Gene Therapy and Regulation

A challenge to the realization of the vision of personalized medicine is our lack of understanding of the amount of genetic variation contained in the human species, which has been prohibitively expensive to acquire to date. In this review, we discuss a new system based on massively parallel picotiter plate based sequencing. This approach, now commercially available, delivers in excess of 200,000 individual clonal reads in a single five hour run and hence provides orders of magnitude improvement in speed and cost over conventional Sanger-based sequencing. The system is scalable well beyond its current performance and its emergence makes plausible the routine sequencing of individual human genomes in the near future. In this review, our system is presented in light of recent NHGRI sequencing technology grants, and a number of new applications enabled by ready access to hundreds of thousands of individual clonal reads are discussed with a particular focus on their potential impact on human health.

Gene targeting to selected chromosomal loci is greatly stimulated when free DNA ends are created that initiate double-strand break repair. Gene therapy reagents can be developed by engineering DNA endonucleases that cleave genomes at desired target sequences. Homing endonucleases are naturally occurring rare-cutting enzymes that have well understood DNA binding and DNA cleavage properties. Rational design methods as well as directed evolution strategies that involve genetic selections and screens using combinatorial libraries generate homing endonucleases with altered sequence specificities. Molecular switches are being introduced into these enzymes to regulate their activity. This article reviews the progress that has been made in constructing homing endonucleases for gene therapy and genome engineering, and discusses the challenges that remain.

Engineered DNA-binding proteins, in particular zinc finger proteins (ZFPs), have broad-ranging applications in gene therapy. An engineered ZFP transcription activator targeted to the VEGF locus is currently undergoing clinical trials for the induction of angiogenesis. A number of ZFP gene switches have been developed which allow highly controllable regulation of therapeutic transgene expression based on small molecule inducers/repressors. Finally, engineered ZFP nucleases have been used to correct a gene sequence in a living cell by stimulating homologous DNA recombination, suggesting a new, highly targeted approach to gene therapy. All these approaches rely on DNA-binding protein engineering, which in the past has mainly been achieved by selection using phage display. However, a convenient cell-free selection method known as in vitro compartmentalization (IVC) has previously been used to engineer DNA-binding proteins with enzymatic activities (e.g. polymerase and methylase), and the method has recently been extended to the engineering of sequence-specific ZFP DNA-binders. Below we describe the IVC procedure and review the progress made in applying this to the problem of facilitating the engineering of DNA-binding proteins.

A fundamental problem in cancer research is the identification of the cell type capable of initiating and sustaining the growth of the neoplastic clone in vivo. The key to solving this problem lies on the observation made over 40 years ago that tumors are heterogeneous and thus might be maintained only by a rare subset of cells called "cancer stem cells" (CSCs). However, the proof of this principle was only possible after the development of modern research tools for investigating the behavior of defined cell populations in vivo. The blood-related cancer leukemia was the first disease where human CSCs, or leukemic stem cells (LSCs), were isolated. The development of quantitative xenotransplantation assays using immune-deficient mouse recipients to detect primitive human hematopoietic stem cells (HSCs) with in vivo repopulating ability and the adaptation of this model to leukemia have been instrumental. Leukemia can now be viewed as aberrant hematopoietic processes initiated by rare LSCs that have maintained or reacquired the capacity for indefinite proliferation through accumulated mutations and/or epigenetic changes. Yet, despite their critical importance, much remains to be learned about the developmental origin of LSC and the mechanisms responsible for their emergence in the course of the disease. This report will review our current knowledge on normal and leukemic stem cell development and finally demonstrate how these discoveries provide a paradigm for identification of CSCs from solid tumors. By a careful comparative analysis of the properties of CSCs and of their normal counterparts, it should be possible to pinpoint critical features amenable to efficient anti-CSC therapies.

The ability of neural stem cells (NSCs) to home to diseased areas of the brain and their capacity to differentiate into all neural phenotypes provides a powerful tool for the treatment of both diffuse and localized neurologic/oncogenic disorders. NSCs are the most immature neural precursor cells in the nervous system and are defined by their ability to self-renew by symmetric division as well as to give rise to more mature progenitors of all neural lineages by asymmetric division. A full understanding of the molecular mechanisms regulating their migratory properties and their choice between various differentiation programs is essential if these cells are to be used for therapeutic applications. This review focuses on summarizing the factors and signaling molecules that are involved in migration and differentiation of neural stem cells and also gives an insight into therapeutic potential of these cells with an emphasis on glioma therapy.