good traction focusing on one especially hard-to-treat lymphoma. …