Cas9

Accepted Manuscript Title: Delivery and Therapeutic Applications of Gene Editing Technologies ZFNs, TALENs, and CRISPR/Cas9 Author: Justin S. LaFountaine Kristin Fathe Hugh D.C. Smyth PII: DOI: Reference:

S0378-5173(15)30126-5 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.08.029 IJP 15112

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

30-6-2015 5-8-2015 9-8-2015

Please cite this article as: LaFountaine, Justin S., Fathe, Kristin, Smyth, Hugh D.C., Delivery and Therapeutic Applications of Gene Editing Technologies ZFNs, TALENs, and CRISPR/Cas9.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.08.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Delivery and Therapeutic Applications of Gene Editing Technologies ZFNs, TALENs, and CRISPR/Cas9 Justin S. LaFountaine1, Kristin Fathe1, and Hugh D. C. Smyth1* 1

The University of Texas at Austin, 2409 University Avenue, Austin, TX, 78712, USA.

Cor r esponding Author * Address: The University of Texas at Austin, College of Pharmacy, Division of Pharmaceutics, 2409 University Avenue A1920, Austin, TX, 78712, USA. E-mail: [email protected]

Graphical abstract

Abstract

In recent years, several new genome editing technologies have been developed. Of these the Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the CRISPR/Cas9 RNA-guided endonuclease system are the most widely described. Each of

these technologies utilizes restriction enzymes to introduce a DNA double stranded break at a targeted location with the guide of homologous binding proteins or RNA. Such targeting is viewed as a significant advancement compared to current gene therapy methods that lack such specificity. Proof-of-concept studies have been performed to treat multiple disorders, including in vivo experiments in mammals and even early phase human trials. Careful consideration and investigation of delivery strategies will be required so that the therapeutic potential for gene editing is achieved. In this review, the mechanisms of each of these gene editing technologies and evidence of therapeutic potential will be briefly described and a comprehensive list of past studies will be provided. The pharmaceutical approaches of each of these technologies are discussed along with the current delivery obstacles. The topics and information reviewed herein provide an outline of the groundbreaking research that is being performed, but also highlights the potential for progress yet to be made using these gene editing technologies.

KEYWORDS zinc finger nucleases, transcription activator-like effector nucleases, CRISPR/Cas9, gene editing, viral vectors, non-viral vectors

1. I NTRODUCTI ON The initial promises of gene therapy as a broad-spectrum treatment for a variety of diseases waned due to several tragic setbacks. Early gene therapy trials successfully cured 17 children with severe combined immunodeficiency-X1 (SCID X-1) (Cavazzana-Calvo et al., 2000; Fischer et al., 2010), however, four of the patients later developed leukemia-like symptoms (Hacein-Bey-Abina et al., 2008; Hacein-Bey-Abina et al., 2003; Thomas et al., 2003a). The development of leukemia-like symptoms was attributed to the retroviral-mediated gene delivery that inserts the corrective gene into an unpredictable location within the

chromosome. In some cases, insertional mutagenesis can occur in a part of the human genome that regulates cell growth and division, and results in the uncontrolled proliferation of cells (Check, 2002; Kaiser, 2003). In another gene therapy trial for the deficiency of ornithine transcarbamylase, a patient died due to an immunological response to the adenovirus vector used to deliver the corrective gene (Thomas et al., 2003a). In both of these tragic incidences, the therapeutic delivery method had a role in the adverse outcomes, whether due to a severe immune response or a nonspecific insertion. Recently, there is renewed enthusiasm for gene therapy with the discovery of three new types of gene editing technologies: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) – CRISPR associated (Cas9) system (CRISPR/Cas9) (Cai and Yang, 2014; Gaj et al., 2013; Kim and Kim, 2014). These three gene editing technologies have been the subject of many recent primary research papers and reviews, however in this manuscript we will provide a comprehensive outline of their mechanisms, various applications, strategies for therapeutic delivery, current set backs, and future directions of these modern gene editing technologies. Each of these technologies utilizes restriction enzymes to introduce double stranded breaks (DSBs) in DNA at specific locations based on engineered proteins or RNA that target, and specifically bind, to a designated sequence of the genome. These technologies offer great therapeutic potential by significantly reducing the risk of off-target mutagenesis. All three of these new gene editing technologies leverage the endogenous, eukaryotic mechanisms of DSB repair: non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is the primary mechanism of DNA repair in eukaryotic cells (Lieber et al., 2003). Simply, NHEJ repairs DSBs by joining the two broken ends of DNA using specific protein

factors that re-ligate the strand without a homologous DNA template (Hefferin and Tomkinson, 2005). The two ends of DNA may be directly ligated or there can be processing of the DNA that will cause and insertion or deletion event (indel) of nucleic acids in order to promote ligation. In either of these cases, mutations and NHEJ-mediated deletions are common (van Gent and van der Burg, 2007). Alternatively, the repair of a DSB can occur through HDR, a precise repair mechanism that requires a homologous DNA template (endogenous or donor) to serve as a guide to make the repair (Capecchi, 1989; Takata et al., 1998). This technology has been harnessed for years with implications in genetically altered mouse models and stem cell therapies (Gordon and Ruddle, 1981; Hall et al., 2009). A representation of both NHEJ and HDR following nuclease induced DSBs, including the various repair pathways, is shown in Figure 1. NHEJ and HDR are essential endogenous pathways that can be exploited when using new gene editing technologies.

2. M ECHANI SMS OF NEW GENE EDI TI NG TECHNOL OGIES 2.1. Zinc Finger Nucleases ZFNs were the first specific gene editing technique to be developed in the early 2000’s (Bibikova et al., 2003; Bibikova et al., 2002). The fusion of two components forms a ZFN: 1) a sequence of 3 to 6 zinc finger proteins and 2) the restriction enzyme FokI. The first component of ZFNs, zinc finger proteins, are common in eukaryotic cells and are involved with transcriptional regulation and protein-protein interactions (Wolfe et al., 2000). It is their highly specific DNA binding (each zinc finger recognizes a 3 base pair sequence) that initially generated significant interest in zinc fingers as a gene editing tool. Individual zinc fingers have been developed that can bind most 3-bp combinations and various techniques have been

developed for optimal assembly of zinc finger sequences (Urnov et al., 2010). The second component of ZFNs is the FokI nuclease. FokI is a restriction enzyme found in the bacterium Flavobacterium okeanokoites that consists of DNA recognition and cleavage domains (Wah et al., 1998). FokI only cleaves DNA when it forms dimers (Bitinaite et al., 1998), thus gene editing by ZFNs requires two monomers that bind to the top and bottom strands of DNA to induce a DSB. By replacing the natural DNA recognition domain of FokI with a designed zinc finger sequence, a ZFN is formed that has the ability to cleave DNA at a targeted location based on zinc finger specificity. Total target DNA sequences are typically 18 to 36-bps in length as shown in Figure 2. A targeted DSB introduced by ZFNs can result in gene disruption through mutagenesis caused by indels during NHEJ or gene correction/addition through HDR with codelivery of a donor DNA template. 2.2. Transcr iption Activator -L ike Effector Nucleases Shortly after the discovery of ZFNs for specific genome editing, a new class of DNA binding proteins was discovered in gram-negative plant pathogens such as Xanthomonas termed Transcription Activator-Like Effectors (TALEs) (Fujikawa et al., 2006; Wright et al., 2014). Each TAL effector protein contains ~34 amino acids that were found to be largely similar in composition except for two amino acids at positions 12 and 13 (Boch et al., 2009; Moscou and Bogdanove, 2009). A total of four TAL effector proteins with specific domains were found to bind each of the four individual amino acids guanine (G), adenine (A), cytosine (C), and thymine (T), respectively along the major groove of the DNA double helix. This 1:1 binding affinity between TALEs and the four DNA bases allows for the construction of a TALE array that can recognize any DNA sequence.

Based on this code, TALE arrays were later linked to endonucleases, the same Fok1 as ZFNs, to form hybrid TALE Nucleases (TALENs) for gene editing purposes. Similarly to ZFNs, TALENs create specific DSBs in target sequences of DNA around 30 to 40-bps, as shown in Figure 3. Greater detail on the mechanism and design of TALENs has been the topic of multiple reviews (Joung and Sander, 2013; Sun and Zhao, 2013; Wright et al., 2014). 2.3. CRI SPR/Cas9 The newest of the three gene editing technologies reviewed here, CRISPR/Cas9, was adapted from a recently discovered immune system found in prokaryotes (van der Oost et al., 2009). It was established that archaea and bacteria have evolved a defense mechanism against viruses and plasmids in which a segment (~20 bp) of invading DNA is copied into the host genome at a locus of clustered regularly interspaced short palindromic repeats (CRISPR) (Terns and Terns, 2011). This locus in the host genome serves as a genomic memory of invading pathogens. Upon future invasions, a corresponding crispr RNA (crRNA) strand is transcribed from the specific locus. This crRNAwill recognize and bind to the foreign DNA through base pairing and together with an endogenous CRISPR-associated endonuclease (Cas), a DSB will be introduced in the pathogenic DNA, inhibiting integration and replication of the pathogen. Shortly after this discovery, researchers began to evaluate this novel CRISPR/Cas system in eukaryotic cells. The Cas9 endonuclease was optimized to include nuclear localization signals for human cells and single guide RNA (sgRNA) can be synthesized to target any 20-bp DNA sequence (Mali et al., 2013). The following details of the DNA recognition and subsequent double stranded cleavage by CRISPR/Cas9 have been the subject of many reviews (Doudna and Charpentier, 2014; Liu and Fan, 2014; Sander and Joung, 2014). An illustration of the CRISPR/Cas9 system is shown in Figure 4.

The CRISPR/Cas9 system represents a departure from the technologies of ZFNs and TALENs. The Cas9 endonuclease operates as a monomer to induce DSBs, whereas the FokI in ZFNs and TALENs operates as a dimer. The enzymatic machinery remains the same for any intended target; only the guide RNA provides DNA binding affinity and therefore targeting. Thus, CRISPR/Cas9 requires no protein engineering for any change in target, only synthesis of a new guide RNA. This simplicity has dramatically reduced the time needed to conduct genome engineering experiments. 3. EVI DENCE OF THERAPEUTI C POTENTI AL Each of the three technologies described above have spent variable amounts of time being tested for therapeutic efficacy and potential. In this section, we will explore the most relevant data and results from the various studies involving ZFNs, TALENs, and CRISPR/Cas9. 3.1. Zinc Finger Nucleases The use of ZFNs for therapeutic purposes has primarily been focused on common inherited monogenic disorders such as SCID and cystic fibrosis as well as acquired infections such as HIV and HBV. Studies have been conducted in a variety of animal and human cell lines and have included gene disruption mechanisms by NHEJ and gene correction by HDR through the use of donor templates. ZFNs are among the most mature gene editing technologies with successful completion of early phase human clinical trials for the treatment of HIV (Tebas et al., 2014). A list of known therapeutic applications that have been investigated by ZFNs is shown in Table 1.

Some of the earliest investigations using ZFNs for therapeutic applications were in cells containing a mutation in the IL2R?gene that results in X-linked SCID (Urnov et al., 2005). In these studies, two ZFN binding domains were developed to recognize 24-bps surrounding the mutated site and were transfected into K-562 (human bone marrow chronic myelogenous leukemia) and human CD4+ T-cells, along with a donor plasmid that carried a single point mutation in the IL2R?fragment. After only 4 days, the corrective point mutation was observed in 18% of the cells, confirming HDR had occurred with the donor plasmid. Analysis showed the cells were stably transfected and the genetic modifications were passed onto the daughter cells, demonstrating the clinical utility of ZFNs to correct inherited monogenic disorders. The use of ZFNs for the treatment of X-linked SCID were further verified in other studies that showed high rates of gene addition in several human cell types through delivery by integrase-defective lentiviral vectors (IDLVs) (Lombardo et al., 2007). Future studies demonstrated the efficacy of ZFNs to correct genetic disorders in vivo. Hemophilia B is a genetic disorder affecting liver cells causing a deficiency in the production of the blood coagulation factor IX. Most mutations of the F9 gene responsible for the production of factor IX are distributed between exons 2-8 of the gene, thus targeting any single allele with a ZFN would not be effective. However, researchers have demonstrated that targeting the first intron upstream of the mutations with ZFN and inducing HDR with a donor-template encoding factor IX leads to higher expression of the protein (Li et al., 2011). This strategy was demonstrated in vivo in transgenic hemophilic mice, where treatment increased concentrations of circulating factor IX and a significantly shortened clotting time in an activated partial thromboplastin time test when compared to untreated mutants.

An alternative approach was used to demonstrate the in vivo efficacy of ZFNs in another X-linked hereditary disease, Duchenne muscular dystrophy (DMD). DMD is caused by a mutation in the dystophin gene, which codes for a protein that is essential for muscle function. Loss of dystophin results in the progressive wasting of muscles and eventual loss of life. A mutation in exon 51 of the gene accounts for 13% of patients with the disease (Kinali et al., 2009). In a novel approach, researchers used two pairs of ZFNs that would flank each side of exon 51 in order to permanently delete the exon after NHEJ and restore protein expression (Ousterout et al., 2014). This approach was successful in DMD myoblasts, which were then implanted into the limbs of mice to demonstrate continued expression in vivo after transplantation. The strongest support for ZFNs as a therapeutic agent lies in the positive results obtained in human clinical trials for treatment of HIV thus far. HIV often requires a specific receptor, the C-C chemokine receptor type 5 (CCR5), found on the surface of CD4+ T-cells in order to facilitate viral integration into cells. It has been found that the absence of the CCR5 receptor does not result in any significant adverse events in healthy humans, and it has even been established that certain portions of the population possessing a null CCR5 genotype are resistant to HIV infection (Samson et al., 1996). Sangamon Biosciences has leveraged the ability of ZFNs to disrupt the expression of CCR5 by introducing DSB-induced indels on the CCR5 gene. Mutated CD4+ T-cells are formed ex-vivo and then reintroduced into patients with HIV infection in order to confer resistance to HIV replication (Perez et al., 2008). A positive phase I trial of this approach in HIV-positive patients has been completed and a Phase 2 clinical trial is ongoing (Tebas et al., 2014). Sangamon Biosciences has taken the synergistic technologies of hiPSCs and ZFNs and shown the value and versatility of these tools together. Recently, they demonstrated that mutations that lead to DNA repair deficiency syndromes such as Fanconi

Anemia could be corrected with the use of ZFNs and integrase defective lentiviral vectors to reprogram hiPSCs. These reprogrammed cells showed the potential to become hemopoetic progenitor cells, which produced further generations of corrected cells (Rio et al., 2014). Among the three gene editing technologies, ZFNs are the first to complete a phase of human clinical trials, representing an important milestone for these new technologies. 3.2. Transcr iption Activator -L ike Effector Nucleases Similar to ZFNs, the therapeutic potential of TALENs has been demonstrated in cellular and animal models for a variety of genetic and acquired disorders. A list of known therapeutic investigations using TALENs is provided in Table 2. Examples of both gene disruptions and gene corrections by NHEJ and HDR, respectively, have been reported. While TALENs are not as mature as ZFNs, and no clinical trials have begun, a number of the investigators in Table 2 report the increased specificity and less laborious engineering of TALENs relative to ZFNs.

A majority of the published studies focus on treating inherited monogenic disorders including SCID, DMD, A1ATD, PV, RDEB, sickle cell anemia, and hemophilia A. TALEN technologies have progressed such that they can even correct most common point mutation that is responsible for the condition of cystic fibrosis in hiPSCs. The procedure using TALENs has become so robust that it has been published as a methods paper using hiPSCs (Sargent et al., 2014). For diseases such as DMD and hemophilia A, it was shown that TALENs could be used to correct a mutation in the absence of providing a donor DNA template for repair. This represents an interesting case as most cases of hemophilia A are characterized by a chromosomal inversion in the F8 gene. To tackle this, researchers developed a pair of TALEN dimers that

would target two ends of a 140-kbp chromosomal sequence in hemophilia A model hiPSCs (created by the same pair of TALENs) (Park et al., 2014). After transfection with the TALEN plasmids, reversion to wild type was observed in 1.3% of cells. Additionally, when the iPSCs were differentiated into endothelial cells, expression of the F8 protein necessary for blood coagulation was also observed. This study suggests that gene editing technologies such as TALENs can be used for more complicated genome errors, such as long sequence chromosomal inversions. In other disease models, gene correction by HDR was performed by including a donor DNA template with TALEN-encoding plasmids (Ma et al., 2013). In order to correct the mutation in the -globin gene responsible for -thalassemia disorders, researchers developed donor templates that contain a 2.4-kb 5 homology arm encoding the entire wild type -globin gene and introduced the donor with TALEN vectors into hiPSCs by electroporation. This strategy resulted in very high efficiencies of 68% and 40% in hiPSCs from two different patients, respectively. The hiPSCs remained pluripotent and when differentiated were able to express normal -globin protein. TALENs can also be effective at targeting diseases resulting from mutations in mitochondrial DNA (mtDNA). Mitochondria are unique organelles that have their own circular DNA containing 37 genes (Chan, 2006). The accumulation of mutations in mtDNA is heavily associated with the process of ageing and also can be transferred to germ cells (Ross et al., 2013). Mutations in these genes can cause disorders such as mitochondrial myopathy, MERRF syndrome, and Leigh syndrome. Researchers have engineered TALENs to specifically target mtDNA by including mitochondrial localization sequences rather than nuclear localization sequences (Bacman et al., 2013). This strategy was effective at correcting a point mutation that

causes Leber’s optic neuropathy resulting in increased wild type mtDNA and a normal biochemical phenotype in patient-derived cells. Since mtDNA mutations are heteroplasmic, and typically require a high mutation level to be problematic, even small increases in wild type mtDNA can be effective to restore normal function. Strategies employed by ZFNs to combat acquired diseases have also been replicated and expanded with the use of TALENs. Targeting of the CCR5 gene necessary for HIV replication was performed in hiPSCs successfully (Ye et al., 2014). Similarly, another cellular protein required for HIV integration, LEDGF/p75, was knocked out in Jurkat cells, which then had confirmed inhibition of HIV-1 integration (Fadel et al., 2014). TALENs have also been developed to target the viral DNA of HBV and HPV in order to reduce infection (Bloom et al., 2013; Chen et al., 2014; Hu et al., 2014b). In the latter case, in vivo targeting of HPV oncogenes E6 and E7 in mice resulted in apoptosis and growth inhibition of malignant cells, demonstrating a potentially therapeutic use of TALENs for treating both HPV infection and cervical cancer. 3.3. CRI SPR/Cas9 Even though the first reported uses of the CRISPR/Cas9 system as a gene editing technique occurred in January 2013, significant progress has been made in the last two years demonstrating therapeutic potential (Table 3). This is likely attributed to the simplicity and relative affordability of the system that makes it very accessible to researchers. Looking at the disease targets for CRISPR/Cas9, along with ZFNs and TALENs, there is a clear trend in evaluating these three new gene editing technologies to treat inherited monogenic disorders and for antiviral therapy. For the CRISPR/Cas9 system, the path to clinical testing may still be years away, but recent in vivo studies including some performed in non-human primate embryos (Niu et al., 2014) continue to rapidly progress this technology towards that goal.

Several monogenic disorders such as sickle cell anemia, DMD, and A1ATD have now been evaluated by all three gene editing technologies with varying efficiencies. In the case of A1ATD, head-to-head evaluations comparing NHEJ and HDR efficiencies have been performed between TALENs and CRISPR/Cas9 (Smith et al., 2014). In evaluating the ability of each technique to target the AAT locus and introduce indels by NHEJ, CRISPR/Cas9 was found to result in ~ 100-fold more indels compared to TALENs in hiPSCs. When a donor DNA template was added to induce HDR, CRISPR/Cas9 and TALENs were found to comparably introduce the corrected gene. This trend was also observed for two other gene targets (Jak2 and the safeharbor locus AAVS1), suggesting that the intended DNA repair pathway may be a determining factor in selecting one particular gene editing technique over another. Interestingly, CRISPR/Cas9 was effective in correcting the mutation in the CFTR gene (deletion of F508) that causes cystic fibrosis in patient-derived intestinal stem cells (Schwank et al., 2013). Patient organoids were transfected with sgRNAs that targeted exon 11 and intron 11 of the CFTR gene along with a donor plasmid encoding the wild type CFTR sequence. Several organoids were isolated that contained the corrected wild-type CFTR gene. These cells were then expanded and shown to regain wild-type function based on forskolin-induced surface area increases. While these results are promising, the multi-organ nature of cystic fibrosis does not make it an attractive candidate for stem cell based therapies, and other in vivo approaches may be necessary. CRISPR/Cas9 gene editing has also been utilized to correct a genetic disorder in mice that results in cataracts. A dominant mutation in the Crygc gene was corrected by co-injecting Cas9 mRNA, a single guide RNA, and a donor oligonucleotide into zygotes (Wu et al., 2013) or

spermatogonial stem cells (SSCs) (Wu et al., 2015). In the former, some of the fertilized embryos developed to term with normal phenotypes and the change went germline. In the case of SSCs, healthy offspring could be produced with 100% efficiency by generating spermatids from corrected lines for fertilization. It can be envisioned that a similar procedure could be applied to human SSCs to treat paternal dominant disease alleles and X-linked dominant genetic diseases. Proof of concept has also been demonstrated for CRISPR/Cas9-mediated antiviral therapy. Similar to ZFNs and TALENs, CRISPR/Cas9 can efficiently knock out the CCR5 gene in order to prevent HIV-1 integration (Ye et al., 2014). Conversely, CRISPR/Cas9 has been used to target the integrated HIV-1 genome directly in human cells, eradicating the virus in tested cell lines (Hu et al., 2014a). Direct targeting of viral genome sequences with no host-genome homology is a strategy that has also been employed for EBV and HPV in order effectively treat viral infections with reduced chances of off-target mutations in human DNA (Wang and Quake, 2014; Zhen et al., 2014). Finally, CRISPR/Cas9 gene editing has been evaluated as a potential alternative to statin therapy to prevent coronary heart disease (Ding et al., 2014). PCSK9 is a protein expressed in liver cells that acts as an antagonist to low-density lipoprotein (LDL) receptors, resulting in higher levels of LDL cholesterol (LDL-C). Individuals with a loss-of-function mutation in PCSK9 have between 30 and 80% reductions in LDL-C concentrations with seemingly no adverse effects. Thus, knockout targeting of PCSK9 was performed in mice by adenovirus delivery of CRISPR/Cas9. Four days after administration, PCSK9 mutations were as high as 50% in liver cells with 35 to 40% reductions in plasma cholesterol levels. This suggests a onetime treatment using CRISPR/Cas9 could lead to a therapeutic effect, offering a substantial

benefit over therapies that must be taken chronically, often resulting in reduced patient compliance. 4. CURRENT DEL I VERY STRATEGI ES AND THERAPEUTI C OBSTACL ES In order for these technologies to become clinically viable for a wide range of diseases, they will need to be efficiently delivered to target cells ex vivo and in vivo. These gene editing technologies can be delivered to target cells by viral and non-viral vector-mediated delivery of an expression cassette, which will allow for their translation into functional enzymes within the cell. As will be reviewed, in some cases the proteins can also potentially be directly delivered to target tissues with or without assistance of exogenous lipids. If gene addition is required via exogenous HDR, the donor sequence also needs to be incorporated into the delivery vehicle, adding complexity to the challenge of achieving efficacious and safe therapeutics. Beyond therapeutic potential, the delivery of these enzyme systems has become a valuable tool for genomic modeling that is becoming readily available in easy to use kits through commercial sources. 4.1. Deliver y Challenges and Str ategies for Zinc Finger Nucleases 4.1.1. Challenges The sequence encoding a ZFN monomer is relatively short (~1kb) thus they are not likely to be limited by vector capacities. The greatest limitations for ZFNs relative to other gene editing technologies are their limited targeting density, or the limited number of sites that they can effectively and selectively target. This decrease in the overall amount of DNA that can be targeted by ZFNs is due to insufficient 3-bp combinations of ZFPs (only around one site in every 100 base pairs exists) (Kim and Kim, 2014) and their reported cytotoxicity because of the off site

effects that may occur when not exactly directed (Porteus and Baltimore, 2003). However, the targeting density is still sufficient for most genes and advances to ZFN design have been made over the years to reduce cytotoxicity (Bach et al., 2014; Sander et al., 2011; Wilson et al., 2013). DNA targeting specificity has been found to be the major determinant of both the activity and toxicity of ZFNs (Cornu et al., 2008). Efforts to improve the specificity of ZFNs have included the development of heterodimer cleavage domains (Miller et al., 2007; Szczepek et al., 2007), optimizing the spacer requirements between the two target DNA sections through linker design (Handel et al., 2009), and enhancing cleavage activity through directed evolution of the FokI nuclease (Doyon et al., 2011). The development of heterodimer nucleases is perhaps the most significant contribution to increasing their specificity. Previously, ZFN homodimers (e.g. two left monomer or two right monomers) could bind and cleave the DNA at an off-target site, as illustrated in Figure 5. By engineering obligate heterodimers, individual ZFNs were found to interact with a larger amount of DNA and therefore confer a greater specificity (Miller et al., 2007) (Cornu and Cathomen, 2007) (Hurt et al., 2003). 4.1.2. Viral Deliver y Viral vectors are the most common delivery vectors evaluated for ZFNs (and delivery of nucleic acids overall (Ginn et al., 2013)) due to viruses’natural ability to enter cells and propagate their genome. Preferably, ZFNs will be transiently within a cell just long enough to produce the desired DSB, thus non-integrating viral vectors are ideal, as they produce episomes that eventually degrade (Apolonia et al., 2007; Basu and Willard, 2005). As was previously described in the introduction, lentiviral vectors that directly incorporate into the host DNA have been avoided because of multiple tragedies involving patient death in earlier clinical trials (Thomas et al., 2003b). Therefore modified viral vector such as integration-deficient lentiviral

vectors (IDLV), adenoviral vectors, and adeno-associated viral (AAV) vectors have been heavily explored in recent years. Two groups of researchers have evaluated the delivery of a ZFN expression cassette via integration-deficient lentiviral vectors.(Cornu and Cathomen, 2007; Lombardo et al., 2007) In both cases, a donor sequence was also delivered to induce gene correction through HDR. Gene corrections were as high as 6 and 12% of cells in each study depending on the vector dose and cell type evaluated, however unintended integration associated with the IDLVs was also observed. Additionally, both of these studies were performed in vitro and thus it remains to be seen if IDLV will be a suitable delivery method for ZFN in vivo. IDLVs remain an attractive option going forward due to continued advances in reducing the risk of insertional mutagenesis, their ability to transfect dividing and non-dividing cells, their low immunogenicity, and limited pre-existing immunity (Crommelin and Sindelar, 2004; Matrai et al., 2010). Adeno-associated viral vectors and recombinant adeno-associated viral (rAAV) vectors are attractive options for delivery of ZFNs and have been investigated for this purpose by multiple investigators (Ellis et al., 2013; Gellhaus et al., 2010; Metzger et al., 2011; Rahman et al., 2013). AAVs can transfect a variety of dividing and non-dividing cell types and are nonpathogenic. The major limitation of traditional AAVs is their relatively small genome size (~ 4.7 kb), which restricts an expression cassette to ~4.5 kb. Fortunately, the sequences to express ZFN monomers are ~ 1 kb and thus, with careful design, two monomers along with an optional donor DNA template can be accommodated in a single vector. Delivering all of the components in a single vector results in greater specificity and reduced off-target effects compared to cotransduction of multiple AAVs. rAAV vectors containing two ZFN monomers, as well as a donor DNA template, have been successfully demonstrated in mouse models (Ellis et al., 2013).

Interestingly, the transduction efficiencies were further improved by up to 6-fold with coadministration of FDA approved proteasome and histone deacetylase inhibitors (Ellis et al., 2013). Other studies have reported using adenovirus vectors for highly efficient delivery of ZFNs (Perez et al., 2008), however the strong immune response elicited by these vectors remains a clinical concern (Gregory et al., 2011). Additionally, it has been noted that AAVs work more efficiently in some tissues, but are less effective in others (Giacca and Zacchigna, 2012). 4.1.3. Non-Viral Deliver y Cationic liposomes and cationic polymers such as polyethylenimine (PEI) can be used as non-viral alternatives to deliver DNA plasmids into cells. The cationic charge of the particle is attracted to the anionic charge of cell membranes, thereby inducing cellular uptake (Almofti et al., 2003; Xu and Szoka, 1996). Non-viral vectors are not limited by the expression cassette size within the plasmid and their use has increased in gene therapy trials over the years after some initial setbacks with viral-mediated delivery vectors (Ginn et al., 2013). However, non-viral vectors tend to be less efficient and can exhibit toxicity at high concentrations (Lappalainen et al., 1994; Olson et al., 1982). Cationic liposomes have shown the most promise in hiPSCs, when utilized like conventional cationic lipid delivery systems in cell culture. There are several examples of researchers using cationic liposomes to transfect ZFN plasmids into cells in-vitro (Cradick et al., 2010; Perez et al., 2008; Urnov et al., 2005), but to the best of our knowledge there are no reports of researchers developing non-viral vectors for in vivo delivery of ZFNs. As an alternative to vector-based delivery of ZFNs, direct delivery of ZFN proteins into cells has also been investigated (Gaj et al., 2012). Typically, proteins have poor cell-penetrating activity and methods to improve cell penetration of proteins are an active area of research (Fonseca et al., 2009; Mae and Langel, 2006; Torchilin, 2005). In order for proteins to penetrate

cells directly, they must have a minimum level of lipophilicity to cross freely through the cellular membrane (Camenisch et al., 1998). Additionally, direct protein delivery is accompanied by other issues such as pH sensitivity and degradation by endogenous proteases (Morishita and Peppas, 2006). Surprisingly, it was found that ZFN are inherently cell-penetrating due to the positive charge of the Cys2− His2 zinc finger domains (Gaj et al., 2012). Direct delivery of ZFN protein was found to efficiently transfect and disrupt the CCR5 gene in HEK293 and HDF cells (>24% gene disruption with 3 doses of 2 μM ZFN) and human CD4+ T-cells (>8% gene disruption with 3 doses of 0.5 μM ZFN). Off-target mutagenesis was also significantly reduced compared to plasmid delivery of ZFN due to the shorter half-lives of ZFN proteins. These findings are the first step to support direct in vivo delivery of ZFN proteins as a viable alternative to DNA-based expression methods for therapeutic applications. In addition to the ability that ZFNs have to directly penetrate cell membranes, ZFN proteins have also been directly delivered as cargo in lentiviral particles (Cai et al., 2014). Delivery of left and right ZFN proteins, co-packaged in lentiviral particles, was shown to be feasible resulting in gene disruption efficiencies ranging from 13 to 24% in HEK293 cells, human dermal fibroblasts, and primary human keratinocytes. Furthermore, gene editing was also demonstrated even with a donor sequence also packaged in the lentiviral particles (Cai et al., 2014). Gene editing frequencies of up to 8% were observed in treated cells, offering a viable alternative to CPP-mediated delivery and repair. 4.2. Deliver y Challenges and Str ategies for Transcr iption Activator -L ike Effector Nucleases 4.2.1. Challenges

A number of advantages of TALENs have been reported including reduced cytotoxicity in comparative studies with ZFNs (Mussolino et al., 2011), relative ease of engineering, and greater design flexibility owing to their 1:1 DNA binding domain. However, some of these advantages may be hampered by additional challenges in cellular delivery and mutation rates (Chen et al., 2013; Gaj et al., 2013). This can partly be attributed to the increased size of TALENs. Each TALEN monomer requires an expression cassette of ~3-kb (Kim and Kim, 2014). While AAV vectors are promising delivery vehicles for nucleic acids, including ZFN transgenes, their capacity (~4.8-kb) would be too small to accommodate a pair of TALEN monomers (> 6-kb). Additionally, TALENs themselves are large proteins with highly repetitive gene sequences. Highly repetitive DNA sequences are known to exhibit instability during replication, repair, and recombination (Bzymek and Lovett, 2001; Pearson et al., 2005), which has further limited the vectors that are capable of successfully transferring an intact TALEN gene into human cells (Holkers et al., 2013). This was evident in studies attempting to use IDLVs to introduce vector genomes encoding TALEN proteins into HeLa cells (Holkers et al., 2013). IDLV delivery resulted in rearranged TALEN sequences and further testing by PCR, Southern blot, and DNA sequencing attributed these rearrangements to recombinant events in these repeat sequences (Holkers et al., 2013). Introduction of rearranged TALEN sequences is certainly undesirable and the extent of the ramifications could be severe. This study further highlights the limitations in delivering TALEN containing vectors, with both IDLVs and adeno-associated viral vectors potentially excluded. 4.2.2. Viral Deliver y

Given the challenges associated with IDLV and AAV delivery of TALENs, viralmediated delivery has largely been focused on adenoviral vectors. In the same article describing the deficiencies of IDLVs, adenoviral vectors were shown to efficiently transfer intact, functional TALEN transgenes into HeLa cells (Holkers et al., 2013). The resulting TALEN proteins were additionally highly efficient at producing the desired DSB at the target chromosomal location. In a follow up study by the same group, adenoviral vectors were found to be highly accurate at introducing targeted gene insertions by HDR when delivering TALENs and donor DNA (Holkers et al., 2014). Furthermore, it was found that protein-capped adenovirus genomes led to more precise gene correction by HDR templates compared to un-capped linear templates. Given the high loading capacity of adenoviral vectors, their ability to transfect dividing and nondividing cells, and rapid, transient expression, they are highly attractive for viral delivery of TALENs. However, the strong immune response elicited by these viruses may limit their potential in clinical settings and efforts to tame this response (e.g. coadministration of immunosuppressive agents, engineering recombinant viruses, and pegylation) remains an area of intense research interest (Ahi et al., 2011). 4.2.3. Non-Viral Deliver y Non-viral delivery of TALEN encoding plasmids is also relevant given the large genomic size of the cargo. In one example, TALEN plasmids complexed with cationic polymers were found to be therapeutically efficacious when delivered in vivo (Hu et al., 2014b). HPV-targeting TALEN plasmids were complexed with TurboFect®, a proprietary blend of cationic polymers, and directly applied to the cervix of transgenic mice displaying HPV infection and cervical cancer. The therapy successfully reduced viral loads and tumor size. Off-target mutations were not detected and the treatment was well tolerated with no signs of inflammatory response, as

characterized by a lack of elevated leukocytosis levels or appearance of mast cells, macrophages, or neutrophils. Direct delivery of TALEN proteins, rather than transgene delivery, has also been evaluated. Unlike ZFNs, which were found to be naturally cell-penetrating, TALEN proteins were found to be incapable of penetrating cellular membranes when administered alone (Ru et al., 2013). Thus, the use of cell-penetrating peptides (CPPs) has been evaluated to promote cellular uptake. The HIV-1 TAT protein, which is highly cationic and hydrophilic, was fused with TALEN proteins and incubated with HeLa and hiPSC cells at 37°C for three-one hour treatments. The fusion proteins adequately crossed the cellular membranes and the TATTALEN disrupted the targeted CCR5 gene in 3% and 5% of HeLa and hiPSC cells, respectively. In a similar study, poly-Arg9 peptides (R9 CPP) were conjugated to TALEN proteins, which were released by disulfide bond reduction upon systolic entry into cells (Liu et al., 2014). The conjugated R9-TALEN proteins were able to transfect and knockout CCR5 and BMPR1A genes in HeLa and HEK293 cells, respectively, at levels that were comparable to vector based delivery. As previously discussed for ZFN protein delivery, lentiviral delivery of TALEN proteins has been evaluated (Cai et al., 2014). However, viral cleavage by the HIV-1 proteases of the TALEN subunits was observed, limiting the efficiency of this approach. Gene disruption and gene editing frequencies were only up to 6.5% and 0.2%, respectively. If lentiviral delivery is the desired method of delivery for TALENs, further studies will be needed to reduce or eliminate internal cleavage sites, and improve disruption or editing frequencies to efficient levels. 4.3. Deliver y Challenges and Str ategies for CRI SPR/Cas9 4.3.1. Challenges

The genome encoding for Cas9 and a sgRNA is ~4.3-kb, the largest monomer between the trio of gene editing technologies described here (Kim and Kim, 2014). However, since Cas9 introduces DSBs as a monomer, in contrast to ZFN and TALEN dimers, the Cas9 genome is intermediate in size between ZFN and TALEN technologies. In relation to viral vector capacities, this size may be prohibitive for use in AAV vectors (~4.7 kb genome) when the promoter and localization sequences are considered (Ding et al., 2014). This is especially true if multiple sgRNAs are needed for multi-genome targeting and/or if donor DNA templates are needed for HDR-mediated gene correction. A few reported cases of off-target mutagenesis based on CRIPSR/Cas9 in human cells have led to concerns of limitations in target-specific delivery. In one study evaluating the specificity of CRISPR/Cas9 in U20S, HEK293, and K562 cell lines, off-target mutagenesis was identified in sequences with up to 5-bp mismatches with frequencies comparable or higher to those of the target sequence (Fu et al., 2013). Substantial off-target mutagenesis, including gross chromosomal deletions, was further observed in additional in vitro studies where it was suggested that CRISPR/Cas9 may be less specific in comparison to ZFNs and TALENs due to the relatively shorter targeting sequence, the promiscuous binding efficiencies of the sgRNA at sites further from the enzyme sequence, and non-Watson Crick base pairing (Cradick et al., 2013). However, further studies have shown limited to no off-target mutagenesis in vitro and in vivo with optimization of the guide RNA (Cho et al., 2014; Niu et al., 2014; Wu et al., 2015). Thus, further research into the specificity of CRISPR/Cas9 and possible optimization strategies, such as investigations using pairs of Cas9 nickases (Ran et al., 2013), will be needed to progress the technology for therapeutic applications. 4.3.2. Viral Deliver y

Despite the apparent limitations on the use of AAV vectors in relation to the size of Cas9 transgenes, engineered cytomegalovirus (CMV) AAV vector constructs have been developed to efficiently incorporate Cas9 and sgRNA (Senis et al., 2014). To reduce the overall size, minimal termination signals and promoter sequences have been evaluated and shown to not effect editing frequencies. Furthermore, it was demonstrated that different promoters could be introduced to target specific cell lines in vitro and in vivo. Alternatively, others have developed separate AAV vectors for Cas9 and sgRNA, respectively, with shortened neuron-specific promoters in order to alleviate capsid size restrictions, whilst leveraging the ability of AAVs to specifically target brain cells in mice (Swiech et al., 2015). The same group also took the reverse approach, and instead discovered a smaller Cas9 enzyme from the bacteria Staphylococcus aureus (SaCas9), which is more than 1 kb shorter than the typical Cas9 enzyme from Streptococcus pyogenes, and thus is readily able to be packaged into AAV vectors (Ran et al., 2015). AAV delivery of SaCas9 and its sgRNA expression cassette targeting the cholesterol regulatory gene Pcsk9 into mice resulted in efficient gene modification of up to 40% after 1 week and a corresponding ~40% decrease in total serum cholesterol. This increased efficiency observed when using the smaller Cas9 enzyme is a significant finding and may enable the use and multiple benefits of AAV delivery. To accommodate larger payloads, other viral vectors with larger capacities including adenovirus and lentivirus have also been investigated by various researchers for CRISPR/Cas9 delivery, especially for multiplex genome editing where several different guide RNAs are needed (Ding et al., 2014; Kabadi et al., 2014). 4.3.3. Non-Viral Deliver y

In at least two cases, delivery of naked plasmids with Cas9 expression cassettes, sgRNA, and, in one case, donor ssDNA has been performed in mice (Xue et al., 2014; Yin et al., 2014b). In both studies, hydrodynamic tail vein injections of saline DNA solutions were administered, resulting in efficient genetic corrections or targeted mutations. Hydrodynamic delivery is a method in which a large volume of fluid is injected into an target, resulting in hydrodynamic forces that permeabilize cellular membranes by generating pores, offering a temporary window for large molecules to permeate cells (Suda and Liu, 2007). In mice, hydrodynamic injection causes temporary cardiac dysfunction, increased blood pressure, and expansions of the liver, but these symptoms eventually recover after a period of time (Yin et al., 2014b). More research on non-vector based delivery of the CRISPR/Cas9 system will be needed, as many of the unwanted side-effects of gene therapy have been a result of the delivery vector. Additionally, non-viral delivery of Cas9 expression plasmids could be mediated by cationic lipid or polymer nanoparticles as with the other therapies, albeit with a speculated lower efficiency (Yin et al., 2014a). Direct delivery of Cas9 proteins and guide RNA has also been investigated. Similar to a method employed with TALENs, cell-penetrating peptides (CPP) have been used to promote cellular uptake of CRISPR/Cas9, though additionally complicated by the requirement to deliver both a protein and RNA (Ramakrishna et al., 2014). In these studies, the Cas9 protein was conjugated with an m9R CPP and the sgRNA was complexed with a similar 9R CPP through charge interactions to form condensed, positively charged nanoparticles. Delivery of the two components resulted in mutation frequencies ranging from 2.3% to 16% in a range of cultured human cells including embryonic stem cells, dermal fibroblasts, HEK293, HeLa, and embryonic carcinoma cells. In another study, an alternative to CPP-mediated delivery of Cas9 protein and sgRNA was proposed that may facilitate additional protection of the proteins in vivo from

extracellular or endosomal proteases (Zuris et al., 2014). It was demonstrated that cationic lipids could efficiently delivery Cas9 proteins complexed with polyanionic sgRNA, using the same commercially available lipid transfection agents that are typically used to delivery nucleic acids. This method resulted in up to 80% gene modification in cultured human cells and up to 20% gene modification in vivo when injected into the inner ear of mice to restore hair cell growth. In both the cationic lipid-mediated and CPP-mediated delivery methods, much higher specificity was achieved in comparison to plasmid transfection methods, which was attributed to the reduced working time of the protein. Direct delivery of proteins that can cross cell membranes is a viable approach to future in vivo therapeutic applications. 5. CANDI DATE DI SORDERS AND FUTURE PROSPECTI VES Reviewing the indications for current gene therapy clinical trials can provide a window into the types of disorders that may be more efficiently treated by new gene editing technologies in the future. Current gene therapy trials are overwhelmingly focused on treating cancer, followed by monogenic disorders, infectious diseases, and cardiovascular diseases, as shown in Figure 6 (Ginn et al., 2013). Recent advances in immunotherapy, in which a patient’s own immune system is trained to target and destroy tumor cells, have furthered the focus of gene therapy trials on cancer (Cross and Burmester, 2006). Initial proof of concept studies for gene editing technologies ZFNs, TALENs, and CRISPR/Cas9 have primarily focused on treating inherited monogenic disorders, such as those listed in Figure 6, and viral infections (e.g. HIV, HPV, and HBV). Depending on the particular disorder, or affected tissues, there are two possible therapeutic uses of these technologies namely ex vivo and in vivo administration. Ex vivo administration consists of first isolating and culturing target cell lines from the affected individual, followed by in vitro genetic correction. Corrected cells are then genetically

and morphologically screened and administered back to the patient in vivo. These cells may further multiply in vivo, thereby partially or fully restoring the wild type phenotype. Using this approach, cells are pre-screened for any off-target mutations that may lead to adverse events and a higher level of transfection, and genetic correction, is often achieved. Traditionally, this approach is only viable for tissues and cells that are readily accessible and proliferative such as in the blood, skin, and muscle. Recent advances in using hiPSCs have opened even more doors for ex vivo based gene therapies (Filareto et al., 2013; Jung et al., 2012; Mali et al., 2013). The ability to take a convenient sample of any human tissue, and reprogram it into stem cells that can subsequently be differentiated into any type of human cell for therapeutic reasons, greatly expands the utility of ex vivo therapies. Recently, CRISPR technology was used to modify and establish a line of hematopoietic stem and effector cells (Mandal et al., 2014). This establishment of a line of modified stem cells illustrates the great potential that CRISPR/Cas9 holds for personalized medicine. When considering another publication titled “Liver in a Dish,” the possibilities of these gene editing technologies expand exponentially. The ability to create organs in a dish that are recognized as self, yet corrected for any genetic error that an individual may have, open up endless possibilities of treatment, but also the ability to reduce the amount of animal and human trials that are necessary in pharmaceutical development (Ding and Cowan, 2013). Currently, this approach is still rather expensive, however the research done in this field represents the beginning of truly personalized medicine. For cellular and tissue targets that would be too invasive for ex vivo therapies (e.g. liver, brain, etc.), in vivo delivery mediated by viral or non-viral vectors may be more applicable. Specific examples of ex vivo and in vivo delivery of ZFNs, TALENs, and CRISPR/Cas9 have been discussed in this review. Viral-mediated delivery of genetic constructs, in general, continues to be the primary method used in gene therapy trials, though non-viral mediated

delivery of genetic constructs have continued to increase over the last decade (Ginn et al., 2013), likely as a result of initial safety concerns with viral delivery. As discussed in Section 4, viral mediated delivery of these different technologies has historically met various challenges including adverse outcomes in clinical trials. Progress continues to be made in both categories (viral and non-viral) to improve safety and increase payloads, to further advance these new technologies to commercialization. Even considering the long timeframe that it will take for these technologies to progress into human therapies, they still have immediate impacts on the field of medicine. Animal models are necessary for both basic science research and preclinical testing of pharmaceuticals. The ability to create animal models of various genetic disorders has proven to be invaluable since the first report of germ line transmission of a genetic modification made to a pluripotent cell (Bradley et al., 1984). For decades this method of genetic mutation was used to create specific model mice that allowed researchers to explore biochemical pathways and new drug formulations. Gene editing technologies such as Crispr/Cas9 represent a new, one-step method to generate mouse models, including multiple allele changes, knock-outs, and conditional animals (Wang et al., 2013; Yang et al., 2013). The ease of creation of model mammals by these technologies will allow for dramatic growth in the ability to investigate genetic modifications and model human diseases. This is exemplified in a study where gene editing technologies have allowed for the creation of primates with specific genetic mutations, which could serve as an invaluable tool for investigation into human conditions (Niu et al., 2014). 6. CONCL USI ON Despite some initial positive data, it will be several years before ZFNs, TALENs, or CRISPR/Cas9 are approved and marketed as therapeutic entities. Many more studies are needed

to prove efficacy and safety in humans as advancements are made in delivery and characterization methods. In terms of delivery methods for these gene editing technologies, the current literature indicates that much can be learned from the large body of knowledge obtained already for nucleic acid delivery, protein delivery, and technologies developed for specific routes of administration. In addition to the reviewed technologies, additional gene editing technologies are also likely to be considered, such as recently reported triplex-forming peptide nucleic acids described for gene modification (McNeer et al., 2015). From the analysis provided in this review, it is clear that these technologies hold great promise as next generation gene therapy options, however it will be necessary to progress cautiously in these endeavors to safely advance to human trials. Meanwhile, these technologies will continue to offer insight in medical and pharmaceutical sciences by furthering our understanding of biochemical pathways in diseases, generating new disease targets for high-throughput drug discovery screening, and simplifying the creation of transgenic model animals to replicate human diseases. Author Contr ibutions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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Figure captions Fig. 1 After a double stranded break (DSB), DNA repair can occur by two endogenous mechanisms: non-homologous end joining (NHEJ) or homology directed repair (HDR). With NHEJ, gene insertion or deletion mutations (indels) can occur potentially resulting in gene disruption. With HDR, a donor template enables insertion or substitution edits in the genome. Fig. 2 An illustration of a Zinc Finger Nuclease (ZFN) pair is shown. A ZFN consists of left and right monomers of typically 3 to 6 Zinc Finger Proteins (ZFPs) and the FokI restriction enzyme, which cleaves DNA when a dimer is formed. Each ZFP recognizes a target 3 base pair DNA sequence. Fig. 3 An illustration of a Transcription Activator-Like Effector Nuclease (TALEN) pair is shown. A TALEN consists of left and right monomers of TALE proteins and the FokI restriction enzyme, which cleaves DNA when a dimer is formed. Each TALE protein recognizes single DNA base pair. Fig. 4 An illustration of the CRISPR/Cas9 endonuclease is shown. The Cas9 endonuclease targets a 20-bp DNA sequence upstream of the PAM sequence (NGG) based on a designed single guide RNA sequence with homology to the target DNA sequence. After a match is made between the sgRNA and target DNA sequence, a DSB is introduced by Cas9 monomer 3-bp upstream of the PAM, as shown.

Fig. 5 Illustration of original ZFN homodimer architecture (left panel), which could bind and then cleave an off-target DNA site when two-left or two-right monomers interact with matching DNA sequences. By engineering obligate heterodimers (right panel), homodimer binding cannot occur, which reduces the frequency of off-target cleavage. Fig. 6 Gene therapy trials as of June 2015 by indication. Cancer diseases represent the largest proportion of current trials, followed by inheritable monogenic disorders. A list of inheritable monogenic disorders that have been the study of gene therapy trials is provided. Constructed with data from The Journal of Gene Medicine Gene Therapy Clinical Trials Database, June 2015 update, http://www.wiley.com//legacy/wileychi/genmed/clinical/.

Table 1. Reported Therapeutic Applications of ZFNs Disease

Tar get Gene / Sequence

Study Type

Cell L ine(s) / Species

Refer ence(s)

Sickle cell anemia

β-globin

in vitro

hiPSCs

(Sebastiano et al., 2011)

Hemophilia B

hF9

in vitro

K-562, Hep3B

(Li et al., 2011)

in vivo

Mouse

in vitro

Human DMD myoblasts

Duchenne Muscular Exon 51 of Dystrophy (DMD) dystrophin gene

in vivo

(Ousterout et al., 2014)

Mouse Cystic Fibrosis (CF)

CFTR

in vitro

HBE and CFTE

(Lee et al., 2012)

Severe combined immune deficiency X-1 (SCID)

IL2R γ

in vitro

K-562, hCD4+ T cells, HEK-293, lymphoblastoid cells, Jurkat cells, hESCs

(Lombardo et al., 2007; Urnov et al., 2005)

Paroxysmal nocturnal hemoglobinuria (PNH)

PIG-A

in vitro

hiPSCs, hESCs

(Zou et al., 2009)

X-linked chronic granulomatous disease (XCGD)

Gp91phox donor to AAVS1

in vitro

hiPSCs

(Zou et al., 2011)

Ataxia, retinitis pigmentosa (NARP), and Leigh’s syndrome

T8993G mutation in mitochondrial DNA

in vitro

NARP cells

(Minczuk et al., 2006)

a1-antitrypsin deficiency (A1ATD)

A1AT

in vitro

hiPSCs

(Yusa et al., 2011)

in vivo

Mouse

Down’s syndrome

XIST

in vitro

hiPSC

(Jiang et al., 2013)

Parkinson’s diseases

A53T mutation in a-synuclein gene

in vitro

hiPSC

(Soldner et al., 2011)

Human immunodeficiency virus (HIV-1) resistance

CCR5

in vitro

CD4+ T cells

in vivo

Mice, human

(Holt et al., 2010; Perez et al., 2008; Tebas et al., 2014)

Hepatitis B Virus (HBV)

Various

in vitro

Huh7 hepatoma cells, pTHBV2 transfected

(Cradick et al., 2010)

hiPSC = human induced pluripotent stem cells, hESC= human embryonic stem cells

Table 2. Reported Therapeutic Applications of TALENs Disease

Tar get Gene / Sequence

Study Type

Cell L ine(s) / Species

Refer ence(s)

Sickle Cell Anemia

β-globin (HBB)

in vitro

hiPSCs

(Ma et al., 2013; Sun and Zhao, 2014)

Hemophilia A

F8

in vitro

hiPSCs

(Park et al., 2014)

Duchenne Exon 51 of Muscular dystrophin gene Dystrophy (DMD) Exon 45 of dystrophin gene

in vitro

Skeletal myoblasts, dermal fibroblasts

(Ousterout et al., 2013),(Li et al., 2014)

in vitro

hiPSCs

Muscular dystrophy

MSTN

in vitro

HT1080, BAEC, NIH3T3, C2C12

(Xu et al., 2013)

α1-antitrypsin deficiency (A1ATD)

SERPINA1

in vitro

hiPSCs

(Smith et al., 2014)

Polycythemia vera JAK2 (PV)

in vitro

hiPSCs

(Smith et al., 2014)

Recessive dystrophic epidermolysis bullosa (RDEB)

COL7A1

in vitro

Human fibroblasts

(Osborn et al., 2013)

Severe combined immune deficiency X-1 (SCID)

IL2Rγ

in vitro

Jurkat

(Matsubara et al., 2014)

Mitochondrial diseases

m.8483_13459d

in vitro

Human (Bacman et al., osteosarcoma cells 2013)

MT-ND6

143B/206 cells Human PSIP1 immunodeficiency CCR5 virus (HIV-1) resistance

in vitro

293T, Jurkat

in vitro

hiPSCs

Hepatitis B Virus (HBV) replication

in vitro in vivo

Huh7, HepG2.2.15 (Bloom et al., 2013; Chen et al., Mouse 2014)

HPV infection and E6, E7 cervical cancer

in vitro

SiHA, S12, HeLa

in vivo

Mouse

Various cancers

in vitro

HeLa, Hek-293, Ramos lymphoma cells

C, S, an pol of HBV

miR-21

(Fadel et al., 2014; Ye et al., 2014)

(Hu et al., 2014b)

(Chen et al., 2015)

Malaria

TEP1 (mosquito genome)

in vitro

Mosquito germ cells

(Smidler et al., 2013)

Table 3. Reported Therapeutic Applications of CRISPR/Cas9 Disease

Target Gene / Sequence

Study Type

Cell L ine(s) / Species

Refer ence(s)

Sickle Cell Anemia

β-globin (HBB)

in vitro

hiPSCs

(Song et al., 2014; Xie et al., 2014)

Duchenne Muscular Dystrophy (DMD)

Exon 45 of dystrophin gene

in vitro

hiPSCs

(Li et al., 2014; Long et al., 2014)

Exon 23 of dystrophin gene

in vivo

mdx mice

Cystic Fibrosis

CFTR

in vitro

SI and LI stem cells

(Schwank et al., 2013)

α1-antitrypsin deficiency (A1ATD)

SERPINA1

in vitro

hiPSCs

(Smith et al., 2014)

Polycythemia vera (PV)

JAK2

in vitro

hiPSCs

(Smith et al., 2014)

Cataracts

Crygc

in vivo

Mouse

(Wu et al., 2013; Wu et al., 2015)

Barth syndrome

TAZ

in vitro

hiPSCs

(Yang et al., 2014)

Hereditary Tyrosinemia Type I (HTI)

Fah

in vivo

Mouse

(Yin et al., 2014b)

Human immunodeficiency virus (HIV-1) resistance

CCR5

in vitro

hiPSCs

(Ye et al., 2014)

Human immunodeficiency

LTR loci of integrated viral

in vitro

CHME5, HeLa- (Ebina et al., 2013; TZM-bI, U1, and Hu et al., 2014a;

virus (HIV-1) infection and immunization

genome, T10.

J-lat T-cells

Zhu et al., 2015)

Hepatitis B Virus (HBV)

Multiple

in vitro

Huh7, HepG2, HepAD38, HepaRG

in vivo

Mouse

(Dong et al., 2015; Kennedy et al., 2015; Lin et al., 2014; Liu et al., 2015; Ramanan et al., 2015; Seeger and Sohn, 2014; Zhen et al., 2015)

Epstein-Barr Virus Multiple (EBV)

in vitro

Raji

(Wang and Quake, 2014)

Human papillomavirus (HPV) and cervical cancer

E6 and E7 oncogenes

in vitro

SiHa and Caski

in vivo

Mouse

(Hu et al., 2014c; Yu et al., 2015; Zhen et al., 2014)

Osteosarcoma

CDK11

in vitro

KHOS and U20S

(Feng et al., 2014)

Cardiovascular disease

Pcsk9

in vivo

Mouse

(Ding et al., 2014)

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6