Review Article Theme: Emerging Drug Delivery Technologies Guest Editor: Dexi Liu Nonviral Gene Delivery: Principle, Limitations, and Recent Progress Mohammed S. Al-Dosari1,3 and Xiang Gao2 Received 15 June 2009 accepted 14 September 2009 published online 16 October 2009 Abstract. Gene therapy is becoming a promising therapeutic modality for the treatment of genetic and acquired disorders. Nonviral approaches as alternative gene transfer vehicles to the popular viral vectors have received significant attention because of their favorable properties, including lack of immunoge- nicity, low toxicity, and potential for tissue specificity. Such approaches have been tested in preclinical studies and human clinical trials over the last decade. Although therapeutic benefit has been demonstrated in animal models, gene delivery efficiency of the nonviral approaches remains to be a key obstacle for clinical applications. This review focuses on existing and emerging concepts of chemical and physical methods for delivery of therapeutic nucleic acid molecules in vivo. The emphasis is placed on discussion about problems associated with current nonviral methods and recent efforts toward refinement of nonviral approaches. KEY WORDS: gene delivery gene therapy lipoplex nonviral vectors polyplex transfection. INTRODUCTION Gene transfer, the technique to introduce new genetic materials to hosts, has become an invaluable experimental tool to study gene function and its regulation, to establish various disease models, to acquire DNA-based immunization, and finally, to explore potential therapeutic applications to various acquired or inherited diseases. Naked DNA mole- cules do not enter cells efficiently because of their large size and hydrophilic nature due to negatively charged phosphate groups. In addition, they are very susceptible to nuclease- mediated degradation. Therefore, the primary challenge for gene therapy is to develop carriers (commonly called vectors) and physical methods that facilitate gene transfer to targeted cells without degradation of the delivered gene. Recombinant viruses such as retrovirus, lentivirus, adenovirus, adeno-associated virus, and herpes simplex virus have been widely utilized as vectors for gene transfer (1). Viruses mediate efficient gene transfer through their favor- able cell uptake and intracellular trafficking machineries. However, viral vectors have several intrinsic drawbacks including difficulty in production, limited opportunity for repeated administrations due to acute inflammatory response, and delayed humeral or cellular immune responses. Inser- tional mutagenesis is also a potential issue for some viral vectors that integrate foreign DNA into the genome. The nonviral gene delivery methods, on the other hand, use synthetic or natural compounds or physical forces to deliver a piece of DNA into a cell. The materials used are generally less toxic and immunogenic than the viral counter- parts. In addition, cell or tissue specificity can be achieved by harnessing cell-specific functionality in the design of chemical or biological vectors, while physical procedures can provide spatial precision. Other practical advantages of nonviral approaches include ease of production and the potential for repeat administration. Nonviral methods are generally viewed as less efficacious than the viral methods, and in many cases, the gene expression is short-lived. However, recent develop- ments suggest that gene delivery by some physical methods has reached the efficiency and expression duration that is clinically meaningful. The purpose of this article is to provide an update and concise review in the field of nonviral gene delivery. Particular emphasis will be on the rate-limiting steps that affect the overall transfection and current efforts and strategies to overcome these limitations. EXTRA- AND INTRACELLULAR BARRIERS FOR GENE DELIVERY Several anatomical and cellular barriers limit the overall efficiency of gene transfer by nonviral methods (Fig. 1). Anatomical barriers are epithelial, endothelial cell linings and the extracellular matrix surrounding the cells that prevent direct access of macromolecules to the target cells. Profes- sional phagocytes such as Kupffer cells in the liver and residential macrophages in the spleen are largely responsible for the clearance of DNA-loaded colloidal particles adminis- tered through blood circulation. In addition, various nucleases existing in blood and extracellular matrix can 1 Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box 2457( Riyadh 11451, Saudi Arabia. 2 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh( Pennsylvania 15261, USA. 3 To whom correspondence should be addressed. (e-mail: msdosari@ yahoo.com) The AAPS Journal, Vol. 11, No. 4, December 2009 (# 2009) DOI: 10.1208/s12248-009-9143-y 671 1550-7416/09/0400-0671/0 # 2009 American Association of Pharmaceutical Scientists

rapidly degrade free and unprotected nucleic acids following systemic administration. Crossing plasma membrane is con- sidered the most critical limiting step for an efficient DNA transfection. Nucleic acids typically cannot pass through cell membrane unless their entry is facilitated by creating transient holes by physical meanings (2), or through various active cell uptake mechanisms such as endocytosis, pinocy- tosis, or phagocytosis (3). Upon being taken up via endocytosis, macromolecules captured within the endosomes usually transform into digestive lysosomes unless some escape mechanisms are used to intercept this maturation process. Two escape mechanisms have been explored. The first involves the use of membrane active or fusogenic molecules such as fusion peptides (4) or lipid components with acid-sensitive bond and large hydrophobic portion of the molecules to rupture the endosome membrane (5). The other mechanism acts on building up osmotic pressure within the endosomal compartment that eventually triggers the swelling or burst of endosomal vesicles (6). Weak amine compounds such as chloroquine and cationic polymers (poly- ethyleneimines (PEI) and partially degraded polyamidoamine dendrimers) absorb protons and slow down the acidification process that is essential for endosome���lysosome transition (7). Consequently, the influx of chloride counter ions builds up osmotic pressure inside the endosomes. For polyester-based carriers such as poly(lactic-co-glycolic acid), the breakdown products by hydrolysis can also build up the osmotic pressure inside the endosome which leads to the release of the contents trapped therein. Several other attempts have been used to increase the rate of endosome release, among which are codelivery of inactivated viral particles or recombinant viral capsule proteins that possess endosomolytic activities (8), and the use of photochemically generated free radicals to cause membrane damage (9). Upon their release from endosomes, DNA molecules in their free form or as complexes must travel through cytoplasmic space filled with viscous protein solution and a network of cytoskeleton matrix toward the nucleus where transcription takes place. Observations made by direct intracellular microinjection of naked DNA prove that the movement by diffusion is slow and inefficient, and the resulting levels of gene expression are very weak (10). The nuclear envelope represents an important barrier for the entry of DNA. This double-membrane envelope is interrupted by large protein structures called nuclear pore complexes (NPC) which regulate transport through nuclear envelope. NPC have diameters of ���9 nm, which allow free diffusion of ions and molecules of small to medium sizes, such as proteins of up to 40���60 kDa, or nucleic acids of up to ���300 bp, but restrict larger macromolecules passing through freely (11). For resting cells, nuclear uptake of large proteins is an active transport process mediated through sequence-specific recognition of nuclear localization signal peptide (NLS) sequence in their structures by importin proteins (12). Protein-NLS/importin complexes dock at the NPC to allow nuclear entry of DNA. The entry is achieved indirectly through the NLS sequences of transcription factors that are associated with the DNA molecules. For replicating cells, most DNA molecules enter the nuclei through the process of dissolution and reorgan- ization of the nuclear envelope during mitosis (13). Finally, the unpacking of DNA���carrier complexes could constitute yet another rate-limiting step after transfection. Cationic lipids dissociate from DNA through lipid mixing and exchange with host cell lipids at the cytoplasm entry step, while DNA complexes formed with cationic polymers, such as PEI, remain stable after endosome escape. An interest- ing concept has been reported recently, under which the intracellular trafficking of DNA-loaded nanoparticles is coupled with microtubule-directed transport mechanism (14). The polymer���DNA complexes disintegrate later in nucleus (15). Fig. 1. Schematic representation of barriers limiting nonviral gene transfer in vivo 672 Al-Dosari and Gao