ChemBioChem 2004, 5, 53 ± 54 DOI: 10.1002/cbic.200300766 ¹ 2004 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 53

Gene therapy may be described as the useof genes as medicines to treat disease, ormore precisely as the delivery of nucleicacids by means of a vector to patients forsome therapeutic purpose. Gene therapyhas enormous promise but clinical trialshave failed to deliver.[1] The primaryreason is the inadequacy of the vectorsused. Researchers have driven for clinicalapplications long before vector technolo-gies have been adequately developed orunderstood. Predictably, there has been asignificant decline in scientific and publicperceptions of gene therapy. This trend isunhelpful but can be reversed bya period of patient, logical, tech-nical and scientific developmentof new vector systems, prior toany major second round of clin-ical trial activity.[1]

But which type of new vectorsystem would be most appropri-ate to develop: viral or nonviral,synthetic or physical? Opinionsmay vary, but a balanced assess-ment suggests that syntheticnonviral vectors should be themain vector systems of choice forroutine gene therapy in the fu-ture. Synthetic nonviral vectorsystems have many potentialadvantages compared with viralsystems, including significantlylower toxicity/immunogenicityand potential for oncogenicity,size-independent delivery of nu-cleic acids (from oligonucleotidesto artificial chromosomes), sim-pler quality control, and substan-tially easier pharmaceutical andregulatory requirements. Increas-

ing public alarm, particularly with thetoxic side effects of virus use, is alsostrengthening these significant advantag-es. So what is holding us back? Quitesimply, synthetic nonviral vector systemsdo not appear to be stable enough inbiological fluids, neither are they suffi-ciently reproducible or efficient enough atmediating transfection (i.e. , gene deliveryand expression).[2] There is a clear need fortechnical breakthroughs.

The recent paper by Aoyama et al.[3]

may represent one such breakthrough.While many synthetic nonviral vector

systems have relied upon the centraluse of cationic lipids or cationic polymers,Aoyama et al. describe a novel class ofglycoconjugate amphiphiles (glycoclus-ter amphiphiles) (Scheme 1). These form

micelle-like pentameric aggregates in sol-ution known as glycocluster nanoparticles(GNPs) (Scheme 1), whose size, shape anddimensions resemble those of a viralcapsid protein. Glycocluster 1a was stud-ied in detail and shown to complex andcondense plasmid DNA completely ata host/base or host/phosphate ratio(1a/P)�0.5 to form glycoviral particles(approx. 50 nm) with low negative(� �10 mV) to zero � potentials (depend-ing upon the exact 1a/P ratio) that appearto contain only a single plasmid DNAmolecule per particle (Figure 1). Remark-

ably, while these glycoviral particles con-tain no cationic charges, they are still ableto mediate the transfection of HeLa, CHO,Huh-7, HepG2 or COS cells in vitro (serum-free or 10%-serum conditions) giving

[a] Prof. A. D. MillerImperial College Genetic Therapies CentreDepartment of ChemistryFlowers Building, Armstrong RoadImperial College LondonLondon SW7 2AZ (UK)Fax: (�44)20-7594-5803E-mail : a.miller@imperial.ac.uk

Scheme 1. Structures of glycocluster amphiphiles and schematic drawing to show formation of a pentamericglycocluster nanoparticle (GNP) from glycocluster amphiphiles.[3]

Gene Therapy Needs Robust Synthetic NonviralPlatform TechnologiesAndrew D. Miller*[a]

A. D. Miller

54 ¹ 2004 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chembiochem.org ChemBioChem 2004, 5, 53 ± 54

acceptable levels of gene expression,competitive with cationic liposome-medi-ated gene delivery in vitro.

Recent research, our own included, hasamply demonstrated that attempts atsystematic improvements of syntheticnonviral vector systems are destined tobe fruitless unless the most fundamentalproblems associated with achieving re-producible and scalable formulations,resistance to aggregation, long termstorage and properly reproducible trans-fection outcomes are convincingly solvedprior to future attempts at systematicimprovements.[2] By definition, any truesynthetic nonviral vector platform tech-nology must embody solutions to all ofthese fundamental problems. Such size-defined, self-assembly virus-like nanopar-ticles have been much sought after in thefield of synthetic nonviral vector systems,not least because they can lay claim topresenting solutions to the fundamentalproblems above. Good recent examplesare the stabilised plasmid-lipid particle(SPLP),[4, 5] and liposome/mu/DNA (LMD)systems (Figure 1),[6, 7] which comprise atleast some cationic lipids. Both are pri-mary vehicles that have been constructedfrom primary toolkits of well-definedchemical components. Both have been

shown to mediate transfection in highserum conditions and even in vivo. Bothcan now be developed in a sequential andlogical fashion for expanded use in vivoby means of modular adaptations/mod-ifications by using secondary toolkits ofchemical components, designed to en-hance the transfection process and over-come specific barriers to transfection.[8]

The glycoviral particles of Aoyama et al.could well be another valuable syntheticnonviral vector platform technology, val-uable especially for being of neutralcharge and comprising novel classes ofdefined chemical components. Evidently,Aoyama et al. will need to provide furtherevidence to demonstrate that glycoviralparticles may be formulated in a reprodu-cible and scalable manner, be amenableto long-term storage and give properlyreproducible transfection outcomes.Moreover, assuming that this informationwill be provided, then Aoyama et al. willalso need to demonstrate more than arudimentary biological efficacy for theirparticles. Transfections in the presence ofhigh levels of serum in vitro and trans-fections in vivo will surely be the acid testfor these exciting new particles. Never-theless, the novelty and potential of thisresearch should not be underestimated,

and Aoyama et al. are to be congratulatedfor breaking potentially impressive newground in the quest for the ideal, clinicallyviable, synthetic nonviral vector systemfor gene therapy.

Keywords: DNA ¥ gene expression ¥gene therapy ¥ nucleic acids ¥ platformtechnologies

[1] A. D. Miller, Global Outsourcing Rev. 2002, 4, 8.[2] A. D. Miller, Curr. Med. Chem. 2003, 10, 1195.[3] Y. Aoyama, T. Kanamori, T. Nakai, T. Sasaki, S.

Horiuchi, S. Sando, T. Niidome, J. Am. Chem.Soc. 2003, 125, 3455.

[4] J. J. Wheeler, L. Palmer, M. Ossanlou, I. MacLa-chlan, R. W. Graham, Y. P. Zhang, M. J. Hope, P.Scherrer, P. R. Cullis, Gene Ther. 1999, 6, 271.

[5] Y. P. Zhang, L. Sekirov, E. G. Saravolac, J. J.Wheeler, P. Tardi, K. Clow, E. Leng, R. Sun, P. R.Cullis, P. Scherrer, Gene Ther. 1999, 6, 1438.

[6] M. Keller, R. P. Harbottle, E. Perouzel, M. Colin, I.Shah, A. Rahim, L. Vaysse, A. Bergau, S. Moritz,C. Brahimi-Horn, C. Coutelle, A. D. Miller, Chem-BioChem 2003, 4, 286.

[7] T. Tagawa, M. Manvell, N. Brown, M. Keller, E.Perouzel, K. D. Murray, R. P. Harbottle, M. Tecle,F. Booy, M. C. Brahimi-Horn, C. Coutelle, N. R.Lemoine, E. W. F. W. Alton, A. D. Miller, GeneTher. 2002, 9, 564.

[8] E. Perouzel, M. R. Jorgensen, M. Keller, A. D.Miller, Bioconj. Chem. 2003, 14, 884.

Received: September 9, 2003 [H766]

Figure 1. Electron microscopy of particles. a) Transmission electron microscopic (TEM) images of glycoviral particles formed in water from 1a and plasmid DNA ina 1a/P ratio of 0.7, with uranyl acetate as a negative stainer.[3] b) Enlargment from (a). c) Cryoelectron microscopy image of liposome/mu/DNA (LMD) particles forcomparison.[7]