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New Zinc-Therapy Gene Therapy: Targeted gene knockout by direct delivery of zinc-finger nuclease proteins
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News Release

Scripps Research Institute Scientists Develop Alternative to Gene Therapy

The Technique Points to Safer, Simpler Potential HIV Treatment

LA JOLLA, CA - July 1, 2012 - Scientists at The Scripps Research Institute have discovered a surprisingly simple and safe method to disrupt specific genes within cells. The scientists highlighted the medical potential of the new technique by demonstrating its use as a safer alternative to an experimental gene therapy against HIV infection.

"We showed that we can modify the genomes of cells without the troubles that have long been linked to traditional gene therapy techniques," said the study's senior author Carlos F. Barbas III, who is the Janet and Keith Kellogg II Professor of Molecular Biology and Chemistry at The Scripps Research Institute.

The new technique, reported in Nature Methods on July 1, 2012, employs zinc finger nuclease (ZFN) proteins, which can bind and cut DNA at precisely defined locations in the genome. ZFNs are coming into widespread use in scientific experiments and potential disease treatments, but typically are delivered into cells using potentially risky gene therapy methods.

The Scripps Research scientists simply added ZFN proteins directly to cells in a lab dish and found that the proteins crossed into the cells and performed their gene-cutting functions with high efficiency and minimal collateral damage.

"This work removes a major bottleneck in the efficient use of ZFN proteins as a gene therapy tool in humans," said Michael K. Reddy, who oversees transcription mechanism grants at the National Institutes of Health's (NIH) National Institute of General Medical Sciences, which helped fund the work, along with an NIH Director's Pioneer Award. "The directness of Dr. Barbas's approach of 'simply' testing the notion that ZFNs could possess an intrinsic cell-penetrating ability is a testament to his highly creative nature and further validates his selection as a 2010 recipient of an NIH Director's Pioneer Award."

Questioning Assumptions

ZFNs, invented in the mid-1990s, are artificial constructs made of two types of protein: a "zinc-finger" structure that can be designed to bind to a specific short DNA sequence, and a nuclease enzyme that will cut DNA at that binding site in a way that cells can't repair easily. The original technology to make designer zinc finger proteins that are used to direct nucleases to their target genes was first invented by Barbas in the early 1990s.

Scientists had assumed that ZFN proteins cannot cross cell membranes, so the standard ZFN delivery method has been a gene-therapy technique employing a relatively harmless virus to carry a designer ZFN gene into cells. Once inside, the ZFN gene starts producing ZFN proteins, which seek and destroy their target gene within the cellular DNA.

One risk of the gene-therapy approach is that viral DNA-even if the virus is not a retrovirus-may end up being incorporated randomly into cellular DNA, disrupting a valuable gene such as a tumor-suppressor gene. Another risk with this delivery method is that ZFN genes will end up producing too many ZFN proteins, resulting in a high number of "off-target" DNA cuts. "The viral delivery approach involves a lot of off-target damage," said Barbas.

In the new study, Barbas and his colleagues set out to find a safer ZFN delivery method that didn't involve the introduction of viruses or other genetic material into cells. They experimented initially with ZFN proteins that carry extra protein segments to help them penetrate cell membranes, but found these modified ZFNs hard to produce in useful quantities. Eventually, the scientists recognized that the zinc-finger segments of ordinary ZFNs have properties that might enable the proteins to get through cell membranes on their own.

"We tried working with unmodified ZFNs, and lo and behold, they were easy to produce and entered cells quite efficiently," Barbas said.

New Strategy Against HIV

Next, the team showed how the new technique could be used in a ZFN-based strategy against HIV infection.

The AIDS-causing retrovirus normally infects T cells via a T cell surface receptor called CCR5, and removing this receptor makes T cells highly resistant to HIV infection. In 2006, an HIV patient in Berlin lost all signs of infection soon after receiving a bone marrow transplant to treat his leukemia from a donor with a CCR5 gene variant that results in low expression of the receptor. Disrupting the CCR5 gene in T cells with a ZFN-based therapy might be able to reproduce this dramatic effect.

"The idea is to protect some of the patient's T cells from HIV, so that the immune system remains strong enough ultimately to wipe out the infection," said Barbas.

A gene therapy that uses ZFNs to disrupt CCR5 genes in T cells and reinfuses the modified T cells into patients is currently in clinical trials. Barbas and his team showed that they could achieve the same effect with their simpler ZFN-delivery method. They added ZFN proteins directly to human T cells in a culture dish and found that within hours, a significant fraction of the ZFN-treated cells showed sharp reductions in CCR5 gene activity.

After several applications of ZFNs, aided by a special cooling method that improves the ability of the proteins to get across cell membranes, the scientists were able to inactivate CCR5 genes with an efficiency approximating that of the gene therapy-based approach, Barbas said.

The new approach also appeared to be safer. A DNA-based method the team used for comparison or the viral-based methods reported in the literature by others ended up producing ZFNs for up to several days, causing a significant amount of off-target DNA damage. But the directly delivered ZFN proteins remained intact within cells for only a few hours, causing minimal off-target damage.

"At some off-target locations where the gene therapy approach frequently causes damage, we saw no damage at all from this new technique," said Barbas.

Hope for 'Tiny Factories' of Health

The team tested its direct ZFN-delivery technique with a variety of other cell types and found that it works with particularly high efficiency in human skin "fibroblast" cells. Researchers now are working on advanced therapies in which they harvest such fibroblasts from patients and reprogram the cells' gene-expression patterns so that they effectively become stem cells. These induced stem cells can then be modified using ZFNs and other genome-editing techniques. When reinfused into a patient, they can produce millions of therapeutic progeny cells over long periods.

Such techniques may one day be used to treat a vast array of diseases. Barbas, who has been developing anti-CCR5 strategies for more than a decade, wants to start with a ZFN-based therapy that disrupts the CCR5 gene in hematopoietic stem cells. These blood-cell-making stem cells, reinfused into an HIV patient, would become tiny factories for producing HIV-resistant T cells.

"Even a small number of stem cells that carry this HIV-resistance feature could end up completely replacing a patient's original and vulnerable T cell population," he said.

The other authors of the paper, "Targeted gene knockout by direct delivery of ZFN proteins," are first author Thomas Gaj, and Jing Guo, Yoshio Kato, and Shannon J. Sirk, all members of the Barbas laboratory during the study.

The research was funded by NIH grants R01GM065059, DP1OD006990, and T32GM080209 and by the Skaggs Institute of Chemical Biology at Scripps Research.

About The Scripps Research Institute

The Scripps Research Institute is one of the world's largest independent, not-for-profit organizations focusing on research in the biomedical sciences. Over the past decades, Scripps Research has developed a lengthy track record of major contributions to science and health, including laying the foundation for new treatments for cancer, rheumatoid arthritis, hemophilia, and other diseases. The institute employs about 3,000 people on its campuses in La Jolla, CA, and Jupiter, FL, where its renowned scientists-including three Nobel laureates-work toward their next discoveries. The institute's graduate program, which awards Ph.D. degrees in biology and chemistry, ranks among the top ten of its kind in the nation. For more information, see

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Targeted gene knockout by direct delivery of zinc-finger nuclease proteins

Nature Methods 01 July 2012

Thomas Gaj1-3, Jing Guo1-3, Yoshio Kato1-4, Shannon J Sirk1-3 & Carlos F Barbas III1-3

1The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA. 2Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA. 3Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA. 4Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Higashi, Tsukuba, Japan

Zinc-finger nucleases (ZFNs) are versatile reagents that have redefined genome engineering. Realizing the full potential of this technology requires the development of safe and effective methods for delivering ZFNs into cells. We demonstrate the intrinsic cell-penetrating capabilities of the standard ZFN architecture and show that direct delivery of ZFNs as proteins leads to efficient endogenous gene disruption in various mammalian cell types with minimal off-target effects.

ZFNs are fusions of the nonspecific cleavage domain from the FokI restriction endonuclease with custom-designed Cys2-His2 zinc-finger proteins (ZFPs)1. These chimeric nucleases induce sequence-specific DNA double-strand breaks (DSBs) that can be repaired by error-prone nonhomologous end joining (NHEJ) to yield small alterations at targeted genomic loci. This strategy has enabled highly efficient gene disruption in numerous cell types2, 3 and model organisms4, 5 and has facilitated the progress of targeted gene therapy in humans6, 7. Despite these advances and more recent methodological improvements8, 9, 10, there remains a need for new methods that can improve the utility of these enzymes. The development of safe and effective ZFN delivery methods is of particular importance, as the deficiencies of ZFN gene-delivery systems may hinder the continued advancement of this technology. In particular, viral vectors11 are time consuming to produce and can be associated with undesirable side effects, such as insertional mutagenesis, whereas nonviral DNA and mRNA delivery systems are restricted to certain cell types and have been reported to show toxicity12, 13 and low efficiency14. To address this problem, we set out to develop a simple alternative to conventional ZFN delivery systems by investigating the direct delivery of purified ZFN proteins to cells.

We began by introducing protein transduction domains into the established ZFN architecture. For this, we genetically fused either the cell-penetrating peptide sequence from the HIV-1 TAT protein or a polyarginine motif to the N termini of ZFNs designed to target the human chemokine (C-C motif) receptor 5 (CCR5) gene6. These ZFNs, however, were consistently difficult to express or purify in quantities sufficient for analysis in cell culture (data not shown). Following these results, and based on the observation that ZFP DNA-binding domains carry a net positive charge (Fig. 1a), we hypothesized that ZFNs might penetrate the cell in the absence of additional modification. We expressed in Escherichia coli ZFNs designed to target the CCR5 gene and lacking any transduction domain and then purified them to homogeneity from either the soluble or the insoluble fractions (Supplementary Fig. 1). In vitro analysis confirmed that functional ZFN proteins with similar DNA cleavage profiles could be obtained by either method (Supplementary Fig. 2 and Supplementary Note).

To determine the ability of ZFN proteins to penetrate cells and stimulate mutagenesis, we generated a fluorescence-based reporter system to measure ZFN-induced DSBs (Fig. 1b). This system uses an integrated EGFP gene whose expression has been interrupted by a frameshift mutation introduced by a strategically placed ZFN cleavage site. ZFN proteins that penetrate reporter cells can induce DSBs at this target site and drive the introduction of small insertions and deletions in the EGFP locus by NHEJ. Because NHEJ is a stochastic process, approximately one-third of these mutational events (+2, +5, +8,... bp or -1, -4, -7,... bp) will restore the frame and EGFP function.

Direct application of ZFN proteins to reporter cells resulted in a dose-dependent increase in EGFP fluorescence, with maximum activity (6% EGFP-positive cells) achieved after treatment with 2 μM ZFN proteins (Fig. 1c). By comparison, transient transfection of ZFN expression plasmids under saturating conditions resulted in ~7% EGFP-positive cells (Supplementary Fig. 3). We observed no difference in activity between ZFN proteins purified from the soluble fraction or inclusion bodies (Supplementary Fig. 4). At all ZFN concentrations evaluated, the use of transient hypothermic culture conditions9 enhanced the efficiency of mutagenesis nearly twofold (Fig. 1c,d). Extended periods of incubation (>60 min) did not increase the frequency of genome editing (Supplementary Fig. 5). Consecutive protein treatments, however, did increase the percentage of EGFP-positive cells (Fig. 1d,e). Notably, repeated treatment with ZFN proteins over 3 d using transient hypothermic conditions yielded ~12% EGFP-positive cells (Fig. 1e). Sequence analysis of isolated EGFP-positive cells verified targeted mutagenesis, confirming the presence of the anticipated ZFN-induced insertions and deletions in the EGFP locus (Fig. 1f).

To determine the contribution of each ZFN component to cellular penetration, we incubated cells with fluorescently labeled ZFN or FokI cleavage domain proteins (Supplementary Fig. 6). We observed fluorescence in cell lysate following treatment with ZFN-in the presence or absence of a nuclear localization sequence-but not with the FokI cleavage domain, suggesting that zinc-finger domains facilitate cellular internalization.

We evaluated the efficacy of this approach for the disruption of endogenous genes by treating human embryonic kidney (HEK) 293 and human acute monocytic leukemia (THP1) cell lines, as well as primary adult human dermal fibroblast (HDF) and primary CD4+ T cells, with ZFN proteins targeting the CCR5 gene. These ZFNs used the high-activity Sharkey cleavage domain10. Analysis of DNA isolated from each cell type with the Surveyor nuclease assay showed efficient and dose-dependent disruption of the endogenous CCR5 gene (Fig. 2a). HEK293 and HDF cells subjected to three consecutive treatments with 2 μM ZFN proteins showed gene-disruption frequencies >24%, whereas CD4+ cells subjected to three consecutive treatments with 0.5 μM ZFN proteins had gene-disruption frequencies >8%. As observed in the reporter system, the frequency of gene disruption increased with repeated protein treatments (Supplementary Fig. 7). Sequence analysis of cloned CCR5 alleles amplified from each treated cell type confirmed the presence of ZFN-induced insertions and deletions in the CCR5 gene (Supplementary Fig. 8).

To investigate the cleavage specificity of ZFNs using this approach, we evaluated the activity of the CCR5 ZFN proteins against nine previously described6, 15 off-target cleavage sites in HEK293 cells (Supplementary Fig. 9). In direct comparison to Lipofectamine-mediated transient transfection of ZFN expression plasmids, we found that cells subjected to consecutive protein treatments showed a marked decrease in ZFN activity at every off-target site, including the CCR2 locus. Notably, there was no detectable ZFN activity at three of these loci. Western blot analysis showed complete degradation of delivered ZFN proteins <4 h after application, whereas cells transfected with ZFN expression plasmids produced high-levels of protein continuously from 16 h to 72 h after transfection (Supplementary Fig. 10). These results indicate that the differences in cleavage specificity could be attributable to the short half-lives of transduced ZFN proteins and that limiting the duration of ZFN exposure inside cells is a viable method for minimizing toxicity16. Consistent with these degradation kinetics, cells treated with ZFN proteins showed maximum activity at 8 h, whereas cells expressing ZFNs from plasmid DNA showed maximum activity at 48 h (Supplementary Fig. 10).

To examine the breadth of this technique, we treated Chinese hamster ovary (CHO) cells with ZFN proteins designed to target the dihydrofolate reductase (DHFR) gene2. These ZFNs used various specialized cleavage domains, including Sharkey and the evolutionarily optimized DS/RR obligate heterodimeric architecture10. We observed reduced levels of functional DHFR protein, as determined by fluorescein-labeled methotrexate-based flow cytometry analysis, in CHO cells following three consecutive treatments with DHFR ZFN proteins (Fig. 2b). Notably, CHO cells incubated with ZFNs containing Sharkey mutations showed a >12% reduction in functional DHFR. Sequence analysis of cloned DHFR alleles amplified from cells treated with ZFN proteins validated these percentages and confirmed the presence of ZFN-induced insertions and deletions in the DHFR gene (Supplementary Fig. 11). Examination of DHFR protein levels in expanded clonal populations indicated biallelic DHFR gene-disruption frequencies >7% (Supplementary Fig. 12), showing that constitutive ZFN expression from plasmid DNA is not required for high-frequency biallelic modifications and can instead be achieved using directly applied ZFN proteins.

We observed no appreciable toxicity in HEK293 or HDF cells treated with ZFN proteins (Fig. 2c) or in CHO cells incubated with ZFN proteins containing either the wild-type cleavage domain or the DS/RR architecture (Fig. 2d). However, we measured decreased proliferation in CHO and THP1 suspension cells incubated with >1 μM ZFN proteins containing Sharkey mutations (Fig. 2c,d). We also observed, qualitatively, toxicity in CD4+ cells subjected to consecutive treatments with >1 μM ZFN proteins, suggesting that sensitive cell types may require protein to be administered in consecutive low doses to minimize potential toxic effects.

We have demonstrated the intrinsic cell-penetrating capabilities of the standard ZFN architecture. Furthermore, we have shown that direct delivery of ZFNs as proteins can be used to disrupt the expression of endogenous genes in a variety of mammalian cell types, including primary CD4+ T cells and primary adult human dermal fibroblasts, which are frequently used to generate induced pluripotent stem cells. In contrast to methods that require ZFN expression from DNA, ZFN protein delivery leads to comparatively fewer off-target cleavage events and does not carry the risk of insertional mutagenesis. Thus, this method is suitable for genome-editing applications in which minimizing cellular toxicity or maintaining genetic integrity is of particular importance, such as the in vitro modeling of human diseases and the ex vivo modification of nontransformed human cell types. We show that this method can also be used to modify difficult-to-transfect cell types, including patient-derived leukemia cell lines and primary human lymphocytes, supporting the use of this technique in place of viral-mediated gene delivery for inducing gene knockouts in cultured cells for reverse genetics and drug discovery. As methods for engineering cell permeability into proteins improve, we anticipate that protein delivery and the benefits afforded therein will be extended to other designer nucleases, including TALENs.

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