Gene Therapy and Regulation

The deregulation of gene expression is found in virtually every malady afflicting humans. From cancer to HIV-1 the uncontrolled expression or loss of gene expression is prevalent. Clearly, the ability to specifically control transcription would prove exceedingly useful with regards to approaches to avert disease. It has been known for several years now that small antisense non-coding RNAs can induce transcriptional gene silencing in humans suggesting that a mechanism is operative whereby non-coding RNAs exert transcriptional control of gene expression. Only recently was it observed that long antisense non-coding RNAs function as the endogenous epigenetic regulators of transcription in human cells, thus explaining why small antisense RNAs were observed early on to modulate transcription. These observations present an interesting paradigm where it is now possible to either stably silence transcription by targeting small antisense non-coding RNAs to particular gene promoters, or modulate increases in transcription by targeting the degradation of the regulatory long antisense non-coding RNA. This review will highlight the mechanism of action whereby antisense non-coding RNAs modulate transcriptional gene silencing as well as discuss the realized potential to therapeutically regulate the expression of virtually any gene, i.e., turn it on or off as desired.

Random integration of retroviral transgenes is a major oncogenic hazard that hampers both current clinical gene therapy trials and the genesis of clinical-grade induced pluripotent stem (iPS) cells. Noticeably, we have devised a universal stem cell gene therapy platform aimed at eliminating random integration of therapeutic DNA, at reversing inherited diseases including mitochondrial (mt)DNA ones by reestablishing wild-type genomic homeostasis, and at tackling both inherited and acquired/aging disorders through long-term or transient gene therapy protocols culminating in synergistic combinations. Our platform has a transient epigenetic arm directed at long-term transcriptional inactivation/activation of endogenous genes through the transient action of promoter-specific siRNAs or sense/antisense oligonucleotides, thereby opening an attractive avenue for hazard-free reprogramming of therapeutic iPS cells. Therefore, in addition to its obvious original applications such as long-term transcriptional silencing of autosomal-dominant mutant alleles, the transient epigenetic gene therapy arm could initiate the clinical iPS cell era whereby epigenetic reprogramming of patient somatic cells will not only drive regenerative medicine sensu stricto but also the other arms of our universal stem cell gene therapy platform. Both product and drive of our platform, such hazard-free iPS cells and their derivatives should accommodate all kinds of protocols including 1) long-term gene therapy mediated by endonuclease-boosted gene targeting (gene repair/alteration or targeted transgene integration) or cybridization (mtDNA transfer), and 2) transient regenerative gene therapy aimed at synergizing both regenerative cell therapy sensu stricto and long-term/epigenetic gene therapy through the magnification of the homing/regenerative/differentiative potential of therapeutic stem cells. Our approach is thus discussed 1) in light of emerging concurrent transgene-free reprogramming protocols that rely on protein transduction, small molecules or unintegrating vectors and 2) in terms of somatic cell genetic-like breakthrough in which somatic cells can be safely reprogrammed ex vivo into patient-specific pluripotent stem cells, amplified, engineered through standard/personalized protocols, subjected to potential clonal selections and lineage commitments, and then returned to the patient, thereby driving effective therapeutic repopulative/regenerative dynamics into relevant niches (long-term engraftment) and tissues (basic and regenerative cell turn-over).

To support the initiation of a clinical trial in humans, the immunogenicity of a plasmid DNA vaccine encoding the hemagglutinin (HA) of the swine-origin influenza A (H1N1) virus (S-OIV) isolate, A/California/04/09, formulated with the adjuvant, Vaxfectin^®, was administered intramuscularly to mice and rabbits at 100 μg and 1 mg DNA per injection, respectively, on Days 0 and 21. In hemagglutination inhibition (HI) assays using the reassortant virus, A/CA/07/09 NYMC X-179A, the titers for all animals were < 10 before vaccination. Three weeks after the first vaccination, 88% of mice and 75% of rabbits reached an HI titer of ≥ 40 with geometric mean titers (GMT) of 73 and 95, respectively. Two weeks after the second vaccination, 100% of mice and rabbits reached an HI titer of ≥ 40 with GMTs of 987 (range: 320–2560) for mice and 1522 (range: 640–2560) for rabbits. Sera from vaccinated mice and rabbits were also tested for HI titers against related S-OIV isolates. For mouse sera at Day 42, the HI GMTs against A/California/07/09, A/Texas/15/09 and A/Mexico/4108/09 were 905, 1280, and 1174, respectively, and for rabbit sera at Day 35 were 1522, 1280, and 1280, respectively. The results of this study indicate that the Vaxfectin^®-adjuvanted S-OIV DNA vaccine is immunogenic and elicits HI antibodies that are reactive with related S-OIV.

Pluripotent stem (PS) cells have the ability to replicate themselves (self-renew) and to generate virtually any given cell type in the adult body (pluripotency). Human PS (hPS) cells are therefore considered promising sources for future cell replacement therapy. Embryonic stem (ES) cells are the major type of PS cells that are derived from blastocyst embryos. Germ cells from testis can also become PS cells when cultured for a long period with a combination of growth factors. Alternatively, differentiated somatic cells can also be converted to PS cells by the method called nuclear reprogramming. This includes somatic cell nuclear transfer where a somatic cell nucleus is injected into an enucleated oocyte giving rise to a reprogrammed PS cell, as well as the recently developed technique of reprogramming differentiated somatic cells into induced pluripotent stem (iPS) cells by introducing defined transcription factors. Regardless of the sources and generation methods, PS cells share common epithelial structures and maintain tight cellular interactions. Although the molecular mechanisms that regulate self-renewal and pluripotency of PS cells have been extensively studied, the basic cellular interactions that govern how PS cells control cell-cell and cell-matrix adhesions are still not fully understood. In addition, there are several obstacles in the current culture methods for hPS cells that need to be overcome in order to achieve the highest safety and consistency required for clinical applications. A Rho-mediated signaling axis has recently been determined to be the core machinery that integrates cellular interactions between PS cells. By chemically engineering this axis, hPS cells are able to self-renew under completely defined conditions while maintaining their multi-differentiation capacities. When combined with the rapid progress in research focusing on iPS cells, these studies on cell-cell and cell-matrix adhesion in PS cells may not only contribute to further understanding PS cell biology, but also lead to the development of novel technologies enabling the derivation and growth of clinically relevant hPS cells for regenerative therapies.

The success of somatic cell reprogramming using defined transcription factors has laid the groundwork for generation of patient-specific induced pluripotent stem (iPS) cells for potential therapeutic purposes. Despite the promises iPS cell technology holds for regenerative medicine, current iPS cell derivation methods are still in infancy stages and have yet to be optimized for the safe and reliable production of clinical-grade pluripotent cells. Since the inception of iPS cell technology in 2006, rapid progress has been made to address the drawbacks implicated in iPS cell genesis methods. In this article, we review the key studies which have shaped the direction of the field, compare the various methods of iPS cell genesis and look ahead to the possibilities iPS cells offer for both research and clinical purposes.