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Polymer Technology for Gene Therapy Daniel W. Pack University of Illinois at Urbana-Champaign Gene therapy can be defined as the treatment of human diseases by the trans- fer of genetic material into specific cells to elicit a desired therapeutic phenotype. It is not difficult to envision treating monogenic diseases, such as hemophilia, muscular dystrophy, and cystic fibrosis, by replacing errant genes within the affected cells. Gene therapies are also being developed, however, for treating car- diovascular, neurological, and infectious diseases, wound healing, and cancer, by delivering genes to augment naturally occurring proteins, to alter the expression of existing genes, or to produce cytotoxic proteins or prodrug-activating enzymes. Because of the broad potential of gene therapy, it has been heavily inves- tigated during the past 30 years. The first clinical trial of gene therapy, for the treatment of severe combined immunodeficiency (SCID), was initiated in 1990 (Blaese et al., 1995), but it took until April 2000 before the first clinical success was reported by Cavazzana-Calvo et al. (2000) of the treatment of two infants with γc-SCID. Also that year, Kay et al. (2000) reported positive data, including increased circulating levels of factor IX in a hemophilia clinical trial, and Khuri et al. (2000) reported a successful Phase II trial using a combination of gene therapy and traditional chemotherapy to treat recurrent squamous-cell carcinoma 25

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26 FRONTIERS OF ENGINEERING of the head and neck. However, despite more than 1,300 clinical trials to date, no products have been FDA-approved. At the same time, tragic setbacks, including the deaths of patients in two trials, have hindered progress. A severe inflammatory response caused by the adenovirus used in a 1999 trial for the treatment of ornithine transcarbamylase deficiency was proved to be the cause of death and resulted in a temporary halt to all gene therapy trials. In addition, at least 2 of the 11 children in the Cavazzana- Calvo γc-SCID trial developed leukemia as a result of retroviral insertion of the therapeutic sequence in or near a gene associated with childhood leukemias. Thus a key limitation to the development of human gene therapy remains the lack of safe and efficient methods of gene delivery (Verma and Somia, 1997). Current gene-delivery methods comprise recombinant viruses, which are used in the majority of clinical trials, and synthetic materials, including peptides, polymers, and liposomes. Although viruses are the most efficient vectors, they often initiate immune responses, are limited in the size of genetic material they can carry, are difficult to produce and purify, and exhibit limited target-cell specificity (or often nonspecificity). Cationic polymers (Felgner and Rolland, 1998; Pack et al., 2005; Smith et al., 1997) have the potential to be nontoxic and nonimmuno- genic, are chemically and physically stable, are relatively easy to produce in large quantities, and can be targeted to desired cell types; but in general, they are not efficient enough for clinical use. Even the most efficient polymers are orders of magnitude less efficient than viruses (micrograms of DNA are required to achieve transgene expression comparable to that resulting from a virus suspension contain- ing about 10 picograms of genetic material). The Gene-Delivery Problem To ������������������������������������������������������������������������������� escort genes from a solution (e.g., in a vial) to the cell nucleus, gene-deliv- ery vectors must navigate a series of obstacles, both extracellular and intracellular. Viruses have evolved functions to address each of these challenges, but synthetic vectors generally lack one or several of these functions. These obstacles must all be taken into consideration for the rational design of new materials. The first set of barriers facing gene-delivery vectors appears in transporting genes from the test tube to the membrane of a target cell. First, the vector must bind and condense plasmid DNA to a sufficiently small size to allow efficient cel- lular internalization and protect the genes from nuclease degradation. Polycations and DNA spontaneously form tight complexes (polyplexes) through entropically driven electrostatic interactions. The resulting particles typically comprise several DNA molecules and hundreds of polymer chains and range in size from a few tens to several hundred nanometers in diameter. Second, the polyplexes must form a stable solution under physiological conditions, which can often be achieved by coating them with a hydrophilic polymer, such as polyethylene glycol. Third, for

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POLYMER TECHNOLOGY FOR GENE THERAPY 27 FIGURE 1  Schematic illustration of the main steps in the intracellular processing of polymer-DNA gene-delivery vectors. Pack Figure 1 many indications, it is critical that vectors recognize specific cells by displaying R01394 cell-specific ligands (e.g., small molecules, peptides, proteins, or antibodies). Bitmapped fixed image Following internalization, gene-delivery vehicles must overcome a set of intracellular obstacles (Figure 1), which means the vector must have a functional- ity to overcome each one. Polyplexes are generally internalized by endocytosis, and once they are in the endocytic pathway, they are routed through a series of vesicles. The typical endpoint of this journey is the lysosome, an acidic vesicle filled with degradative enzymes including nucleases. It is critical, therefore, that DNA and the vector escape these compartments into the cytoplasm. Next, the vector must escort the DNA through the cytosol toward the nucleus. Because particles as large as typical polyplexes cannot passively diffuse in the cytosol, they require a means of active transport. The genes must then enter the nucleus, with or without the vector material. Although the nuclear envelope con-

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28 FRONTIERS OF ENGINEERING tains pores for transporting biomolecules into and out of the nucleus, the process is very tightly regulated to keep out undesirable species, including exogenous genes. Finally, DNA and the vector must separate from one another to allow transcription of the therapeutic gene. The location at which this unpackaging occurs, however, is not generally known. Although more than 95 percent of cells in culture may internalize vectors (on the order of 100,000 copies per cell), typically less than 50 percent express the transgene, suggesting that the majority are lost at one of these steps. Progress in the Design of Gene-Delivery Materials Many early studies of gene delivery employed commercially available poly- mers (Figure 2). Polylysine was one of the first cationic polymers used in the modern era of gene-delivery research (Wu and Wu, 1987; Zauner et al., 1998). Although these studies were promising, it appears unlikely that polylysine-based polyplexes will be clinically useful because of their lack of efficiency. Polyethylenimine (PEI), however, is one of the most effective gene-delivery polymers (Boussif et al., 1995). Its effectiveness is believed to be due in large part to its efficient escape from the endocytic pathway via the “proton-sponge” mechanism. Because nitrogen represents every third atom in the PEI backbone, this polymer exhibits a very high density of amines, only 15 to 20 percent of which are protonated at physiological pH. As endocytic vesicles are acidified, polyplexes containing PEI (or other proton-sponge materials) are able to buffer the vesicle lumen, leading to an influx of counter ions, osmotic swelling, and vesicle rupture. PEI-mediated gene delivery has been hindered, however, by the polymer’s rela- tively high cytotoxicity in many cell lines, both in culture and in vivo. In the past two decades, many new types of polymers have been synthesized specifically as gene-delivery vectors. Because polymer-mediated intracellular trafficking is poorly understood, however, many of these designs are based on unproven hypotheses. Results, therefore, have been mixed, with few materials providing highly efficient gene delivery. A current focus in the field, therefore, is developing a new understanding of intracellular processing and polymer struc- ture-activity relationships. Because of space limitations, only a small selection of relevant studies will be described here. One important approach has been to focus on the synthesis of biocompat- ible, nontoxic gene-delivery agents, including materials such as poly[α-(4-ami- nobutyl)-L-glycolic acid] (PAGA), a biodegradable mimic of polylysine (Lim et al., 2000), polyurethanes, disulfide-linked polymers, and poly(β-amino esters) (PBAEs) (Figure 2). As one example of the latter, Forrest et al. (2003) cross- linked low-molecular-weight PEI—which is nontoxic, but ineffective for gene delivery—with small diacrylates (Green et al., 2008). The resulting materials exhibited initial molecular weights sufficient to tightly bind and condense DNA,

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POLYMER TECHNOLOGY FOR GENE THERAPY 29 FIGURE 2  Structures of representative gene-delivery polymers. Pack Figure 2 R01394 bitmapped fixed image

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30 FRONTIERS OF ENGINEERING but they degraded within 8 to 24 hours to nontoxic by-products. These degradable PEI derivatives were as much as 16-fold more efficient than the analogous non- degradable commercial PEI of comparable molecular weight. PBAEs also have been exploited in combinatorial syntheses in which a panel of diacrylates and amines are cross-linked to generate more than 2,000 unique polymers (Green et al., 2008). These materials have been screened for gene-deliv- ery activity and other important properties, including toxicity. The best polymers were more efficient than the top commercial transfection reagents and, in some situations, were comparable to adenoviruses. Perhaps most important, by corre- lating gene-delivery activity with polymer/polyplex properties, investigators may begin to extract structure-activity relationships to guide future polymer designs. Because PEI is an off-the-shelf material, one may also expect that its buffer- ing capacity is not optimal. In fact, Forrest et al. (2004) modified the protonation profile of PEI by reaction with acetic anhydride to convert the primary and sec- ondary amines to secondary and tertiary amides, respectively (Figure 2); such a change should make a poorer proton sponge by decreasing the number of pro- tonable nitrogens in the polymer. Surprisingly, gene-delivery activity dramatically increased upon acetylation, and the polymer with acetylation on about 57 percent of the primary amines was as much as 60-fold more efficient than unmodified PEI (Gabrielson and Pack, 2006). Subsequent investigation of the mechanisms leading to this unexpected enhance- ment revealed that PEI acetylation also decreases polymer-DNA binding strength, resulting in enhanced “unpackaging” of polyplexes within target cells. This report was significant in that it identified polymer DNA as a critically important design criterion for gene-delivery materials. Conclusions A variety of polymers has been used in gene-delivery studies, but they are still orders of magnitude less effective as gene-therapy vectors than viral vectors. As a result, polymers are generally considered unacceptable for clinical applications. Even though the important extra- and intracellular barriers to efficient gene deliv- ery are known, the poor performance of polymer gene-delivery vectors is attribut- able to a lack of functionality for overcoming at least one of these barriers. Based on the large number of studies of off-the-shelf gene-delivery polymers, much has been learned about the structure-function relationships of polymer vectors. This knowledge has been applied to the design and synthesis of new polymers, tailor-made for gene delivery, and a number of promising candidates have been reported in recent years. As our understanding of polymer gene-delivery mechanisms improves, it is likely that polymer-based gene-delivery systems will become an important tool in human gene therapy.

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