Converging Paths of Viral and Non-viral Vector Engineering
Authors: Ernst Wagner
Journal: Molecular Therapy (2007)

Vector design is the dominant technical domain of gene therapy research. Diverse ideas and strategies exist for engineering an ideal vector, because vector requirements are specific to particular gene therapy applications. However, these divergent starting points and design directions have begun to converge on one another to promote the entire vector science field. Over the past two decades, viral vectors have been developed that are very effective at gene transfer, and synthetic vectors have been developed that are nonimmunogenic and very flexible in payload. It was apparent early on that both viral and nonviral vectors possess positive and negative characteristics. More recently, investigators have started to combine aspects of each vector type into hybrid vectors that capitalize on the positive characteristics. In this issue, Kreppel and Kochanek1Kreppel F Kochanek S Modification of adenovirus gene transfer vectors with synthetic polymers: a scientific review and technical guide.Mol Ther. 2008; 16: 8-15Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar review the chemical modification of adenoviral vectors with synthetic polymers, and Wolff and Rozema2Wolff JA Rozema DB Breaking the bonds: non-viral vectors become chemically dynamic.Mol Ther. 2008; 16: 16-29Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar review the development of chemically dynamic synthetic vectors that can be viewed as viral vector mimics. Are vector scientists now converging on a common destination from divergent origins? Nature has evolved adenoviruses into nanoparticles that efficiently make their way into target cells, where they disassemble in a chemically programmed manner. After target receptor binding and endocytosis, the receptor-binding adenovirus fiber is dismantled within the endosome. The endosomal environment triggers the release of a peptide fragment of protein VI, which destabilizes the endosomal membrane and promotes escape of the virus into the cytosol, where the virus capsid hitches a ride on motor proteins along microtubules to the nuclear pore; once there, it disassembles and releases the DNA into the nucleus. However, important extracellular hurdles, such as poor specificity for the target tissue and strong immune and inflammatory responses, limit the exploitation of adenovectors for in vivo gene therapy. As outlined by Kreppel and Kochanek,1Kreppel F Kochanek S Modification of adenovirus gene transfer vectors with synthetic polymers: a scientific review and technical guide.Mol Ther. 2008; 16: 8-15Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar chemical coating of the adenovirus surface with simple hydrophilic polymers such as polyethylene glycol (PEG) or poly-hydroxypropylmethacrylamide significantly reduces innate immune responses. In addition, chemically shielded adenoviruses can evade anti-adenovirus immunity and can also be retargeted to show more specific tropism. Very simple chemical modifications can complement genetic retargeting strategies. The ease of chemistry has fueled the development of synthetic vectors. Cationic lipids or polymers spontaneously self-assemble with nucleic acid into nano-sized complexes with morphologies that often resemble viral particles. Production scales can be several log units beyond viral particle production. However, transfection efficiency per particle can be several log units below viral transduction. Two distinct strategies have been used to optimize synthetic vectors. First, targeting and shielding molecules have been incorporated into the carrier formulations for enhanced and more specific performance, as with chemical virus modification. Second, empirical screening of various cationic lipids and polymers has resulted in greatly improved formulations with high gene transfer efficiency and low cytotoxicity. A favorable formulation has to be effective at the many steps of the delivery process, including complexation and protection of the nucleic acid payload, cell-surface binding, translocation across lipid barriers and/or release from endosomes, and delivery to the cytosol or nucleus. No single polymer or lipid excels at every step. This led to the "synthetic virus" approach, whereby various delivery functions are segregated into separate modules, as is the case with native viruses. Proof-of-concept cases have been reported wherein incorporation of single-entry functions (targeting ligands, shielding agents, endosomal release agents, nuclear delivery agents) have markedly improved the properties of synthetic carriers. The simultaneous incorporation of two or several delivery functions, however, rarely resulted in additive or synergistic effects. Sometimes functions were even found to be incompatible. Shielded particles showing improved systemic circulation and targeting can show reduced transfection activity, for example, and formulations with improved endosomal escape can give rise to enhanced toxicity. At the root of the problem is the static or constitutive incorporation of functions in a poorly organized manner. To solve such problems, two additional critical aspects must be considered in the design of "synthetic viruses": proper assembly of multifunctional particles during preparation and timely responsiveness or activation of the various delivery functions during infection. As with natural viruses, synthetic vectors must change their chemical properties with time; they must be chemically dynamic. As nicely reviewed by Wolff and Rozema,2Wolff JA Rozema DB Breaking the bonds: non-viral vectors become chemically dynamic.Mol Ther. 2008; 16: 16-29Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar such functional dynamics can be triggered in several ways. For example, the acidification process in endosomes can be used to trigger chemical changes, such as the cleavage of pH-labile covalent bonds to allow the removal of a PEG shield and/or the activation of endosome-destabilizing domains by removing a masking group. Alternatively, endosomal acidification may also trigger conformational changes that reversibly activate delivery functions. Many other natural or artificial triggers from the microenvironment can be exploited, including cleavage by target-specific proteases, intracellular reduction of disulfide bonds, and photochemical cleavage. Natural viruses build on very similar chemistries; the membrane-destabilizing terminal peptides of viruses from diverse classes are generated by proteolytic cleavage from precursor proteins and, for a large subclass of endocytic viruses, are activated by endosomal conditions. Synthetic vectors are currently formed by simple mixing or slightly more advanced formulation processes, building on self-assembly into larger supramolecular structures. In generating virus particles, nature uses a far more sophisticated assembly process that includes precursor proteins, the protein-folding machinery, and/or cellular lipids as construction scaffolds. Comparing current self-assembly of synthetic viruses with the assembly of natural viruses is like comparing the building of a sand castle by a child with the refined construction of a Gothic cathedral. In perspective, it is interesting to ponder whether with time the perfect "chemically modified virus" may begin to resemble the perfect "synthetic virus analog." What will be similar, what different, and what will be the unique selling points of each class of vector? Although it is impossible to speculate on the sequence and speed of developments, some predictions can be made. Both approaches will be applicable for delivery of genes, RNA-mediated interference, antisense molecules, exon-skipping constructs, and homologous recombination-based repair constructs. Only synthetic vectors will be suitable for delivery of synthetic and chemically modified/stabilized nucleic acids. Presumably they will also be more flexible with regard to size of payload. The highly refined structure of viral vectors has the inherent advantage of being biocompatible and biodegradable—but also the inherent risk of triggering immune responses against viral proteins. Genetic engineering, including incorporation of human sequences and immune-suppressive sequences, micro-RNA-based silencing in immune cells, chemical shielding, and other chemical modifications may allow us to overcome such limitations. Moreover, intended conditional replication of viral vectors will be an exploitable feature that synthetic analogs will never possess. In contrast to biology, which uses primarily lipids, carbohydrates, and proteins as building blocks, synthetic chemistry is far more diverse in the space of physicochemical properties of molecules. This diversity includes very simple structures (for example, PEG) with interesting biological properties that nature cannot synthesize. This expanded repertoire of building blocks will make it possible to design chemical analogs to viral protein functions with much simpler and more chemically accessible structures than the corresponding protein domains. However, it will require considerable effort to identify the proper structures and functions. This leaves current vector scientists with much room for exploring the converging paths of the development of chemically modified viral vectors and chemically dynamic synthetic vectors.