Gene Editing Trials Poised to Expand, but Hurdles Remain

Ricki Lewis, PhD

November 30, 2015

Gene editing has already garnered headlines, financial buzz, and even a few preliminary clinical successes. Thus far, however, few formal clinical trials are in progress, but improvements in the technology are expected to accelerate the pace of clinical development as early as 2016. In advance of that projected expansion, experts are gathering this week in Washington, DC, to address the technical and ethical considerations raised by the technique, which has the potential to alter a patient's germline DNA or the genes of an early embryo.

Gene editing uses DNA-cutting enzymes, called nucleases, to remove, alter, or replace specific DNA sequences in both strands of the molecule. As such, it has the potential to treat any condition for which the underlying molecular pathology is well understood, including single-gene disorders, infectious diseases, and cancer.

In early November this year, clinicians made headlines worldwide with the case of Layla Richards. The 1-year-old girl was treated at Great Ormond Street Hospital in London, United Kingdom, for aggressive acute lymphoblastic leukemia (ALL), using a gene editing product being developed by Cellectis called a chimeric antigen receptor (CAR). A CAR is an engineered receptor, typically expressed on a T cell that is part antibody, part signaling molecule. Binding of cells bearing CARs to target cells, such as the B cells of ALL, evokes an immune response that destroys the cancer cells.

Layla's case is exciting, but physicians should be cautious if patients ask about it, according to Bruce Levine, PhD, the Barbara and Edward Netter Professor in Cancer Gene Therapy at the Perelman School of Medicine at the University of Pennsylvania in Philadelphia, who helped develop the CAR technology. "To answer patient inquiries about gene editing based on Layla's story, physicians should say that it was a report of one child who was treated because they had cells available. Her treatment and others [like it] won't enter controlled clinical trials until probably mid-2016, and at a small number of centers," he told Medscape Medical News.

Cellectis is funding clinical trials to test the CAR cells in larger groups of patients with ALL, set to begin early in 2016, according to a news release from the hospital where Layla was treated.

CAR technology is not new, but the T cells that Layla received were novel in two ways: they were the first cells engineered using the gene-editing tool transcription activator-like effector nucleases (TALENs), and the first to use gene-edited donor cells, which opens the possibility of "off-the-shelf" products. Before Layla, in late 2012, 6-year-old Emma Whitehead was near death from ALL when Dr Levine, Carl June, MD, and others at the University of Pennsylvania treated her ALL with a billion autologous CAR T cells, fashioned using a different gene editing tool, zinc finger nucleases (ZFNs). The engineered cells bound the CD19 antigens that expressed on the surface of the cancerous B cells. Emma is doing great, Dr Levine said. Her case was prominent in the media too.

More Precise Than Gene Therapy

Gene editing differs from gene therapy in changing, replacing, or removing  a specific DNA sequence. Gene therapy, in contrast, adds a gene copy: it does not eliminate or replace the existing chromosomal DNA. Gene editing becomes genome editing when it targets more than one gene.

James M. Wilson, MD, PhD, professor of pathology and laboratory medicine and director, Orphan Disease Center, Perelman School of Medicine, and a veteran of many gene therapy trials, explains the distinction: "Traditional gene therapy introduces a fully independent gene cassette into the cell which resides outside the chromosome, for adeno-associated virus vectors, or randomly integrates into the chromosome, for retroviral vectors. Gene therapy does not directly affect the structure or function of the defective gene, as does gene and genome editing."

Gene editing can take several forms. "The most elegant is to specifically modify the structure of the patient's gene and its associated function by either correcting a mutation, for the treatment of recessive diseases, or by ablating its function, in the setting of dominant diseases," Dr Wilson said. Gene editing introduces a gene cassette into a specific location in the chromosome, he added. "This has advantages over traditional gene therapy, such as achieving better control of expression and diminishing oncogenic consequences of random integration."

A Trio of Strategies

The idea to edit DNA dates to the 1980s. "At that time, many scientists appreciated that if there was a way to make double-strand breaks in the genome in a targeted, systematic way, we could plug into natural repair systems to make changes. It would be like making a change in a word document," said Jennifer Doudna, PhD, a Howard Hughes Medical Institute Investigator and professor of molecular and cell biology and chemistry at the University of California, Berkeley.

The three currently available approaches to gene editing (see Box) deploy molecular guides that bind to specific DNA sequences and then use nucleases to generate double-strand breaks, which trigger DNA repair. CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats; CRISPR-associated protein 9) is the newcomer, but its speed, ease, and lower cost are catapulting it into prominence, with global market prediction for 2019 topping $3.5 billion.

CRISPR-Cas9 can knock out or in many genes at once, and applications are vast and eclectic. "CRISPR-Cas9 allows us to perform surgery on genes in any types of cells in any types of organisms," said Emmanuelle Charpentier, PhD, director of the Max Planck Institute of Infection Biology in Berlin, Germany, when receiving the Gruber Genetics Prize at the American Society of Human Genetics annual meeting in October with Dr Doudna.

"We created a simple two-component system harnessed to introduce double-strand breaks anywhere in a genome," added Dr Doudna. Whereas the other two nuclease types, ZFNs and TALENs, are like hardware, she explained, requiring labor-intensive synthesis of key proteins, "CRISPR-Cas9 is more like software; you just change the guide RNA" to target different addresses in the genome.

The new technology began when the two women met at a conference in 2011. Dr Doudna had been investigating the CRISPR locus, whereas Dr Charpentier was studying clinical manifestations of Strep pyogenes infection. The two joined forces to figure out the function of cas9 and subsequently developed the gene editing technique.

Early Trials Ongoing in HIV and Leukemia

Gene editing using ZFNs is tackling HIV infection at the coreceptor CCR5.

Dr June and Dr Levine and colleagues, with Sangamo Biosciences, reported in 2014 that they had used the approach to remove CCR5 from CD4 T cells in 12 patients, mimicking the "Berlin patient" and others who resist HIV infection because of a mutation in their endogenous CCR5 gene. The researchers found that the procedure was safe and increased CD4 T cell counts. In addition, in four of six patients who temporarily stopped taking antiretrovirals, viremia returned and T cell counts fell, but the modified cells persisted ( N Engl J Med. 2014;370:901-910).

Leukemias account for most of the other clinical trials using gene editing, but a few trials venture into other forms of cancer. For example, researchers at Baylor College of Medicine are testing CAR cells to treat glioblastoma, neuroblastoma, and sarcoma, and at Fuda Cancer Hospital in Guangzhou, China, researchers are using CAR cells against HER-2 receptors on breast cancer cells.

Single-Gene Diseases Trials to Launch in 2016

The number of clinical trials that use gene editing will expand in the near future, researchers predict, with single-gene disorders at the top of the list.

Gene editing makes more sense for some single-gene disorders than for others, said Steven J. Gray, PhD, assistant professor, Department of Ophthalmology, Gene Therapy Center, University of North Carolina at Chapel Hill. It "has an advantage when you need to maintain the normal regulation of a gene," he told Medscape Medical News. "Rett syndrome is a good example, where overexpression is detrimental. It would also have an advantage in dividing-cell populations where you want the change to be stable and transmissible. But in cases like hemophilia, where making a lot of protein from a single cell is good, gene editing makes less sense compared to traditional gene therapy approaches."

For example, clinical trials using ZFNs to treat sickle cell disease and β-thalassemia are expected to begin in 2016, sponsored by Sangamo Biosciences partnering with Biogen. The plan is to disrupt the normal silencing of fetal hemoglobin that happens early in development, so the protein can substitute for beta globin. If all goes as planned, patients would undergo a single treatment in which they receive autologous cells that had been edited ex vivo.

Meanwhile, CRISPR-Cas9 Therapeutics, which Dr Charpentier cofounded, is collaborating with Vertex Pharmaceuticals, known for the blockbuster cystic fibrosis drug Kalydeco, to treat that disease. Cystic fibrosis may be the first application of multigene targeting, which is only possible with CRISPR-Cas9, to explore the effects of genes other than CFTR on the phenotype. Genes that modify cystic fibrosis encode lysosomal enzymes, immune system proteins, and proteins that interact with CFTR protein.

Sangamo Biosciences also expects to file Investigational New Drug applications in 2016 for Hunter disease, Gaucher disease, factor XIII deficiency, and another lysosomal storage disease.

Beyond correcting single-gene diseases, George Church, PhD, the Robert Winthrop professor of genetics at Harvard Medical School in Boston, Massachusetts, and co-founder of eGenesis, recently published a report in Science on using CRISPR-Cas9 to inactivate porcine endogenous retroviruses at 62 identical sites in the pig genome. Removing the porcine endogenous retroviruses might make pig parts more suitable for xenotransplantation.

Concerns and Considerations

Although advocates of gene editing are anxious to test the approach in more diseases and disorders, technical and ethical issues must be addressed before the technology can make a big impression in the clinic.

Delivery is a major "bottleneck" for getting CRISPR-Cas9 into the clinic, Dr Doudna said. "How do we deliver it to cells and tissues in whole organisms?" The first CAR manipulations were performed with in cells ex vivo, and then the modified cells reintroduced into the patient. But gene editing need not completely reinvent the wheel in targeting organs. It can tap into the 25-year legacy of gene therapy, which has provided many vehicles for gene transfer, including DNA vectors such as viruses and plasmids, RNA, and lipid nanoparticles.

Other technical considerations are whether a gene edit is sufficient to affect clinical outcome, and the risk of evoking an immune response. "All of the editing nucleases are foreign proteins, which are likely to be quite immunogenic," Dr Gray said.

The Germline Question

But perhaps more critical are the ethical questions surrounding gene editing and its potential for germline modification: manipulating a fertilized ovum so that the change is passed to all cells of an individual, and thus to their offspring.

In January 2015, Dr Doudna called a local meeting on the subject, which included David Baltimore and Paul Berg, who spearheaded the famous Asilomar meetings held in 1975 to discuss the safety of recombinant DNA technology. The January meeting led to publication of a framework for how to move forward ethically, with respect to germline gene modification, and to a call for the International Summit on Human Gene Editing, which starts tomorrow. The summit is sponsored by the US National Academy of Sciences and the National Academy of Medicine, the Chinese Academy of Sciences, and the Royal Society.

The need for the international meeting became even more pressing in April 2015, when researchers from Sun Yat-sen University published a study reporting the use of CRISPR-Cas9 to edit human zygotes. The technique did not work well on the genetically doomed embryos that had poor DNA repair. "Maybe that's good because it provides more time to see whether we should be doing germline manipulation," Dr Charpentier told Medscape Medical News. (Germline manipulation using CRISPR-Cas9 has been done in mice and monkeys.)

The paper from China prompted National Institutes of Health director Francis Collins, MD, PhD, to issue a statement on April 29 regarding National Institutes of Health funding and gene editing in human embryos. He stated, "The concept of altering the human germline in embryos for clinical purposes has been debated over many years from many different perspectives, and has been viewed almost universally as a line that should not be crossed."

The international meeting this week  is expected to continue the dialog on how to limit germline modification via gene editing while allowing it on somatic cells to treat, cure, or even prevent disease. "I'm sympathetic to the view of not using CRISPR-Cas9 on the germline. But if someone said there would be a moratorium on gene editing technology, I'd disagree because it can work for infectious diseases and cancer. For the types of things we are modifying, immune cells, I don't think there would be danger," said Dr Levine.


Three Gene Editing Tools Borrowed From Bacteria


The techniques of gene editing borrow from the natural double-strand DNA break repair systems in bacteria.


In Streptococcus pyogenes, CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats; CRISPR associated protein 9) protects against viral infection. The bacteria integrate bits of infecting viral DNA among the palindromic CRISPR sequence repeats. Bacterial enzymes then transcribe the CRISPR sequence and its interspersed viral sequences into a "guide" RNA. The guide RNA associates with the Cas protein to bind to and then induce a double-strand break at the integration site of the viral DNA in the chromosome, snipping it out. The double-strand break then activates the DNA repair genes, which heal the gap in the chromosome.


Codiscoverer Emmanuelle Charpentier, PhD, describes how CRISPR-Cas9 works: "It is a two-component system. The enzyme Cas9 is programmed with a single guide RNA to target and cleave a specific DNA sequence at two strands." When one wants to edit a sequence, they just "engineer the guide RNA according to the sequence of the gene to be modified."


Zinc finger nucleases (ZFNs) were the first of the systems to be identified and described in the scientific literature. A zinc finger is a small protein motif that folds into an oblong shape when it binds zinc atoms, discovered in the African clawed frog (Xenopus) in 1987. The zinc finger domain binds to a specific DNA sequence that, when joined to a DNA-cleaving domain, forms a scissor-like molecular tool.


The third system, TALENs (transcription activator-like effector nucleases), resembles the ZFNs. The cleavage domains of both ZFNs and TALENs include the endonuclease domain of the restriction enzyme Fok1. The DNA binding domain of TALENs is 33 to 34 amino acids from the plant pathogen Xanthomonas, differing only at residues 12 and 13, which can be tweaked to add specificity. Researchers began developing TALENs as gene-editing tools in 2009; they are considered more efficient than ZFNs.


Dr Doudna and Dr Charpentier hold patents in and have cofounded or are on the boards of companies that are developing CRISPR-Cas9 technology. The other sources have disclosed no relevant financial relationships in the gene editing space.


Comments on Medscape are moderated and should be professional in tone and on topic. You must declare any conflicts of interest related to your comments and responses. Please see our Commenting Guide for further information. We reserve the right to remove posts at our sole discretion.