Current Gene Therapy (v.12, #5)

Editorial (Hot Topic: Vectorizing mRNA and Proteins) by Axel Schambach, Christopher Baum (345-346).

mRNA as a Versatile Tool for Exogenous Protein Expression by Andreas N. Kuhn, Tim Beißert, Petra Simon, Britta Vallazza, Janina Buck, Brian P. Davies, Ozlem Tureci, Ugur Sahin (347-361).
Several viral and non-viral vectors have been developed for exogenous protein expression in specific cells. Conventionally, this purpose is achieved through the use of recombinant DNA. But mainly due to the risks associated with permanent genetic alteration of cells, safety and ethical concerns have been raised for the use of DNA-based vectors in human clinical therapy. In the last years, synthetic messenger RNA has emerged as powerful tool to deliver genetic information. RNA vectors exhibit several advantages compared to DNA and are particularly interesting for applications that require transient gene expression. RNA stability and translation efficiency can be increased by cis-acting structural elements in the RNA such as the 5'-cap, the poly(A)-tail, untranslated regions and the sequence of the coding region. Here we review recent developments in the optimization of messenger RNA as vector for modulation of protein expression emphasizing on stability, transfection and immunogenicity. In addition, we summarize current pre-clinical and clinical studies using RNA-based vectors for immunotherapy, T cell, stem cell as well as gene therapy.

Adenovirus Vectors and Subviral Particles for Protein and Peptide Delivery by Matthias W. Kron, Florian Kreppel (362-373).
Adenovirus vectors belong to the most frequently used vector type in gene therapy approaches. In addition, adenovirus vector particles and adenovirus subviral particles offer a great potential for the direct delivery of proteins into cells. In this review we discuss this potential and the technology of adenovirus as a protein delivery platform for applications ranging from vaccination to gene therapy.

Protein Transduction Domains: Applications for Molecular Medicine by Maliha Zahid, Paul D. Robbins (374-380).
Protein transduction domains (PTD) or cell penetrating peptides (CTPs) are small peptides able to carry proteins, peptides, nucleic acid, and nanoparticles, including viral particles, across the cellular membranes into cells. In general, PTDs can be classified into 3 types: cationic peptides of 6-12 amino acids in length, comprised predominantly of arginine, ornithine and/or lysine residues; hydrophobic peptides such as leader sequences of secreted growth factors and cytokines; and cell-type specific peptides, identified by screening of peptide phage display libraries. These three types of transduction peptides have many different applications including delivery of therapeutic proteins and drugs, delivery of fluorescent or radioactive compounds for imaging, and improving uptake of DNA, RNA and even viral particles. Here we review the potential applications of protein transduction domains.

Efficient therapeutic protein delivery is a challenging task in several disease contexts and particularly when the CNS is concerned. Different approaches for brain-directed delivery have been thus far investigated, including direct injection of molecules or of their coding information carried by dedicated vector systems within the brain parenchyma or in the ventricular space, intravenous systemic administration of molecules/vectors modified to target and cross the blood-brainbarrier, and exploitation of allogeneic and/or autologous and genetically modified cells as vehicles for the therapeutic of interest. Among these, we here review one of the most promising approaches based on hematopoietic stem cells, taking advantage of lysosomal storage disorders as representative disease setting.

Retroviral Protein Transfer: Falling Apart to Make an Impact by Tobias Maetzig, Christopher Baum, Axel Schambach (389-409).
Retroviral vectors represent evolutionarily optimized gene delivery vehicles, which stably integrate their coding DNA into the host cell genome. In contrast to other gene delivery platforms, retroviral entry and integration are relatively efficient due to the utilization of cellular mechanisms for particle transport, DNA repair and gene expression, features that can be exploited for gene therapy and cell modification. Arresting the retroviral life cycle at specific steps, i.e. prior to reverse transcription or integration, allows for the utilization of intermediate structures (mRNA) or by-products (episomes) as tools for transient applications. However, it is often overlooked that retroviral particles are composed of up to 2500 Gag structural proteins, as well as further proteins involved in viral replication, all of which can be harnessed for the transfer of heterologous proteins into target cells. In this review, we describe the general biology of retroviruses and their derived vector systems, and then discuss the potential of engineering their protein components. We focus on lentiviral, gammaretroviral and alpharetroviral vector systems, and address current developments in the visualization of retrovirus-cell interactions (live cell imaging), and potential applications of engineered retroviral particles in biotechnology and biomedical research. Compared to conventional protein transduction techniques, we envisage protein-transducing retrovirus-like particles as a highly flexible platform for the efficient and cell-targeted delivery of designer proteins, even in combination with transduction of retroviral mRNA, episomal DNA or integrating DNA.

Gene delivery/expression vectors have been used as fundamental technologies in gene therapy since the 1980s. These technologies are also being applied in regenerative medicine as tools to reprogram cell genomes to a pluripotent state and to other cell lineages. Rapid progress in these new research areas and expectations for their translation into clinical applications have facilitated the development of more sophisticated gene delivery/expression technologies. Since its isolation in 1953 in Japan, Sendai virus (SeV) has been widely used as a research tool in cell biology and in industry, but the application of SeV as a recombinant viral vector has been investigated only recently. Recombinant SeV vectors have various unique characteristics, such as low pathogenicity, powerful capacity for gene expression and a wide host range. In addition, the cytoplasmic gene expression mediated by this vector is advantageous for applications, in that chromosomal integration of exogenous genes can be undesirable. In this review, we introduce a brief historical background on the development of recombinant SeV vectors and describe their current applications in gene therapy. We also describe the application of SeV vectors in advanced nuclear reprogramming and introduce a defective and persistent SeV vector (SeVdp) optimized for such reprogramming.

New Insights in the Gene Electrotransfer Process: Evidence for the Involvement of the Plasmid DNA Topology by Jean-Michel Escoffre, Biliana Nikolova, Laetitia Mallet, Julien Henri, Cyril Favard, Muriel Golzio, Justin Teissie, Iana Tsoneva, Marie-Pierre Rols (417-422).
Electropermeabilization is a non-viral method that can be used to transfer plasmid DNA (pDNA) into cells and tissues. According the applications and considered tissues, this safe method can be less efficient than the viral approaches. Biophysical mechanisms of gene electrotransfer are not entirely known. Contrary to small molecules that have direct and fast access to the cytoplasm, pDNA is electrophoretically pushed towards the permeabilized membrane where it forms a complex before being transferred into the cytoplasm. In order to understand the biophysical mechanisms of gene electrotransfer and in this way to improve it, we investigated the dependence of the topology of pDNA i.e. linear versus supercoiled on both pDNA/membrane interaction and gene expression. Our results revealed that: i) even if pDNA/membrane interactions are only slightly affected by the topology of pDNA, ii) gene transfer and expression are strongly influenced by it. Indeed, the linearization of pDNA leads to a decrease in the transfection level.

Current Advances in Vehicles for Brain Gene Delivery by Xiang Zhang, Lili Zhao, Jinhui Wu, Hong Dong, Feng Xu, Guangming Gong, Yiqiao Hu (423-436).
Gene therapy is a novel and promising treatment strategy for brain diseases. Yet, its development is largely obscured by various in vivo transport hurdles, especially the special BBB structure of brain. Developing an ingenious delivery vehicle can provide a great solution. Conventional vehicles for brain gene delivery are viral and non-viral vectors. With inherent superiority of gene transfection, researches on viral vectors are mainly focused on problems of brain cell targeting and global brain delivery. Non-viral vectors are more studied for better brain cell entrance either directly delivered to brain or systemically delivered to the body. Novel vehicles are cell vehicles (genetically engineered or nanoparticle- carrying cells) and exosomes. They exhibit distinct and unique features compared to viral and non-viral vectors. This review gives a summarization of current advances in these four kinds of brain gene vehicles. The merits and demerits of them are also pointed out respectively. We are hoping to give a clue to the future development direction of vehicles for brain gene delivery.