Editing set to broaden genetic blood fixes

SAN DIEGO – Fixing genetic diseases such as hemophilia, sickle-cell disease, and certain immunodeficiencies at their root, by replacing or repairing the faulty gene, has gone from dream to drug, with two gene therapies now approved in Europe, and a number of additional approvals expected on both sides of the Atlantic over the next few years.

Gene therapy's maturation was on display at Sunday's plenary session of the 2016 Annual Meeting of the American Society of Hematology, where Spark Therapeutics Inc. presented the newest from its SPK-9001 trial of gene therapy for hemophilia B, reporting that all nine patients had achieved stable expression levels of Factor IX that were at least 12 percent of normal.

Uniqure NV also presented an update on its AMT-0060, revealing that patients from its second, higher dose cohort, showed gene expression of Factor IX of 7 percent of normal – a level sufficient to provide "therapeutic benefit that is significantly superior to existing therapeutic regimes," Uniqure interim CEO Matt Kapusta told BioWorld Today.

And Bluebird bio reported data from a phase I/II trial of Lentiglobin gene therapy in sickle cell disease showing that three patients with transfusion-dependent beta-thalassemia had not needed transfusions since treatment, while a patient with severe sickle cell disease (SCD) was producing sufficient amounts of healthy globin to prevent sickling.

At Sunday's plenary session, Mount Sinai School of Medicine's Christopher Walsh, who introduced the SPK-9001 presentation by the University of Pennsylvania's Lyndsey George, said that "These are heady times in the field of gene therapy," which "has the potential to revolutionize" the treatment of hematological diseases, especially hemophilia.

Even as gene therapy is becoming a more mature technology, the promise of gene editing, or making more or less precise changes to a cell's own DNA rather than delivering a transgene, was being explored as well.

Genome editing is still new enough to have issues that need working out at every stage of the process.

In terms of the actual editing, the key problem is that CRISPR can spur the cell to edit its genome in two ways. One is via nonhomologous end-joining (NHEJ), an imprecise repair process which Weiss characterized as a cell's "frantic attempt" to fix itself once it has detected a DNA break. NHEJ can be used to knock out genes.

For precision edits, though, cells need to repair their DNA break via homology-directed repair (HDR), in which they use a template – which can be provided by the experimenter – to make precise, defined alterations in their genome. Getting cells to use HDR, though, is easier said than done.

NHEJ "in most cells is more efficient – even if you try hard to get HDR, you almost always get more NHEJ," Weiss told BioWorld Today.

And if that weren't enough, "even with successful HDR, you've created a sequence that only differs from the original sequence by one nucleotide," Massachusetts General Hospital's Keith Young told the audience at the meeting. The repair machinery, in turn, can't necessarily tell the difference between gene sequences that differ only by a single base pair. A sequence that has been successfully repaired from the point of view of the experimenter might still be targeted again by the cell – once again with a higher chance of being subjected to NHEJ than HDR, of course.

A poster being presented by scientists from Delaware State University at the conference on Monday showed that the team was able to correct SCD in about a quarter of cells – but all cells had at least one indel mutation, showing that all had been edited via NHEJ as well.

Because NHEJ is error-prone, it can also make bad situations worse – introducing further mutations that might exacerbate the SCD, or give a patient beta-thalassemia on top of the SCD.

Long-term, Weiss said, there are hopes that the balance between NHEJ and HDR could be shifted, for example by treating cells with NHEJ-inhibiting drugs during the gene correction.

But for now, it is still much easier to break genes than fix them with gene editing. The good news is that sometimes breaking a gene can be used to fix a disease.

Weiss, who is chairman of the department of hematology at St. Jude Children's Research Hospital, and colleagues recent published work in Nature Medicine showing that by taking out a short genome stretch that controls the switch from fetal to adult hemoglobin – essentially, breaking a regulatory mechanism – they were able to produce healthy blood cells from patients with SCD. Because "you can get by with fetal [hemoglobin] your whole life," the approach could fix a genetic disease by mimicking a benign genetic condition.

And in a Sunday poster, researchers by the German Goethe University reported they were able to achieve a high rate of correction of a frameshift mutation that led to X-linked chronic granulomatous disease by introducing another frameshift mutation via NHEJ.

Another issue is that gene correction rates in blood stem cells, "the only cell type you can treat that will cure the disease lifelong," Weiss pointed out, are still very low.

Correction rates do not need to be anywhere near 100 percent to make a clinical difference, as Spark's and Uniqure's work has shown. In October, researchers from UC Berkeley, the University of Utah, and the Children's Hospital Oakland Research Institute reported on a blood stem cell correction rate of two to five percent in preclinical models of sickle-cell disease and argued that given the survival advantage of corrected cells, that rate would be enough to have a clinical impact. (See BioWorld Today, Oct. 18, 2016.)

At the meeting, researchers reported improvements to the rate using several different approaches. "A lot of attention is being paid to process development," Weiss said.

Blood stem cells are more susceptible to damage during the editing process, and a team that included scientists from Sangamo Biosciences Inc. presented data at the conference suggesting that a temporary expression of prosurvival factors might help to increase editing efficiency

In what may deserve the title of cutest ASH presentation, a team from SQZ Biotechnologies Inc. reported that deformation of stem cells or colloquially speaking, squeezing them – led to high rates of CRISPR-based correction of the HIV co-receptor CCR5.

And by tweaking their methods, including the use of chemically modified guide RNAs and AAV to express HDR templates, a team from Stanford University reported they were able to achieve stem cell correction rates of 40 to 50 percent in the beta-globin gene.

"With this editing, the devil is in the details," Weiss said, "and if this is reproducible, it's going to have a big impact."

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