Cell gene engineering is emerging as a field with outstanding impact, not only in medicine/biology, but also, and perhaps most importantly, in agriculture and in all those food sciences involved in the fight against world hunger. Lentivirus vector-based technologies represent the last frontier in the development of powerful and reliable methods for both in vitro and in vivo gene transfer in eukaryotic animal cells. Although the design of lentivirus vectors is closely reminiscent of those already successfully applied to the construction of oncoretroviral vectors, some unique features, e.g., the ef- ciency in transducing both postmitotic and stem cells, render the use of lentivirus vectors invaluable. It has been a great pleasure to edit Lentivirus Gene Engineering Pro- cols, owing in part to the high level of enthusiasm that the authors dem- strated in contributing to this book. The fact that so many outstanding scientists engaged in lentivirus vector research have provided articles renders it so- thing more than a technical handbook. In addition to detailed descriptions of the most innovative methodologies, the reader may find very informative ov- views concerning both theoretical and practical aspects of the origin and the development of diverse lentivirus vector types. This, in my opinion, rep- sents a unique added value of this volume, which should help our work resist the passage of time, to which books such as this are particularly sensitive.

This volume provides current methods and protocols for gene and protein delivery based on both lentivirus-generated and spontaneously released nanovesicles. Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools guides readers through methods on macromolecule delivery and chapters describe the LV-based protocols of gene engineering. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools aims to ensure successful results in the further study of this vital field.

For the first time a compilation of chapters that depict the biological bases underlying the development of lentiviral vectors, the techniques involved in the manufacture of this new gene delivery tool, and its most promising applications.

Viral Vectors for Gene Therapy: Methods and Protocols consists of 30 ch- ters detailing the use of herpes viruses, adenoviruses, adeno-associated viruses, simple and complex retroviruses, including lentiviruses, and other virus systems for vector development and gene transfer. Chapter cont- butions provide perspective in the use of viral vectors for applications in the brain and in the central nervous system. Viral Vectors for Gene Therapy: Methods and Protocols contains step-by-step methods for successful rep- cation of experimental procedures, and should prove useful for both experienced investigators and newcomers in the field, including those beginning graduate study or undergoing postdoctoral training. The “Notes” section contained in each chapter provides valuable troublesho- ing guides to help develop working protocols for your laboratory. With Viral Vectors for Gene Therapy: Methods and Protocols, it has been my intent to develop a comprehensive collection of modern molecular methods for the construction, development, and use of viral vectors for gene transfer and gene therapy. I would like to thank the many chapter authors for their contributions. They are all experts in various aspects of viral vectors, and I appreciate their efforts and hard work in developing comprehensive chapters. As editor, it has been a privilege to preview the development of Viral Vectors for Gene Therapy: Methods and Protocols, and to acquire insight into the various methodological approaches from the many different contri- tors.

With their advantages including wide tropism, high efficiency in gene transfer to both dividing and non-dividing cells, and stable and long-term expression of transgenes, lentiviral vectors have been attractively used for genes therapies and widely used for basic biomedical researches where gene transfer is required. As expression of multiple genes from the same target cell is required in such applications, this research work focused on designing novel multicistronic lentiviral vectors to develop gene therapy of diabetes through regulated insulin delivery from skin cells and live cell arrays for analyzing gene expression in a high-throughput and real-time manner.^Specifically, first, lentiviral vectors were engineered to produce a fusion protein between the furin cleavable proinsulin gene and the self-dimerization mutant of FK506-binding protein to yield bioactive insulin in keratinocytes that could be released following exogenous administration of a small organic molecule, rapamycin. The engineered keratinocytes retained normal morphology and grew similar to lentiviral-treated control cells. Epidermal keratinocytes in culture and in stratified bioengineered epidermis released insulin within 30 min after addition of rapamycin, and secretion slowed or stopped within 2-3 hours after removal of the inducer. When the cells were implanted into athymic mice that were rendered diabetic with streptozotocin, insulin was detected in the plasma within 1 hr after addition of rapamycin. Concomitantly, glucose decreased to normal levels even in diabetic animals suffering severe hyperglycemia. Repeated rapamycin administration yielded similar results.^These experiments provide proof-of-concept that insulin released from the skin in a regulatable manner can be an effective treatment for diabetic patients. Second, a lentiviral vector carrying two transcriptional units was designed to reach independent and high level dual-gene expression. The two transcriptional units were separated by polyadenylation, terminator and insulator sequences in order to eliminate promoter interference existing between the transcriptional units. With this design, the expression level of both genes was as high as that yielded from lentiviral vectors containing only a single transcriptional unit. Similar results were observed with several promoters and cell types including epidermal keratinocytes, bone marrow mesenchymal stem cells and hair follicle stem cells.^Notably, this research work also demonstrated quantitative dynamic monitoring of gene expression in primary cells with no need for selection protocols suggesting that this optimized lentivirus may be useful in high-throughput gene expression profiling studies. Last, using the novel double-promoter lentiviral vector scalable live-cell microarrays were developed to measure gene expression dynamics in a real-time and high-throughput manner. To this end, dual-promoter lentiviral vectors were prepared harboring a transcriptional regulatory element encoding for green fluorescence protein to monitor cell activation in response to exogenous stimuli and a constitutive promoter driving red fluorescence protein for internal signal normalization.^Lentivirus preparations were immobilized in a microarray format and after transduction on the array surface target cells were treated with cytokines and interrogated in real-time using automated fluorescence microscopy, providing rich dynamic information over a period of several days. Data normalization by red fluorescence intensity eliminated errors due to spot-to-spot variability in transduction efficiency or changes in cell proliferation upon cytokine treatment. These results suggest that the LVA can monitor gene expression in real-time and high-throughput manner thereby providing a useful tool for quantitatively measuring gene expression dynamics and deciphering gene regulatory networks.

This volume in the prestigious Methods in Enzymology series discusses methods currently used in preclinical and clinical gene therapy. Subjects covered in this book, such as the use of adeno-associated virus delivery for treatment of Parkinson's disease, are topical and are presented in the methods-oriented style popularized by this series. Discusses methods currently used in preclinical and clinical gene therapy Covers the use of adeno-associated virus delivery for treatment of Parkinson's disease