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Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

Artificial endonucleases consisting of a FokI cleavage domain tethered to engineered zinc-finger DNA-binding proteins have proven useful for stimulating homologous recombination in a variety of cell types. Because the catalytic domain of zinc-finger nucleases (ZFNs) must dimerize to become active, two subunits are typically assembled as heterodimers at the cleavage site. The use of ZFNs is often associated with significant cytotoxicity, presumably due to cleavage at off-target sites. Here we describe a structure-based approach to reducing off-target cleavage. Using in silico protein modeling and energy calculations, we increased the specificity of target site cleavage by preventing homodimerization and lowering the dimerization energy. Cell-based recombination assays confirmed that the modified ZFNs were as active as the original ZFNs but elicit significantly less genotoxicity. The improved safety profile may facilitate therapeutic application of the ZFN technology.

Unexpected failure rates for modular assembly of engineered zinc fingers

Nat. Methods 5, 374–375 (2008); corrected after print 29 May 2008. In the version of this correspondence initially published, the two previously published datasets analyzed were labeled with incorrect references in Figure 1b. Reference 2 should be associated with the second column (80 sites), and reference 3 should be associated with the third column (96 sites).

Zinc-finger nucleases: the next generation emerges.

Methods of modifying the human genome precisely and efficiently hold great promise for revolutionizing the gene therapy arena. One particularly promising technology is based on the homologous recombination (HR) pathway and is known as gene targeting. Until recently, the low frequency of HR in mammalian cells, and the resulting dependence on selection to identify these rare events, has prevented gene targeting from being applied in a therapeutic context. However, recent advances in generating customized zinc-finger nucleases (ZFNs) that can create a DNA double-strand break (DSB) at preselected sites in the human genome have paved the way for HR-based strategies in gene therapy. By introducing a DSB into a target locus of interest, ZFNs stimulate gene targeting by several orders of magnitude through activation of cellular DNA repair pathways. The capability of this technology to achieve gene conversion frequencies of up to 29% in the absence of selection demonstrates its potential power. In this paper we review recent advances in, and upcoming challenges for, this emerging technology and discuss future experimental work that will be needed to bring ZFNs safely into a clinical setting.Methods of modifying the human genome precisely and efficiently hold great promise for revolutionizing the gene therapy arena. One particularly promising technology is based on the homologous recombination (HR) pathway and is known as gene targeting. Until recently, the low frequency of HR in mammalian cells, and the resulting dependence on selection to identify these rare events, has prevented gene targeting from being applied in a therapeutic context. However, recent advances in generating customized zinc-finger nucleases (ZFNs) that can create a DNA double-strand break (DSB) at preselected sites in the human genome have paved the way for HR-based strategies in gene therapy. By introducing a DSB into a target locus of interest, ZFNs stimulate gene targeting by several orders of magnitude through activation of cellular DNA repair pathways. The capability of this technology to achieve gene conversion frequencies of up to 29% in the absence of selection demonstrates its potential power. In this paper we review recent advances in, and upcoming challenges for, this emerging technology and discuss future experimental work that will be needed to bring ZFNs safely into a clinical setting.

DNA-binding Specificity Is a Major Determinant of the Activity and Toxicity of Zinc-finger Nucleases

The engineering of proteins to manipulate cellular genomes has developed into a promising technology for biomedical research, including gene therapy. In particular, zinc-finger nucleases (ZFNs), which consist of a nonspecific endonuclease domain tethered to a tailored zinc-finger (ZF) DNA-binding domain, have proven invaluable for stimulating homology-directed gene repair in a variety of cell types. However, previous studies demonstrated that ZFNs could be associated with significant cytotoxicity due to cleavage at off-target sites. Here, we compared the in vitro affinities and specificities of nine ZF DNA-binding domains with their performance as ZFNs in human cells. The results of our cell-based assays reveal that the DNA-binding specificity-in addition to the affinity-is a major determinant of ZFN activity and is inversely correlated with ZFN-associated toxicity. In addition, our data provide the first evidence that engineering strategies, which account for context-dependent DNA-binding effects, yield ZFs that function as highly efficient ZFNs in human cells.

Expanding or restricting the target site repertoire of zinc-finger nucleases: the inter-domain linker as a major determinant of target site selectivity.

Precise manipulations of complex genomes by zinc-finger nucleases (ZFNs) depend on site-specific DNA cleavage, which requires two ZFN subunits to bind to two target half-sites separated by a spacer of 6 base pairs (bp). ZFN subunits consist of a specific DNA-binding domain and a nonspecific cleavage domain, connected by a short inter-domain linker. In this study, we conducted a systematic analysis of 11 candidate-based linkers using episomal and chromosomal targets in two human cell lines. We achieved gene targeting in up to 20% of transfected cells and identified linker variants that enforce DNA cleavage at narrowly defined spacer lengths and linkers that expand the repertoire of potential target sites. For instance, a nine amino acid (aa) linker induced efficient gene conversion at chromosomal sites with 7- or 16-bp spacers, whereas 4-aa linkers had activity optima at 5- and 6-bp spacers. Notably, single aa substitutions in the 4-aa linker affected the ZFN activity significantly, and both gene conversion and ZFN-associated toxicity depended on the linker/spacer combination and the cell type. In summary, both sequence and length of the inter-domain linker determine ZFN activity and target-site specificity, and are therefore important parameters to account for when designing ZFNs for genome editing.

Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion

Zinc-finger nucleases (ZFNs) have been successfully used for rational genome engineering in a variety of cell types and organisms. ZFNs consist of a non-specific FokI endonuclease domain and a specific zinc-finger DNA-binding domain. Because the catalytic domain must dimerize to become active, two ZFN subunits are typically assembled at the cleavage site. The generation of obligate heterodimeric ZFNs was shown to significantly reduce ZFN-associated cytotoxicity in single-site genome editing strategies. To further expand the application range of ZFNs, we employed a combination of in silico protein modeling, in vitro cleavage assays, and in vivo recombination assays to identify autonomous ZFN pairs that lack cross-reactivity between each other. In the context of ZFNs designed to recognize two adjacent sites in the human HOXB13 locus, we demonstrate that two autonomous ZFN pairs can be directed simultaneously to two different sites to induce a chromosomal deletion in ∼10% of alleles. Notably, the autonomous ZFN pair induced a targeted chromosomal deletion with the same efficacy as previously published obligate heterodimeric ZFNs but with significantly less toxicity. These results demonstrate that autonomous ZFNs will prove useful in targeted genome engineering approaches wherever an application requires the expression of two distinct ZFN pairs.

Integration Preferences of Wildtype AAV-2 for Consensus Rep-Binding Sites at Numerous Loci in the Human Genome

Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome.

Genome editing technologies: Defining a path to clinic

Recently developed genomic editing technologies have the potential to be powerful tools for gene therapy because of their ability to inactivate genes, correct mutated sequences, or insert intact genes. While the genomic editing field is advancing at an exceptionally rapid pace, there remain key issues regarding development of appropriate preclinical assays to evaluate off-target effects and establish safety. In order to begin a dialogue on these issues, the National Institutes of Health (NIH) Office of Science Policy, in collaboration with several NIH-funded investigators and the NIH Recombinant DNA Advisory Committee, organized a workshop on 10 June 2014, in Bethesda, Maryland, to provide a forum to educate the scientific and oversight communities and the public on different genome editing technologies, clinical experiences to date, and the preclinical assays being developed to examine the precision of these tools and their suitability for clinical application.R developed genomic editing technologies have the potential to be powerful tools for gene therapy because of their ability to inactivate genes, correct mutated sequences, or insert intact genes. While the genomic editing field is advancing at an exceptionally rapid pace, there remain key issues regarding development of appropriate preclinical assays to evaluate off-target effects and establish safety. In order to begin a dialogue on these issues, the National Institutes of Health (NIH) Office of Science Policy, in collaboration with several NIH-funded investigators and the NIH Recombinant DNA Advisory Committee, organized a workshop on 10 June 2014, in Bethesda, Maryland, to provide a forum to educate the scientific and oversight communities and the public on different genome editing technologies, clinical experiences to date, and the preclinical assays being developed to examine the precision of these tools and their suitability for clinical application. Targeted genome modification by designer nucleases is an emerging technology that can be used to investigate gene function and could also be used to treat genetic or acquired diseases. A wide range of genome alterations has been achieved by these nucleases, including localized mutagenesis, local and dispersed sequence replacements, large and small insertions and deletions, and even chromosomal translocations. The nuclease approach to targeted genome editing has been applied successfully to more than 50 different organisms, including crop plants, livestock, and humans.1 Recently developed genome editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and clustered regularly interspaced short Genome Editing Technologies: Defining a Path to Clinic

Ultramicroscopy Reveals Axonal Transport Impairments in Cortical Motor Neurons at Prion Disease

The functional imaging of neuronal circuits of the central nervous system is crucial for phenotype screenings or investigations of defects in neurodegenerative disorders. Current techniques yield either low penetration depth, yield poor resolution, or are restricted by the age of the animals. Here, we present a novel ultramicroscopy protocol for fluorescence imaging and three-dimensional reconstruction in the central nervous system of adult mice. In combination with tracing as a functional assay for axonal transport, retrogradely labeled descending motor neurons were visualized with >4 mm penetration depth. The analysis of the motor cortex shortly before the onset of clinical prion disease revealed that >80% neurons have functional impairments in axonal transport. Our study provides evidence that prion disease is associated with severe axonal transport defects in the cortical motor neurons and suggests a novel mechanism for prion-mediated neurodegeneration.

Impaired Axonal Transport in Motor Neurons Correlates with Clinical Prion Disease

Prion diseases are fatal neurodegenerative disorders causing motor dysfunctions, dementia and neuropathological changes such as spongiosis, astroglyosis and neuronal loss. The chain of events leading to the clinical disease and the role of distinct brain areas are still poorly understood. The role of nervous system integrity and axonal properties in prion pathology are still elusive. There is no evidence of both the functional axonal impairments in vivo and their connection with prion disease. We studied the functional axonal impairments in motor neurons at the onset of clinical prion disease using the combination of tracing as a functional assay for axonal transport with immunohistochemistry experiments. Well-established and novel confocal and ultramicroscopy techniques were used to image and quantify labeled neurons. Despite profound differences in the incubation times, 30% to 45% of neurons in the red nucleus of different mouse lines showed axonal transport impairments at the disease onset bilaterally after intracerebral prion inoculation and unilaterally—after inoculation into the right sciatic nerve. Up to 94% of motor cortex neurons also demonstrated transport defects upon analysis by alternative imaging methods. Our data connect axonal transport impairments with disease symptoms for different prion strains and inoculation routes and establish further insight on the development of prion pathology in vivo. The alterations in localization of the proteins involved in the retrograde axonal transport allow us to propose a mechanism of transport disruption, which involves Rab7-mediated cargo attachment to the dynein-dynactin pathway. These findings suggest novel targets for therapeutic and diagnostic approaches in the early stages of prion disease.