Progress and prospects: nuclear import of nonviral vectors

Gene Therapy (2010) 17, 439–447 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 $32.00 www.nature.com/gt REVIEW Progress and prospects: nuclear import of nonviral vectors AP Lam1 and DA Dean2 1 Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL, USA and 2Departments of Pediatrics and Biomedical Engineering, University of Rochester, Rochester, NY, USA The nuclear envelope represents a key barrier to successful nonviral transfection and gene therapy both in vitro and in vivo. Although the main purpose of the nuclear envelope is to partition the cell to maintain cytoplasmic components in the cytoplasm and nuclear components, most notably genomic DNA, in the nucleus, this function poses a problem for transfections in which exogenous DNA is delivered into the cytoplasm. After delivery to the cytoplasm, nucleic acids rapidly become complexed with cellular proteins that mediate interactions with the cellular machinery for trafficking. Thus, it is these proteins that, in essence, control the nuclear import of DNA, and we must also understand their activities in cells. In this review, we will discuss the principles of nuclear import of proteins and DNA–protein complexes, as well as the various approaches that investigators have used to improve nuclear targeting of plasmids. These approaches include complexation of plasmids with peptides, native and engineered proteins, ligands and polymers, as well as the inclusion of transcription factor-binding sites for general and cell-specific delivery. Keywords:nonviral gene transfer|plasmid|nuclear pore complex|importin|nuclear localization signal|karyopherin. Gene Therapy (2010) 17, 439–447; doi:10.1038/gt.2010.31; published online 4 March 2010 Keywords: Nonviral gene transfer; plasmid; nuclear pore complex; importin; nuclear localization signal; karyopherin In brief Progress  Mechanisms of nuclear localization signal (NLS)mediated protein nuclear import have been elucidated.  Transcription factor-binding sites promote DNA nuclear translocation.  Cell-specific transcription factors drive cell-specific DNA nuclear entry.  Proteomics approaches have been used successfully to study DNA nuclear entry.  NLS peptides complexed with plasmids may enhance DNA nuclear translocation.  Nuclear proteins complexed with plasmids facilitate DNA nuclear entry.  Small molecule ligands bound to DNA can increase nuclear entry.  Nanoparticles and polymers may provide alternative routes to the nucleus.  Modulation of the nuclear pore complex may aid in nuclear delivery of DNA. Prospects  Proteomics will blossom in the area of gene delivery.  Large-scale identification of proteins in the DNA– protein complex will aid in understanding of how transport occurs.  RNA interference will be used increasingly to define key mechanisms of intracellular trafficking of nonviral vectors.  Improvements in in vivo imaging on the single cell level will allow the study of intracellular trafficking of plasmids within tissues of living animals.  Complexation of proteins with DNA will facilitate general nuclear import and gene expression.  Designer proteins containing DNA-binding domains and spatially distinct NLSs may enhance plasmid nuclear import and expression. Introduction Correspondence: Dr DA Dean, Department of Pediatrics, Box 850, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA. E-mail: Received 22 April 2009; revised 3 February 2010; accepted 9 February 2010; published online 4 March 2010 Nonviral plasmid-based gene delivery systems show promise for gene therapy because of their ability to be repeatedly administered and their generally good safety profile. However, their greatest limitation has been the reduced levels of gene transfer and expression compared Nuclear import of nonviral vectors AP Lam and DA Dean 440 with their viral counterparts. Viruses have had millions of years to develop strategies to circumvent cellular barriers to ensure infection of their target cells. Most of these mechanisms involve designing and incorporating proteins into the virus that help stabilize virus–cell interactions and increase internalization, enhance endosomal escape, promote movement through the cytoplasm to the nuclear envelope, improve nuclear entry in dividing and nondividing cells (often by promoting mitosis) and increase transcription. Nonviral vectors have not had the luxury of evolution to aid their delivery and are thus confronted by each of these barriers. Although many researchers disagree about the one single ‘rate-limiting’ barrier to efficient gene delivery, it is clear that trafficking across the nuclear envelope is one of the major barriers. Significant progress has been made over the past 20 years to elucidate the mechanisms of nuclear import and export of proteins and RNAs (such as mRNA, tRNA, 5S RNA). Similarly, over the past 10 years, mechanisms for the nuclear import of plasmids have been described, and methods to optimize delivery and expression based on exploitation of these mechanisms have been developed. It has long been appreciated that the nuclear envelope represents a barrier to efficient gene delivery. Most successful laboratory transfections occur in actively dividing cells. As one of the hallmarks of mitosis is nuclear envelope breakdown, any DNA that has entered the cytoplasm before mitosis would gain access to the nuclear compartment once cells enter the M phase. Indeed, nonviral transfections are cell cycle dependent. This is at least one of the reasons why many primary cells, growth-arrested cells and terminally differentiated cells remain difficult to transfect, and is the reason why a multitude of ‘new and improved’ transfection reagents are constantly being introduced and advertised to the community. Mario Chapecchi showed almost 30 years ago that when plasmids were microinjected into the cytoplasm of mouse fibroblasts, they largely failed to express. By contrast, when the same plasmids were microinjected into the nuclei of the same cultured cells, between 50 and 100% of the cells showed some level of gene expression. More recently, several groups have quantified levels of gene expression and found that it takes between 30 and 100 times more DNA delivered to the cytoplasm than it does to the nucleus to give the same level of gene expression, even in dividing cells.1 It is estimated that after lipoplex- or polyplex-mediated transfection, between 2000 and 100 000 plasmids are delivered to each cell, depending on the applied dose of DNA.2 Depending on the cell type transfected and the methods used for detection, it is estimated that between 1 and 10% of unmodified plasmids delivered to the cell can then be detected in the nuclear fraction, using quantitative PCR, Southern blot or electron microscopy.2,3 Thus, only a fraction of input DNA reaches the nucleus for gene expression. Mechanisms of NLS-mediated protein nuclear import have been (...truncated)