Current Gene Therapy (v.11, #1)

This special issue is devoted to artificial nucleases engineered for targeted genome modification. The discovery of restriction enzymes in the late 60s paved the way for enzymatic manipulation of DNA in the test tube. The novel reagents reviewed in this special issue now promise to usher us into a new era of DNA modification performed directly in living cells. Artificial nucleases are designed to introduce a targeted cut in genomic DNA and allow highly efficient sequence modification in living cells. The most prominent so far are zinc finger nucleases (ZFNs) and they are abundantly discussed in this issue. Other types of nucleases are also becoming available and they are specifically described in two reviews. Many important applications are mentioned and it is becoming clear that engineered nucleases are likely to of benefit to many fields of life sciences, from genetic studies in model organisms to gene therapy in humans. Dana Carroll gives us a highly enjoyable personal perspective on the development of ZFNs and their applications for targeted genome modification. His early vision of the importance of assembling novel nucleases by fusing the endonuclease domain of FokI to artificial zinc fingers led him and his collaborators to perform pioneering studies of targeted genome modification in model organisms. The recent generation of rat, zebrafish, maize and tobacco ZFN-mediated mutants testifies to the influence of their work and the list will continue to expand. Recent studies of Dana Carroll and others also exemplify the importance of better understanding the basic mechanisms of DNA recombination and repair at work when modifying the genome with nucleases. Frederic Paques and collaborators review the development of another family of artificial nucleases, the so-called meganucleases. They are derived from unusual endonucleases with very long target sequences and recent studies demonstrate their efficiency in several important model systems. The paper by George Silva et al also provides a very stimulating account of the state of the art in the emerging field of targeted genome modification and its applications to gene therapy, including the emergence of the novel family of TALE-nucleases. The paper from Toni Cathomen's lab reviews recent advances in ZFN design and use that have improved targeted genome modification activity. Importantly, it also discusses current limitations and key questions that remain to be addressed, especially in the context of therapeutic applications. One major concern is sequence specificity and the development of appropriate strategies to evaluate, predict and improve it. The review of Makoto Komiyama and collaborators describes another class of artificial nucleases that are chemical-based rather than protein-based like ZFNs. They are composed of a DNA recognizing moiety and of a cleaving agent. It focuses on the so-called ARCUT nucleases that make use of two pseudo-complementary PNAs to invade the DNA duplex and of a Ce(IV)/EDTA complex to hydrolyze it near the PNA binding site. Such a strategy is much less validated than the protein-based ones but available results are encouraging. In addition it presents the advantage of easy design and production since DNA target recognition is determined by base pairing. All together these reviews present a great overview of recent developments and applications in the field of artificial nucleases. These sequence-specific cleaving agents allow manipulating the genome sequence with extreme precision and are already considered as novel powerful tools for biological research and biotechnological applications. Therapeutic applications still require additional improvements and validations but are on the way and a Phase 1 clinical trial with an anti-CCR5 ZFN in an adenoviral vector started in 2009 as a novel anti-HIV strategy. Finally we can speculate that important progress will be made to accelerate the design and production of artificial nucleases with the desired sequence specificity.

Zinc-Finger Nucleases: A Panoramic View by Dana Carroll (2-10).
Zinc-finger nucleases (ZFNs) are emerging as very powerful tools for directed genome modifications. Their key features are: a DNA-binding domain comprised of zinc fingers that can be designed to favor very specific targets; a nonspecific cleavage domain that must dimerize to cut DNA - this requirement enhances specificity and minimizes random cleavage. ZFNs have been shown to be effective in a wide range of organisms and cell types. This article reviews discoveries that led to the development of ZFNs, cites examples of successes in genome engineering, and projects how ZFNs may be used in the future, particularly in applications to humans.

Meganucleases and Other Tools for Targeted Genome Engineering: Perspectives and Challenges for Gene Therapy by George Silva, Laurent Poirot, Roman Galetto, Julianne Smith, Guillermo Montoya, Philippe Duchateau, Frederic Paques (11-27).
The importance of safer approaches for gene therapy has been underscored by a series of severe adverse events (SAEs) observed in patients involved in clinical trials for Severe Combined Immune Deficiency Disease (SCID) and Chromic Granulomatous Disease (CGD). While a new generation of viral vectors is in the process of replacing the classical gamma-retrovirus - based approach, a number of strategies have emerged based on non-viral vectorization and/or targeted insertion aimed at achieving safer gene transfer. Currently, these methods display lower efficacies than viral transduction although many of them can yield more than 1and#x25; engineered cells in vitro. Nuclease-based approaches, wherein an endonuclease is used to trigger site-specific genome editing, can significantly increase the percentage of targeted cells. These methods therefore provide a real alternative to classical gene transfer as well as gene editing. However, the first endonuclease to be in clinic today is not used for gene transfer, but to inactivate a gene (CCR5) required for HIV infection. Here, we review these alternative approaches, with a special emphasis on meganucleases, a family of naturally occurring rare-cutting endonucleases, and speculate on their current and future potential.

Zinc-Finger Nuclease Based Genome Surgery: It's All About Specificity by Eva-Maria Handel, Toni Cathomen (28-37).
Therapeutic genome engineering is a hallmark of gene therapy but only recent technological advances have permitted the modification of complex genomes in a targeted fashion. Zinc-finger nucleases (ZFNs) have developed into a major playmaker in the genome engineering field and have been employed to trigger the targeted editing of genomes at over 50 gene loci in 11 model organisms, including fruitfly, zebrafish and rat, with allelic frequencies reaching the double digit percentage range. Moreover, ZFN-mediated genome surgery in primary human cells has become a reality and two phase I clinical trials aiming at knocking out the CCR5 receptor in T cells isolated from HIV patients to protect these cells from infection with the virus have been initiated. Considering that specificity is closely linked to ZFN activity and ZFNassociated toxicity, this parameter has been and will be a key quality in any therapeutic application of the designer nucleases. This review summarizes the technological innovations that have successfully catapulted ZFNs into the genome engineering arena and provides an overview of the current state of the art of these nucleases with reference to human gene therapy.

Homologous recombination is almost the only way to modify the genome in a predetermined fashion, despite its quite low frequency in mammalian cells. It has been already reported that the frequency of this biological process can be notably increased by inducing a double strand break (DSB) at target site. This article presents completely chemistrybased artificial restriction DNA cutter (ARCUT) for the promotion of homologous recombination in human cells. This cutter is composed of Ce(IV)/EDTA complex (molecular scissors) and two strands of peptide nucleic acid (PNA), and contains no proteins. Its scission site in the genome is determined simply by Watson-Crick rule so that ARCUT for desired homologous recombination is easily and straightforwardly designed and synthesized. The site-specificity of the scission is high enough to cut human genome at one target site. The DSB induced by this cutter is satisfactorily recognized by the repair system in human cells and promotes the targeted homologous recombination.

Non-Viral Gene Delivery to Mesenchymal Stem Cells: Methods, Strategies and Application in Bone Tissue Engineering and Regeneration by Jose L. Santos, Deepti Pandita, Joao Rodrigues, Ana P. Pego, Pedro L. Granja, Helena Tomas (46-57).
Mesenchymal stem cells (MSCs) can be isolated from several tissues in the body, have the ability to selfrenewal, show immune suppressive properties and are multipotent, being able to generate various cell types. At present, due to their intrinsic characteristics, MSCs are considered very promising in the area of tissue engineering and regenerative medicine. In this context, genetic modification can be a powerful tool to control the behavior and fate of these cells and be used in the design of new cellular therapies. Viral systems are very effective in the introduction of exogenous genes inside MSCs. However, the risks associated with their use are leading to an increasing search for non-viral approaches to attain the same purpose, even if MSCs have been shown to be more difficult to transfect in this way. In the past few years, progress was made in the development of chemical and physical methods for non-viral gene delivery. Herein, an overview of the application of those methods specifically to MSCs is given and their use in tissue engineering and regenerative medicine therapeutic strategies highlighted using the example of bone tissue. Key issues and future directions in non-viral gene delivery to MSCs are also critically addressed.

The success of gene therapy largely relies on the development of high-efficient and low-toxic gene delivery vectors. Nanovector-based delivery of nucleic acids is a very promising approach for the effective transfer of genetic materials into cells. Compared with encapsulating of nucleic acids inside biodegradable nanoparticles which often suffers from low encapsulation efficiency and degradation of the loaded therapeutic gene, the layer-by-layer self-assembly vectors prepared by the surface adsorption of gene/polycation multilayered films on colloidal particles using layer-bylayer technique are a potent gene delivery system in offering efficient loading of nucleic acids, controlling the release of the loaded gene in physiological environment and targeting to a particular site or a specific cell type in the body. This review focuses on the preparation, advantages, application and the probable associated drawbacks of layer-by-layer selfassembly vectors for gene delivery.