Germline Engineering vis CRISPR; the inevitable journey towards the first genetically modified humans.

Rewriting life – First Human Embryos Edited in U.S.

Researchers have demonstrated they can efficiently improve the DNA of human embryos.

The first known attempt at creating genetically modified human embryos in the United States has been carried out by a team of researchers in Portland, Oregon, MIT Technology Review has learned.

The effort, led by Shoukhrat Mitalipov of Oregon Health and Science University, involved changing the DNA of a large number of one-cell embryos with the gene-editing technique CRISPR, according to people familiar with the scientific results.

Until now, American scientists have watched with a combination of awe, envy, and some alarm as scientists elsewhere were first to explore the controversial practice. To date, three previous reports of editing human embryos were all published by scientists in China.

Now Mitalipov is believed to have broken new ground both in the number of embryos experimented upon and by demonstrating that it is possible to safely and efficiently correct defective genes that cause inherited diseases.

Although none of the embryos were allowed to develop for more than a few days—and there was never any intention of implanting them into a womb—the experiments are a milestone on what may prove to be an inevitable journey toward the birth of the first genetically modified humans.

In altering the DNA code of human embryos, the objective of scientists is to show that they can eradicate or correct genes that cause inherited disease, like the blood condition beta-thalassemia. The process is termed “germline engineering” because any genetically modified child would then pass the changes on to subsequent generations via their own germ cells—the egg and sperm.

Some critics say germline experiments could open the floodgates to a brave new world of “designer babies” engineered with genetic enhancements—a prospect bitterly opposed by a range of religious organizations, civil society groups, and biotech companies.

The U.S. intelligence community last year called CRISPR a potential “weapon of mass destruction.”

Reached by Skype, Mitalipov declined to comment on the results, which he said are pending publication. But other scientists confirmed the editing of embryos using CRISPR. “So far as I know this will be the first study reported in the U.S.,” says Jun Wu, a collaborator at the Salk Institute, in La Jolla, California, who played a role in the project.

Better technique

The earlier Chinese publications, although limited in scope, found CRISPR caused editing errors and that the desired DNA changes were taken up not by all the cells of an embryo, only some. That effect, called mosaicism, lent weight to arguments that germline editing would be an unsafe way to create a person.

But Mitalipov and his colleagues are said to have convincingly shown that it is possible to avoid both mosaicism and “off-target” effects, as the CRISPR errors are known.

A person familiar with the research says “many tens” of human IVF embryos were created for the experiment using the donated sperm of men carrying inherited disease mutations. Embryos at this stage are tiny clumps of cells invisible to the naked eye. MIT Technology Review could not determine which disease genes had been chosen for editing.

“It is proof of principle that it can work. They significantly reduced mosaicism. I don’t think it’s the start of clinical trials yet, but it does take it further than anyone has before,” said a scientist familiar with the project.

Mitalipov’s group appears to have overcome earlier difficulties by “getting in early” and injecting CRISPR into the eggs at the same time they were fertilized with sperm.

That concept is similar to one tested in mice by Tony Perry of Bath University. Perry successfully edited the mouse gene for coat color, changing the fur of the offspring from the expected brown to white.

Somewhat prophetically, Perry’s paper on the research, published at the end of 2014, said, “This or analogous approaches may one day enable human genome targeting or editing during very early development.”

Genetic enhancement

Born in Kazakhstan when it was part of the former Soviet Union, Mitalipov has for years pushed scientific boundaries. In 2007, he unveiled the world’s first cloned monkeys. Then, in 2013, he created human embryos through cloning, as a way of creating patient-specific stem cells.

His team’s move into embryo editing coincides with a report by the U.S. National Academy of Sciences in February that was widely seen as providing a green light for lab research on germline modification.

The report also offered qualified support for the use of CRISPR for making gene-edited babies, but only if it were deployed for the elimination of serious diseases.

The advisory committee drew a red line at genetic enhancements—like higher intelligence. “Genome editing to enhance traits or abilities beyond ordinary health raises concerns about whether the benefits can outweigh the risks, and about fairness if available only to some people,” said Alta Charo, co-chair of the NAS’s study committee and professor of law and bioethics at the University of Wisconsin–Madison.

In the U.S., any effort to turn an edited IVF embryo into a baby has been blocked by Congress, which added language to the U.S. Food & Drug Administration funding bill forbidding it from approving clinical trials of the concept.

Despite such barriers, the creation of a gene-edited person could be attempted at any moment, including by IVF clinics operating facilities in countries where there are no such legal restrictions.

*adopted from Steve Connoa freelance journalist based in the U.K.

The G9a molecule offers new hope for patients with aggressive breast cancer

Researchers at QIMR Berghofer have discovered a set of genes that could be used to predict the survival of breast cancer patients. The findings could in future help to determine which patients would benefit from additional treatments.

The scientists, from QIMR Berghofer’s Control of Gene Expression research group, have also discovered one of the factors that can cause breast cancer to grow more aggressively.

The findings have been published in the Proceedings of the National Academy of Science.

Dr Jason Lee said G9a was a molecule that promoted tumour growth.

“When conditions are normal inside the body, a change occurs to this molecule and it breaks down, making it harmless,” Dr Lee said.

“However, we have discovered that inside a tumour, where there is very little oxygen, that change doesn’t occur to the molecule, meaning it doesn’t break down and instead starts to accumulate.

“This accumulation of the molecule G9a then makes the tumour grow more aggressively.”

Dr Lee said the team had also found that G9a silences certain genes.

“We don’t yet know what these genes control, but we found that when these genes are switched on, patients tend to have better survival rates, and when they’re switched off, patient survival tends to be worse.

“In other words, these genes are predictors of whether patients are likely to experience a recurrence of their cancer.”

Professor Frank Gannon said when the group tested an available drug that targets G9a, they found tumour growth more than halved.

“We are now working with international collaborators to develop a potential new treatment,” Professor Gannon said.

“The available drug we tested appeared to be effective in all types of breast cancer, but particularly in estrogen-receptor-positive (ER+) breast cancer, which is the most common form. While most of these patients respond well to treatment, about a quarter develop resistance to the treatment they receive.

“We hope that in future, we will be able to test patients for the genes controlled by G9a to determine which patients are likely to experience a relapse and need further treatment.

“Only those patients could then receive the treatment, which would be more cost effective and save patients from unnecessary drugs.”

The study involved internal collaborators at QIMR Berghofer Medical Research Institute.

Combination approach improves power of new cancer therapy called ‘Smac Mimetics’

An international research team has found a way to improve the anti-cancer effect of a new medicine class called ‘Smac mimetics’.

The team discovered how a protein called MK2 helps to keep cancer cells alive, making them resistant to the anti-cancer effects of Smac mimetics. The findings provide a rationale for combining inhibitors of MK2 with Smac mimetics as a potentially powerful new combination therapy for cancers with few treatment options, such as acute myeloid leukaemia (AML).

The research, recently published in the journal Molecular Cell, was the outcome of a research collaboration between Dr Najoua Lalaoui, Professor John Silke and colleagues at the Walter and Eliza Hall Institute, Australia; Professor Pascal Meierand colleagues at the Institute of Cancer Research, UK; and Professor Manolis Pasparakis and colleagues at the Cluster of Excellence CECAD at University of Cologne, Germany.

Dr Lalaoui said the research helped to advance her team’s previous discovery that combining the Smac mimetic agent birinapant with another new class of anti-cancer agents, called p38 inhibitors, could offer a new approach to treating AML.

“We knew these two agents could be combined, but didn’t fully understand the how they worked together at the molecular level,” Dr Lalaoui said.

“This latest study has pinpointed the MK2 protein as critical for the combination of Smac mimetics and p38 inhibitors to have a potent anti-cancer effect. As well as understanding our previous discovery better, it also highlights MK2 as an exciting new target for anti-cancer therapies, particularly in combination with Smac mimetics.”

Professor Silke said the research was part of a growing trend in the field, taking ‘rational’ approaches to treating cancer better, particularly through selecting combinations of anti-cancer agents.

“By understanding precisely which molecules are helping cancer cells to survive and evade treatment, we can develop smarter ways to kill these cells,” Professor Silke said.

“In the first place, the rational development of combination therapies has the potential to provide new treatments for cancers, such as AML, that have previously had poor outcomes.”
He said another potential benefit of combined anti-cancer therapies could be using each agent at lower doses.

“With a combined approach, the agents could still kill the cancer cell but with fewer harmful side effects on healthy tissues. Our goal is to develop cancer treatments that are both safer and more powerful than are currently available” Professor Silke said.

The research was supported by the Australian National Health and Medical Research Council, the Victorian Cancer Agency, the Victorian Government Operational Infrastructure Support Program, Breast Cancer Now, World Wide Cancer Research, the UK Medical Research Council and the European Research Council.

A giant neuron found wrapped around entire mouse brain

Like ivy plants that send runners out searching for something to cling to, the brain’s neurons send out shoots that connect with other neurons throughout the organ. A new digital reconstruction method shows three neurons that branch extensively throughout the brain, including one that wraps around its entire outer layer. The finding may help to explain how the brain creates consciousness.

Christof Koch, president of the Allen Institute for Brain Science in Seattle, Washington, explained his group’s new technique at a 15 February meeting of the Brain Research through Advancing Innovative Neurotechnologies initiative in Bethesda, Maryland. He showed how the team traced three neurons from a small, thin sheet of cells called the claustrum — an area that Koch believes acts as the seat of consciousness in mice and humans1.

Tracing all the branches of a neuron using conventional methods is a massive task. Researchers inject individual cells with a dye, slice the brain into thin sections and then trace the dyed neuron’s path by hand. Very few have been able to trace a neuron through the entire organ. This new method is less invasive and scalable, saving time and effort.

Koch and his colleagues engineered a line of mice so that a certain drug activated specific genes in claustrum neurons. When the researchers fed the mice a small amount of the drug, only a handful of neurons received enough of it to switch on these genes. That resulted in production of a green fluorescent protein that spread throughout the entire neuron. The team then took 10,000 cross-sectional images of the mouse brain and used a computer program to create a 3D reconstruction of just three glowing cells.

Well connected

The three neurons stretched across both brain hemispheres, and one of the three wrapped around the organ’s circumference like a “crown of thorns”, Koch says. He has never seen neurons extend so far across brain regions. The mouse body contains other long neurons, such as a nerve projection in the leg and neurons from the brainstem that thread through the brain to release signalling molecules. But these claustrum neurons seem to connect to most or all of the outer parts of the brain that take in sensory information and drive behaviour.

Koch sees this as evidence that the claustrum could be coordinating inputs and outputs across the brain to create consciousness. Brain scans have shown that the human claustrum is one of the most densely connected areas of the brain2, but those images do not show the path of individual neurons.

The claustrum is a good brain region in which to test the new technique because it has been extensively studied in mice and consists of only a few cell types, says James Eberwine, a pharmacologist at the University of Pennsylvania in Philadelphia.

A digital reconstruction of a neuron that encircles the mouse brain.

A digital reconstruction of a neuron that encircles the mouse brain.

Taking stock

“It’s quite admirable,” Rafael Yuste, a neurobiologist at Columbia University in New York City, says of the method. He doesn’t think that the existence of neurons encircling the brain definitively proves that the claustrum is involved in consciousness. But he says that the technique will be helpful for census efforts that identify different cell types in the brain, which many think will be crucial for understanding how the organ functions. “It’s like trying to decipher language if we don’t understand what the alphabet is,” he says.

Yuste and Eberwine would like to see 3D reconstructions of individual neurons compared to analyses of the genes expressed in those neurons. This may offer clues as to the type and function of each cell.

Koch plans to continue mapping neurons emanating from the claustrum, although the technique is too expensive to be used to reconstruct all of these neurons on a large scale. He would like to know whether all the region’s neurons extend throughout the brain, or whether each neuron is unique, projecting to a slightly different area.

Engineering Antibodies: A FAb-ulous Future for Therapeutics

Engineering the elements of an antibody can create potential therapeutics—ones designed to be more effective and safer than ever. But this often requires optimization of not just selectivity and affinity for the given antigen, but also stability, solubility and immunogenicity of the antibody. The familiar Y-shaped structure includes a variable domain called the fragment antigen-binding (Fab) region. The entire Fab or fragments of it can be engineered to change an antibody’s capabilities, and focused research in this area is still evolving. “If there is any general trend, in terms of platform/format, it’s that the number of solutions to the same problem keeps increasing, and there still isn’t much convergence on a single platform,” says David Pearlman, director, biologics software platform, and senior scientist at Schrödinger.

For antibody-based therapeutics, today’s scientists tend to use monoclonal symmetrical antibodies, single-chain antibodies, asymmetric bispecific antibodies, antibody-drug conjugates (ADCs) and so on. For most of these, there is no consensus on how to make them. “In the area of bispecifics, which can simultaneously recognize two different targets, there are now dozens of published platforms,” Pearlman explains. “The proliferation of different proposed solutions arises both from the fact that all have their own clinical advantages and disadvantages and from the commercial need to avoid IP overlap and patent infringement.”

Despite juggling multiple approaches to engineering antibodies, scientists are starting to agree on one thing. “Engineering is critical in the discovery process to yield antibody drugs with the best chances for survival in the clinic,” says Pearlman. Nonetheless, scientists face some significant issues in getting this engineering right.

To move ahead in developing an antibody-based drug, scientists often face challenges from aggregation, immunogenicity and viscosity.

“As with the platform, there is not, as yet, convergence on an efficient and reliable approach that satisfactorily addresses these issues,” Pearlman says. “Some of these properties, such as viscosity and aggregation, are expensive and time-consuming to evaluate.” The immunogenicity can remain uncertain until testing in humans.

Make a wish

At the University of Virginia, Thomas Barker, biomedical engineering professor, and his team used directed evolution to engineer antibodies. Specifically, Barkers shares that they used this process to engineer antibodies “from a parent library following phage screening.”

Getting the best results depends on the complementarity-determining regions (CDRs), which are the molecules in Fab that connect the antibody to a target antigen.

And non-CDRs matter, too. Barker points out that better computational models would improve the ability to predict the non-CDR regions that affect an antibody’s specificity and affinity.

In testing, Barker could also use some improvement in analytical tools. For example, he would like to see “cheaper instrumentation for high-throughput kinetic binding analysis.”

So, there are few wishes for vendors to fulfill.

Computing the connections

Barker mentions the desire for better computational tools, and others agree. “There is growing interest in in silico approaches to assessing protein liabilities,” Pearlman says. “The advantage of such approaches is that they are relatively fast and inexpensive to run, that they can be included in workflows addressing hundreds or even thousands of potential therapeutic candidates, and that they often offer not only predictions that can be used for triage but also insights into where you might engineer changes into the protein to remove or reduce the liabilities.”

Like some of the analytical approaches to engineering antibodies, the in silico side remains in development. However, advances in computation promise ongoing improvements. EpiVax, for instance, offers its “EpiMatrix High Throughput Antibody Immunogenicity Prediction Report.” This uses in silico screening and other technologies to provide what the company calls “an overall assessment of potential clinical immunogenicity for a large set of antibody candidates.” At the University of Melbourne in Australia, David Ascher, group leader for structural biology and bioinformatics, and his team also use computational tools for guided affinity maturation and construct design. Benchmarking the currently available tools has shown that there is still significant room for improvement [1]. Recently, the Ascher group released mCSM-AB, which, Ascher says, outperformed all the currently available commercial and academic tools for in silico optimization; the team made it freely available through a fast and easy-to-use tool [2].

Schrödinger also keeps creating new approaches. As an example, Pearlman says, “We have been focusing on new in silico structure-based techniques that facilitate early-stage liability assessment.” To computationally screen a collection of antibodies, scientists need an easy and automated tool. So, Schrödinger developed an antibody-structure-prediction tool that is robust, fast and reliable. Pearlman explains, “The ability of this tool to provide good predictions was demonstrated in the recent Antibody Modeling Assessment II—a blinded evaluation where our fully automated approach did quite well” [3]. In addition, Charlotte Deane, professor of structural bioinformatics at the University of Oxford in England, and her colleagues recently developed SAbPred, which is a fast and freely available tool for antibody-structure modeling and epitope prediction [4].

Furthermore, Schrödinger is developing tools that predict the potential liabilities in new structures. To build a model like this, the company needed a large experimental dataset to use for training and testing the computational tool. “A huge amount of relevant experimental data exists but is hidden behind corporate firewalls,” Pearlman says. “Recently, Schrödinger entered into several substantial collaborations that have given us access to these hidden data, and this access is making it possible to develop predictive, validated tools for liability assessment.” In brief, this technique created an automated quantitative structure-activity relationship (QSAR) model engine that computes the possible liabilities from a predicted antibody.

Increasing opportunities

Ascher’s team also works on antibodies for therapeutic possibilities. Here, he says, a lot of work in collaboration with Lisa Kaminskas, a research fellow at the University of Queensland in Australia, “has focused on the optimization of biomolecules, including antibodies and Fabs, pharmacokinetic and pharmacodynamic properties, and for this we have found PEGylation to be very useful [5].” However, much remains unknown about the optimal modifications that are needed to obtain the biological properties required.

Beyond antibodies, scientists can also study antigens. By optimally identifying the antigen being targeted by the antibody, Ascher explains, you can “avoid off-target effects and avoid or minimize the development of escape mutations.”

Despite the challenges of engineering antibodies for therapeutics, the promising potential they hold will result in better medicine. “Antibody therapies have enormous benefits and represent a larger proportion of the approved drugs,” Ascher explains. “In fact, if you look at the top-selling therapies approved in the last 10 years, many of them are biological therapies/antibodies, as opposed to the traditional small molecules.”