September 15, 2016 (Vol. 36, No. 16)

MaryAnn Labant

Gene Therapy Is Improving Its Style by Adopting Gene-Editing Tools Such As CRISPR/Cas9

Gene therapy is due for a second draft. As we have known it, gene therapy amounts to gene augmentation, a fairly random procedure whereby a gene is plunked down pretty much anywhere—so long as it lands somewhere in the genome.

Removing or editing a gene is too much to ask of conventional gene therapy, which is imprecise. In fact, using conventional gene therapy to correct specific genes would be like trying to type while wearing boxing gloves.

Gene therapy, however, promises to become more nimble. It is beginning to incorporate gene-editing technology. With CRISPR/Cas9 and other gene-editing technologies, gene therapy is gaining the ability to rewrite the genome at will, correcting bits of DNA where they reside in the genome, bits as small as a single base pair.

Despite its capacity for DNA word smithery, gene editing won’t rewrite gene therapy all at once. Each organ system presents unique challenges that need to be overcome, and in some circumstances, conventional gene therapy will remain the superior therapeutic option.


CRISPR-CAS9 gene editing complex from Streptococcus pyogenes. The Cas9 nuclease protein uses a guide RNA sequence to cut DNA at a complementary site. Cas9 protein: red surface model; DNA fragments: yellow; RNA: blue [Molekuul / Getty Images]

Cystic Fibrosis

Technically a rare disease, cystic fibrosis (CF) ranks as one of the most widespread life-shortening genetic diseases. A chronic, autosomal recessive disease, CF causes excessively produce thick, sticky mucus that affects the respiratory, digestive, and reproductive systems. Particularly problematic in the lungs, the mucus accumulates and blocks the airways, leading to breathing difficulties.

The lung has particular challenges for gene transfer. These include extracellular mucus and CF sputum, cilia beating, and the immune response.

Nonetheless, lung-relevant progress is evident in gene therapy. Pseudotyped lentiviral vectors have been engineered to carry envelope proteins that support entry into airway epithelial cells, and these vectors have been shown to efficiently and persistently transduce lung cells. In addition, removal of CpG islands from plasmid DNA makes DNA less inflammatory, which reduces toxicity and improves gene expression.

Most recently, the completion of a nonviral Phase IIb multidose trial, conducted by the U.K. Cystic Fibrosis Gene Therapy Consortium, showed, for the first time, that gene therapy was able to alter the progression of CF lung disease. Some groups are also investigating genome-editing strategies for CF, but this research is in its early phases, and a number of hurdles need to be overcome.

According to Uta Griesenbach, Ph.D., professor of molecular medicine, National Heart and Lung Institute, Imperial College London, the barriers to increasing the efficiency of gene editing in vivo are similar to those affecting gene transfer vectors. One might argue that gene editing should target airway progenitor cells, but these are buried beneath the surface epithelium, and they are difficult to access with currently available vectors.

Gene-editing strategies are dependent on DNA repair pathways that are most active in dividing cells, so in terminally differentiated or slowly dividing cells, such as the airway epithelium, gene editing may be inefficient. Molecules, such as Cas9, may also induce immune responses in the host, which will affect efficiency following repeated administration.

In contrast to gene therapy, which in principle can genetically complement any CF-causing mutation, gene editing will require mutation-specific reagents, but theoretically may be suitable to correct any mutation.


Cancer Immunotherapy

Work is also ongoing on gene and cellular therapy for disorders that can potentially be treated by regenerative medicine. Genome-editing nucleases and multiple terminally differentiated stem cell populations are utilized with the goal of optimizing ex vivo cellular therapies.

At the University of Minnesota, Mark Osborn, Ph.D., an assistant professor in the department of pediatrics, division of blood and marrow transplantation, is using CRISPR/Cas9 for immunotherapy by engineering T cells for enhanced tumor recognition and eradication. One strategy Dr. Osborn is using involves targeting the components of the TCR complex that consist of alpha and beta chains encoded by the TCR alpha and TCR beta genes, respectively. When the gene sequence of either of these chains is disrupted, the TCR complex is ablated, altering antigen recognition and cellular activation.

A treatment for leukemia is to infuse bone marrow/T cells from an unrelated donor into the patient. This mismatch allows the T cells to recognize cancer cells as foreign. The tumor cells are targeted for destruction as part of the therapeutic graft-versus-leukemia effect. But the T cells can also recognize healthy tissue as foreign, initiating a pathological process, namely, graft-versus-host disease (GVHD).

When CRISPR/Cas9 is used to remove the TCR complex, the T cells are no longer able to recognize any antigen, and the incidence of GVHD is greatly reduced. To restore the tumor recognition, a molecule termed a chimeric antigen receptor (CAR) is introduced that is able to recognize and destroy tumor cells.

“CRISPR/Cas9 allows us to engineer a universal donor population of cells,” says Dr. Osborne. “It can be paired with any CAR and given to any patient.”

CRISPR/Cas9 is also being expanded to include the ability to utilize checkpoint blockade approaches. Tumor cells can express PD-L1, which interacts with PD-1 on T cells, resulting in T-cell inhibition. When PD-1 is removed, the T cells can operate in the tumor environment more effectively. Both this approach and TCR disruption are moving toward the clinic and have passed initial NIH safety reviews.

“We have ongoing work in both the discovery and translational stages including rigorous safety studies to show reagent specificity for a given gene target,” informs Dr. Osbrone. “CRISPR/Cas9 is allowing for novel cellular engineering approaches that will aid in cancer treatments.”


Genome-editing nucleases and multiple terminally differentiated stem cell populations are utilized with the goal of optimizing ex vivo cellular therapies. [lvcandy / Getty Images]

Hearing Loss

The degeneration of hair cells is one of the major causes of hearing loss, which is one of the most common neurosensory deficits. Hearing loss can also be caused by the degeneration of cells in the inner ear, such as ganglion neurons and the cells of the stria vascularis.

Mutations in numerous genes are responsible for genetic hearing loss. For genetic hearing loss, gene therapy is one of the main therapeutic choices. However, traditional gene therapy using adeno-associated virus (AAV) as a delivery vehicle has limitations including restricted insert size, targeted cell types (for example, different AAV serotypes may be needed for different inner ear cell types), the lack of an efficient way to target dominant deafness, as well as long-term safety concerns.

“We are always on the lookout for alternative technologies that could overcome existing limitations, and genome editing has become a focus,” says Zheng-Yi Chen, Ph.D., an associate professor of otology and laryngology at Harvard Medical School and a researcher at Massachusetts Eye and Ear Infirmary. “One of the primary concerns is how to make it as safe as possible. Protein delivery could have the benefit of an editing effect that is permanent, whereas degradation of proteins would make the presence of the Cas9/gRNA complex transient, which greatly improves safety.”

Dr. Chen and his collaborator David Liu, Ph.D., a professor of chemistry and chemical biology at Harvard University, have shown efficient genome editing in the inner ear in vivo. These investigators have further targeted dominant deafness of hair cell origin, and they shown that one-time editing is sufficient to rescue hearing.

Instead of using AAV, which needs to target specific cell types, Drs. Chen and Liu are developing a general strategy to target a majority of inner ear cells for genome editing. This one-shot strategy would make it possible to target genes of genetic hearing loss irrespective of their cellular origins, negating the need to deliver different complexes to different types of cells.

Currently, dominant deafness can be dealt with by means of non-homologous end-joining (NHEJ), a DNA repair pathway that abolishes the mutations quite efficiently (20%). For recessive deafness, improved efficiency is needed. Mutations are corrected via the homology-directed repair (HDR) pathway in vivo.

Another issue is the inefficiency of dealing with many mutations in multiple genes that give rise to genetic deafness. Approaches that make it possible to rescue hearing by targeting one gene at a time instead of one mutation at a time, with the exception of the most common mutations, will need to be developed.

The inner ear delivery route presents yet another hurdle. The inner ear is small and has a complicated anatomic structure. However, the small size of the inner ear also offers a unique model, one that has few cells to target (16,000 hair cells per human inner ear), is separate from other parts of the body, and has a physiological outcome that can be measured precisely.


Harvard Medical School’s Zheng-Yi Chen, Ph.D., and Harvard University’s David Liu, Ph.D., used CRISPR/Cas9-mediated genome editing to modify hair cells in vivo. Top panel: Delivery of Cas9 protein complexed with a guide RNA against a green fluorescent protein (GFP) gene resulted in elimination of GFP in hair cells (boxes). Bottom panel: In a control trial, all hair cells are positive for GFP.

Neurological Disorders

X-linked dystonia-parkinsonism (XDP) is a complex disorder. The exact genetic cause is yet to be determined. One hypothesis is that the disorder results from the insertion of a retrotransposon (SVA) in an intronic region of the transcription factor TAF1. This insertion seems to interfere with the expression of a neuronal splice variant of TAF1 (N-TAF1).

This complexity is seen in many neurological disorders. In general, disease-causing mutations result in degeneration of a very specific population of neurons. Such degeneration can affect entire brain circuits. In XDP, communication in the basal ganglia is disturbed, resulting in dystonic clinical features. Other neurological disorders, such as schizophrenia or autism, have many risk genes associated to them, and the epigenome may also contribute to the disease etiology.

Genome editing can be used to understand neurological disease through the generation of isogenic cell lines. Combined with induced pluripotent stem cell (iPSC) technology, this allows the repair of genetic defects or mutations in patient-derived iPSCs or the introduction of mutations in nondisease-associated healthy control iPSCs in order to study the contribution of a given disease-associated mutation or single nucleotide polymorphism.

The significance of this approach is the absence of potential confounding effects of differences in genetic background on subtle disease phenotypes when comparing cohorts of patient vs. healthy subject iPSCs.

Brain tissue is in general inaccessible for biopsy. The combination of iPSC technology with gene editing provides the opportunity to study human disease “in a dish” and potentially to repair mutations in patient iPSC-derived neuronal progenitors or neurons for use in autologous transplantation.

Neurons are terminally differentiated cells. To date, in vivo gene-editing approaches in diseased neurons have been based on trying to knock out disease genes or pathogenic gene sequences based on NHEJ, since precise repair using HDR is not possible in these cells. An advantage is that delivery of CRISPR/Cas9 using AAV vectors works very well in transducing neurons.

‘With respect to neurological disease, the main application of gene-editing technologies is in disease modeling, specifically, in vitro disease modeling, primarily using human pluripotent stem cell-derived neuronal cells,” explains William Theodorus Hendriks, Ph.D., instructor, Harvard Medical School. “These in vitro models are used for both investigating pathobiology underlying disease and for screening new therapeutics.”

“An example of the power of this approach was the report of the anticonvulsant Retigabine being effective in reducing hyper excitability in ALS iPSC-derived motor neurons in vitro,” Dr. Hendriks continues. “Retigabine is currently in a Phase II clinical trial.”


Using CRISPR for Target Identification and Validation

The discovery of the CRISPR-Cas system in bacteria has initiated an impressive array of innovations that have enabled the use of the RNA-guided Cas9 nuclease in functional genomic screens, As a result, new opportunities for drug target identification and validation have arisen.

For example, Horizon Discovery has been using CRISPR-Cas9 to perform unbiased large-scale functional genomic screens to identify novel synthetic lethal oncology targets, according to Jon Moore, Ph.D., chief scientific officer at the company. Novel cancer-specific vulnerabilities or synthetic lethal interactions with proteins that are frequently mutated in cancer are difficult to predict, but could yield alternative yet effective targets for cancer treatment, he says.

To perform these screens, Horizon built a custom sgRNA library based on the druggable genome (DG+), containing 10 sgRNA sequences per target gene, and the requisite positive and negative control sequences.

Following screen optimization, a panel of standard cancer cell lines supplemented with some isogenic derivatives were infected with the DG+ library and maintained at a minimum of 300-fold coverage for approximately 12 cell doublings.

Results from the isogenic cell lines highlighted the effectiveness of the approach, notes Dr. Moore.

“We identified several genes that appear to be more important in cells harboring mutant PIK3CA, including PIK3CA itself and the downstream kinases AKT1 and AKT2, indicating an increased reliance on this pathway,” he adds.

Conversely, EGFR was found to be selectively essential in DLD1 isogenic cells that retain only the wild-type PIK3CA allele. In contrast EGFR signaling is dispensable in cells with the activating PIK3CA E545K mutation, which is consistent with its operating downstream
of EGFR.

The full poster featuring this and other CRISPR-based screening case studies, presented at SLAS 2016 is available on the Horizon website.


Rapid Screening for Gene Editing

The use of CRISPR/Cas9 as a high-throughput genetic modification tool has increased exponentially, but determining the nature and frequency of intended gene edits remains a key bottleneck. Next-generation sequencing (NGS) remains the gold standard for measuring both, but it’s too expensive for routine use.

To develop a workable solution, researchers at Advanced Analytical Technologies (AATI) have devised an automated capillary electrophoresis assay that pre-screens for gene-editing events. The assay efficiently handles today’s high sample volumes, isolating those that require NGS exploration, according to Kyle Luttgeharm, Ph.D., application scientist at AATI.

“The process takes advantage of heteroduplex cleavage assays that screen for precise and random events and provide an estimated mutation frequency,” says Dr. Luttgeharm. “The method forms duplexes between the PCR products of wild-type and edited sequences of pooled cells, before treating them with an endonuclease that recognizes and cleaves at DNA mismatches.”

The cleaved PCR products are then processed using AATI’s Fragment Analyzer™, maximizing AATI’s “set and forget” automation to quickly analyze fragments and calculate the percentage of cleaved products via capillary gel electrophoresis. Researchers can cost effectively determine which samples should be further investigated by NGS, while also being able to identify gene-editing events in complex samples such as polyploid cell lines, according to Dr. Luttgeharm.

“AATI is optimizing the cleavage assay to determine zygosity of individual mutations in diploid cell lines before sequencing. Once completed, the team will expand the assay to accommodate all cell lines,” he continues. “With this complete CRISPR detection solution, researchers can rapidly and efficiently screen for edited cell lines, animals, and plants.”

























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