Introduction

Transgenes can be delivered to muscle using viral or non-viral systems. Although the pioneer study used naked plasmid DNA for gene delivery,¹ the low efficiency of this approach has led to the focus being placed on viral delivery systems over the last 10 years or so. Several genetically modified viruses, such as retrovirus, herpes simplex virus, Epstein–Barr virus, adenovirus (AdV) and adeno-associated virus (AAV), have all been tested for gene delivery into muscle.^2,3,4,5 Of these, AdV and AAV have been found to be most efficient so far for transducing muscle fibres.^6,7,8 AAV in particular is able to transfect non-proliferating muscle fibres and is considered to be non-pathogenic, as it is not found to be associated with any disease. Gene delivery by AAV vector also appears to be the least immunogenic of the viral vector systems as regards the transgene and can sustain longer periods of transgene expression than other viral vectors.^9,10,11 While the majority of gene therapy experiments and clinical trials currently use viral delivery systems,^12,13,14 several aspects need to be improved to achieve therapeutic benefit in patients. These include better efficiency and reliability of procedures for the manufacture of virus, freedom from helper virus contamination,¹⁵ avoidance of immunogenicity of the viral particle itself, and ways of overcoming the limitation of transgene size.^16,17,18

In several of these respects, non-viral delivery systems have clear advantages over viral delivery systems. The use of plasmid DNA does not pose health risks in principle entailed by viral infection. Plasmid DNA is easy to propagate on a large scale at high quality, and is also able to carry relatively large DNA sequences. Naked plasmid DNA itself has low immunogenicity and can be administered repeatedly although local inflammatory reactions are still a concern.¹⁹ The other major disadvantage reported is that plasmid DNA-mediated gene delivery with a foreign transgene driven by non-muscle specific promoters may evoke more severe immune responses to the transgene product than AAV vectors expressing the same protein.^20,21

The big drawback of plasmid DNA as a vector is its low efficiency for gene delivery. However, this has recently been improved significantly by use of transfection-enhancing reagents such as lipids and polymers. These reagents improve the dissemination of plasmids within the target tissues, penetration through cell membranes, stability of plasmids within the target cells, and entry of plasmids into nuclei. In addition, novel methods have been developed for restricted local as well as systemic delivery of transgenes, including the use of microbubbles and ultrasound, multi-purpose gene delivery injectors, and, perhaps the most promising, electroporation. Another development in non-viral delivery is the use of viral-specific peptide sequences or synthetic peptides, aimed at obtaining high efficiency and tissue specificity without the use of infectious virus or whole viral coat proteins. These developments in combination with advances in vector construction have led to efficiencies of transgene expression with non-viral delivery systems comparable to those with recombinant viral vectors.²² In this review, we will focus on these developments in non-viral delivery systems aimed at using skeletal muscle as a target tissue.

Why skeletal muscle?

Skeletal muscle, by virtue of a number of inherent anatomical, cellular and physiological properties, commends itself as a target tissue for gene therapy, particularly for production of proteins as systemic therapeutic reagents.

1. Skeletal muscle constitutes about 30% of the normal adult body mass and is easily accessible for nearly all gene delivery approaches currently used in gene therapy.
2. Skeletal muscle has an abundant blood vascular supply. An extensive capillary network wraps around every muscle fibre with regular spacing, thus providing an efficient transport system for the carriage of secreted protein into the circulation.
3. Skeletal muscle fibres are terminally differentiated cells, and nuclei within the fibres are post-mitotic. Individual fibres are thought to persist for much of the lifetime of the individual. Even when muscle fibres are damaged, only short segments of individual fibres undergo degeneration, and the myonuclei of surviving segments remain viable.²³ This provides a stable environment as a "factory" for continuous production of transgene.
4. The syncytial nature of muscle fibres provides a mechanism for dispersal of transgene from a limited site of penetration to a large number of neighbouring nuclei within the fibre. Such dispersion within transfected fibres may well be one of the reasons behind the efficient expression of transgenes in muscle.

Skeletal muscle also has great regenerative capacity due to the presence of a population of stem-like precursor cells, called satellite cells, situated between fibre membrane and basal lamina. These cells, while quiescent in normal muscle, can be activated in response to muscle damage, proliferating, migrating and fusing with one another to replace lost fibres, resulting in a regeneration of the lesion (see review by Partridge²⁴). Moreover, satellite cells can be purified from muscles and cultured without losing their ability to differentiate into muscle fibres (Figure 1). A large number of myogenic cells can be produced from a single muscle biopsy and transferred back into the host after ex vivo genetic manipulation (Figure 1), thus providing the potential to expand and bank an individual's own cells expressing any desired transgene. In this context, the recent identification of other stem cell populations within skeletal muscle is also potentially important to gene therapy.^25,26, These stem cells, although their precise nature is not understood, are capable of differentiating into other cell lineages, thus providing an accessible source of multi-potential stem cells for gene therapy.

Figure 1.

(a) An isolated muscle fibre showing a chimeric expression of nuclear-localizing -galactosidase in myonuclei (blue staining and indicated by arrow heads) over half the length (left side) of the fibre. Host myonuclei are stained pink with propidium iodide (indicated by arrows). Donor H2K myoblasts were first infected with retrovirus carrying -galactosidase transgene and the infected cells were then injected into the tibialis anterior muscle. Muscles were removed 4 weeks later and single fibres were prepared and stained for -galactosidase and counterstained with propidium iodide. The blue X-gal nuclear staining quenches the propidium iodide fluorescence. (b) Marker gene, GFP, expression in cultured myotubes and myoblasts. H2K myoblasts were cultured and transfected with plasmid DNA containing the GFP transgene mediated by polyethylenimine 25000 (Sigma). Both myoblasts (indicated by arrow heads) and myofibres (indicated by arrows and multi-nucleation) show expression of GFP. GFP signals were seen throughout the whole length of the myofibres. The nuclei are counterstained blue with DAPI.

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Having highlighted the advantages of skeletal muscle as a target for gene therapy, one needs to bear in mind its possible limitations. For example, post-transcriptional modifications in muscle may differ from other cell types. Arruda et al found that carboxylation, tyrosine sulphation and serine phosphorylation of factor IX differ between liver and muscle, although post-translational modifications critical for biological activity of the factor are similar in these two tissues.²⁷ It should also be noted that there are substantial differences between the efficacies of transfection of myoblasts in vitro and muscle fibres in vivo for any given procedure. Nearly all systems that give efficient plasmid delivery in vitro have so far shown little beneficial effect when used for in vivo gene transfer into skeletal muscle (see below).

Vector construction

The importance of vector construction in gene therapy is unequivocal for both viral and naked plasmid delivery systems. Vectors can influence the level and duration of gene expression, affect the efficiency of gene delivery and confer regulatory functions.

Promoter, enhancer and other elements

The most critical feature of a vector is the 'promoter' which drives the transcription of a transgene. A variety of plasmid backbones containing different promoters have been tested for transgene expression in skeletal muscle with varying success. The most widely used are viral promoters, such as cytomegalovirus (CMV) immediate-early promoter,²⁸ simian virus, SV40 early promoter²⁹ and Rous sarcoma virus promoter.¹ These promoters can drive expression in a wide range of cells and tissues eg CMV promoter can be activated in nearly all cell types. Of the non-viral promoters employed, some such as actin have general activity within eukaryote cells.^30,31 Some promoters control expression in a cell- or tissue-specific manner eg skeletal muscle actin promoter,³² myosin light chain 3F promoter³³ and muscle creatine kinase (MCK) promoter³⁴ which are activated specifically in muscle. The best example of promoters of high cell specificity is the ventricle-specific myosin light chain-2 which is activated exclusively in cardiac myocytes.³⁵

Muscle-specific promoters may have advantages over non-muscle-specific promoters, particularly for gene therapy of muscular dystrophies. Cordier et al, using an AAV vector for the gamma-sarcoglycan construct, observed a much diminished humoral immune response and a more persistent expression when the transgene was under the control of muscle-specific MCK promoter than when it was controlled by the CMV promoter.³⁶ They suggested that it may be important to restrict transgene expression to muscle tissue, so that antigen-presenting dendritic cells are unable to elicit an immune response. This may be crucial for treatment of muscular dystrophy which requires the long-term expression of the transgene. In contrast, Wang et al reported that equally sustained expressions of human mini-dystrophin genes were achieved under the control of both CMV and MCK promoters in otherwise the same AAV-mediated gene transfer vectors.³⁷ In this case, it may be that the highly deleted human dystophin proteins, lacking almost the entire rod domain, contained few epitopes that were not present on murine dystrophin expressed in revertant fibres,³⁸ and might thus benefit from immune tolerance to these proteins. Clearly, we need a better understanding of the interaction of the immune system and transgene products and the influence of various vector and promoter systems.

Natural muscle-specific promoters in general drive relatively lower levels of transgene expression compared to non-tissue-specific viral promoters, such as CMV promoter, thus limiting their potential use in muscle as a gene therapy target. Efforts have been made to increase the strength of muscle-specific promoters while maintaining their tissue specificity. Muscle-specific promoters display complex organization usually involving combinations of several myogenic regulatory elements including E-box, MEF-2, TEF-1 and SRE sites. Li et al assembled these elements randomly into synthetic promoter recombinant libraries and screened hundreds of individual clones for transcriptional activity both in vitro and in vivo.³⁰ Several artificial promoters were isolated whose transcriptional potencies exceed those of natural myogenic and viral gene promoters.

Promoters are not the only important elements in control of transgene expression. Attention must also be paid to enhancers, poly A tails and other regulatory elements such as intronic sequences. The power of a vector to drive gene expression is a combined effect of all these elements and may vary between particular cell or tissue systems. Efforts have been made to construct highly efficient vector systems for transgene expression in muscle by searching for optimal combinations of these elements. Barnhart et al used enhancers and promoters from various muscle-specific genes and created 19 enhancer/promoter chimeras in combination with the human CMV IE enhancer/promoter to drive a luciferase reporter gene.³⁹ The efficiency of reporter gene expression was examined both in vitro by transfection of differentiated C2C12 mouse myoblasts and in vivo by direct intramuscular injection. Only a 170 bp myogenin enhancer sequence inserted in front of CMV promoter increased expression levels in vivo compared with the CMV promoter alone. In other experiments, Li et al combined the CMV promoter/enhancer with the SV40 enhancer and found a 20-fold greater expression in muscle with a single copy of the 72 bp element of the SV40 enhancer placed either 5' of the CMV promoter/enhancer or 3' of the poly A tail.⁴⁰ However, this higher expression was only seen in non-dividing muscle fibres, not in myoblasts. Similarly, Xu et al evaluated the combined effect of different promoters, enhancers and other regulatory elements (including promoters and introns of RSV, human phosphoglycerate kinase and actin), using luciferase as a reporter gene.³¹ The most powerful vector system for gene expression in skeletal muscle was a combination of CMV or actin promoters with SV40 enhancer, together with SV40 poly A and CMV intron A sequence. The consensus of these findings is that direct DNA plasmid injection driven by the CMV promoter/enhancer together with its intronic A sequences, and flanked by an SV40 enhancer with its poly A sequences is the most potent vector construct. Figure 2 depicts this construct, which can serve as the basis for further improvement in vector construction. Further improvements might include nuclear localization elements to enhance transport of plasmids into the nucleus as well as regulatory elements for export, stabilization and translation of transcripts as detailed below.

Figure 2.

Diagrammatic representation of idealized vector for high levels of transgene expression in skeletal muscle. Note the presence of two enhancers, one in front of promoter sequence and the other at the end of poly A tail of the transgene. The additional nuclear localization element (NLE) and other regulatory elements would contribute to high level and regulated production of the transgenic protein.

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Vector construction for enhancing nuclear transport and expression

The potency of transgene expression partly depends on the efficiency with which plasmid DNA is transported into nuclei. Transport of macromolecules between cytoplasm and nucleoplasm is a dynamic process, mediated mainly via the nuclear pore complex by at least three different classes of transport receptor systems.⁴¹ Although the mechanism(s) by which plasmid DNA enters nuclei is not well established, efforts have been made to facilitate the nuclear import of plasmids by utilizing mechanisms employed by viruses or nuclear proteins. Since some viral and non-viral DNA sequences can be recognized by nuclear import machinery, incorporation of such specific DNA sequences into vectors may aid the entry of plasmid DNA into nuclei. For example, the yeast transcription factor GAL4 enhances gene expression through its binding to specific 17 bp DNA sequence and its nuclear localization ability. By incorporating this DNA sequence into a vector together with the GAL4 protein, Chan et al found an increase in transgene expression, which they attributed to GAL4-mediated transport of the plasmid into the nuclei.⁴² Similarly, Dean and colleagues showed that a 72-bp repeat of the SV40 enhancer facilitated nuclear localization of plasmids.^43,44 Other viral promoter DNA sequences, such as oriP which the EBV virus uses for nuclear targeting, could also be beneficial.^45,46 However, not all viral promoter sequences have this capacity:the CMV promoter and the RSV LTR, for example, appear unable to directly target the plasmids into the nucleus.

Expression of transgenes can also be enhanced by co-expression of transactivators with recognition sequences built into the vector. These include serum response elements within CMV and some skeletal muscle promoters, such as alpha-actin promoter. Li et al reported that co-expression of serum response factor (SRF) increased luciferase gene expression by five fold or more when the transgene was under control of skeletal muscle alpha-actin promoter and CMV promoter.⁴⁷

Inducible promoter systems

The advance in vector construction has so far been limited mainly to improvement in efficiency of gene expression. Ideally, vector systems should be able to confer a regulated expression required for different clinical applications. An example of such needs is the expression of insulin for the treatment of diabetes, which requires expression to be coordinated with blood glucose levels. Another example is the expression of erythropoietin (EPO) for the correction of anaemia associated with diseases such as renal failure.⁴⁸

Several systems have been developed for achieving regulated transgene expression.^49,50,50, These approaches use the principle of controlling transgene expression by means of elements or transcription factors designed to bind selectively to the promoter which drives the transgene. Expression of transgenes can thus be regulated by modulating expression of these transcription factors or altering their activity through drug administration. However, regulation of transgene expression in vivo by such approaches is unreliable, mainly due to the leakage of activity in what ought to be inactive states, and low levels of control associated with the complexity of the systems. It is beyond the scope of this review to discuss this issue in detail but it is well covered in reviews by Mansuy et al⁵⁰ and Ozawa et al.⁵¹

Delivery system

It is the inefficiency of naked plasmid DNA that restricts its clinical potential as a vehicle for gene delivery. This arises from a number of limitations most notable of which are: instability in the extra-cellular milieu, poor entry into the cell, tendency to be targeted to intracellular degradation pathways and lack of effective transport into the nucleus. The majority of publications on this topic have been dedicated to means of overcoming these various problems. We will discuss these approaches individually, since many of them tackle more than one of the hurdles.

Lipids, liposomes and polymers

A number of cationic substances, such as liposomes, lipids and polymers, have been investigated for their capacity to improve efficiency of gene delivery.^52,53,54 Cationic lipids and liposomes are the most widely used and several have been shown to raise the efficiency of in vitro gene delivery in many cell types. The mechanism(s) by which lipids and liposomes improve gene transfer is not clearly understood, but several factors, such as the type of cell and their proliferative status, appear to be involved. The positively charged lipids and liposomes are thought to improve transgene delivery mainly through binding to and condensing negatively charged DNA, forming a complex called lipoplex in which the DNA is protected against extracellular degradation. Moreover, the positively charged lipoplex binds to the negatively charged cell surface molecules facilitating endocytosis. Once in the endosome, some lipids may destabilize the endosomal membrane and encourage the release of DNA into the cytosol, thus avoiding the lysosomal degradation pathway.^55,56,57 Such lipoplexes can effectively deliver transgenes to myoblasts in culture and many different types of cells in vivo, including epithelial cells and hepatocytes.^{58,59,60,61,62} However, the effectiveness of lipoplexes on gene transfer in muscle in vivo has been disappointing, both as reported by other groups⁶¹ and in our laboratory. In fact, lipoplexed DNA is often less efficient for transduction of muscle than the same plasmid delivered as naked DNA. The reasons for this ineffectiveness in skeletal muscle in vivo are not clear. One obvious difference between muscle fibres and the cells in tissues such as liver, kidney and skin is that every muscle fibre is surrounded by a layer of mechanically strong extracellular matrix (ECM), a basement membrane rich in glycosaminoglycans (GAGs). It has been suggested that directly injected lipoplexes may bind to those negatively charged ECM components.⁶³ This is supported by the work of Ruponen et al⁶⁴ who demonstrated that the transfection efficiency of several commonly used cationic lipid formulations, such as DOTAP, DOTAP/Chol,⁵⁴ DOTAP/DOPE,⁶⁵ DOSPER,⁶⁶ DOGS,⁶⁷ can be blocked by anionic heparan sulphate. Furthermore, the retardation of lipids in ECM may cause immune and inflammatory responses, which could damage the adjacent transduced fibres.

Cationic polymers have also been disappointing for muscle transfection.^68,69 The commonly used cationic polymers, poly-(L)-ornithine (PLO), polyamidoamine and branched polyethylemine (PEI) and its analogues mediate highly efficient plasmid delivery in vitro into many types of cells, including myoblasts.^70,71,72 In vivo, however, these polymers did not improve transfection efficiency in skeletal muscle. The reason(s) for the failure in vivo may again be the interaction between the positively charged polymers and negatively charged ECM components within skeletal muscle.

In contrast, non-ionic polymers such as poly N-vinyl pyrrolidone (PVP) and some co-polymers have been shown in vivo to enhance transduction of muscle fibres significantly.⁷³ Based on the use of PVP, Rolland and colleagues established the concept of the 'protective, interactive, non-condensing' (PINC) delivery system, and reported up to 10-fold enhancement of transgene expression over naked plasmid alone.⁷³ They have used this delivery system to obtain high levels of biologically active human growth hormone (hGH) in the circulation following intramuscular injection.²⁰

More recently, Lemieux et al reported that an amphiphilic carrier, SP1017, composed of a mixture of the block co-polymers (poloxymers), pluronics L61 and F127, significantly augmented intramuscular expression of several reporter genes.⁶⁸ SP1017 enhanced peak gene expression by about 10-fold compared with injected naked plasmid alone and also led to sustained higher levels of expression. We too have observed an improvement in transgene expression in muscle with naked plasmid delivered in conjunction with such a polymer formulation. An average of more than 200 muscle fibres can be transduced to express the GFP marker gene at the site of single injection (Figure 3), compared with less than 10 fibres in control injections of the plasmid alone. An important advantage of these polymers is that they exhibit a significantly higher efficiency, with an optimal dose of the carrier some 500-fold lower in amount than PVP. The block co-polymers used in SP1017 are listed in the US Pharmacopoeia as 'inactive excipients' and are widely used for drug delivery.^74,75 Toxicity studies have shown that the amount of SP1017 used in gene delivery has a greater than 1000-fold safety margin, implying that they could readily be used in clinical trials. The mechanism(s) by which poloxamers enhance gene transfer is not clear. However, SP1017 has been shown to considerably increase plasmid DNA diffusion within muscle tissue.⁶⁸

Figure 3.

Cross-sections of tibialis anterior muscle of C57 B10 mice transfected with plasmid DNA containing a marker gene expressing cytoplasmic GFP. The plasmid DNA was injected intramuscularly with poloxamers in SP1017 formulation (a) or with saline as a control (b) in a total volume of 30 l at a single site. Mice were killed and muscles sectioned 7 days after injection. GFP expression was viewed by fluorescence microscopy. The background red colour shows the outlines of muscle fibres, whereas the green signals indicate fibres expressing GFP. More than 150 muscle fibres express GFP in this field alone (a). In contrast, less than 10 fibres express GFP in the control muscle (b) injected with the same plasmid at the same concentration but in the absence of the polymers. Note the variation in signal intensity among GFP-positive fibres.

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Proteins and polypeptides

The high efficiency of virus infection is thought in general to be mediated by mechanisms that overcome each of the four hurdles described above. First, the viral envelope stabilizes the viral genome in extracellular compartments. Second, internalization of viral particles via endosomal vacuoles is facilitated by binding of specific viral surface proteins to cognate cell surface receptors. Third, by destabilizing endosomal membranes, viral particles are released into the cytosol thus escaping the lysosomal degradation pathway. Finally, the viral genomes gain access to the nucleoplasm by active targeting, again through specific viral proteins. Since these viral functions are executed by specific domains or sequences of amino acids of viral proteins, there is clearly room for exploitation of these peptides to boost delivery of naked plasmids. Indeed, whole inactive viral particles or viral proteins, particularly those derived from adenoviruses, have been shown to enhance transgene expression.^72,76,77 Although the results are encouraging, the amount of viral protein required presents an impediment to therapeutic usage of this approach for in vivo transfection. However, it may have some applicability for ex vivo gene therapy. For example, Campeau et al used this principle for delivery of the full-length dystrophin gene (12 kb in size) into myoblasts, which were then transplanted into muscle.⁷² Intensive effort has been focused therefore on the design of short synthetic peptides that would mimic the useful functions of viral proteins, while avoiding the disadvantages associated with virus infection, namely, induction of inflammation, immunogenicity and toxicity.⁷⁸

Most peptides designed and tested so far have either originated from earlier studies on drug delivery or have been modified from viral protein sequences. The important features of these polypeptides are thought to be their ability to bind and/or condense DNA, or to destabilize cell membranes (see review by Plank⁷⁹). Poly-L-lysine and its derivatives are the most widely reported polypeptides employed for gene delivery. Positively charged lysine polymers bind strongly to negatively charged DNA, compacting it and possibly protecting it from enzyme degradation. But their ability to improve transfection on their own has proved to be limited.⁸⁰ On the other hand, when such positively charged polymers are used in combination with membrane-destabilizing reagents such as inactive viral particles,⁷⁷ viral fusion peptides,^81,82 or receptor ligands such as transferrin,⁸⁰ transfection efficiency increased significantly. Such combinations have not been tested for gene transfer in muscle.

In search of peptides with selective membrane destabilisation properties to improve gene delivery, Subbarao et al designed a 30-residue amphipathic peptide.⁸³ This peptide, GALA (WEAALAEALAEALAEHLAEALAEALEALAAGGSC), has repeat units of glutamic acid–alanine–leucine–alanine and possesses the property of destabilizing membranes in a pH-dependent manner. Parente et al examined the membrane-destabilizing effect of GALA peptide, on the experimental model of lipid biolayer vesicles of egg phosphatidylcholine in a lipid/peptide molar ratio of 500/1.⁸⁴ At pH 5.0, leakage of contents was detected from 100% of the vesicles, but was abrogated at pH 7.5. This is thought to result from pH-dependent conformational changes of the peptide, which binds to the bilayer at pH 5.0, but not at neutral pH. The drawback of this peptide is that it has the same charge as DNA and consequently binding of the two is weak, resulting in limited effects on gene delivery. However, covalent attachment of the peptide to polyamidoleamine, via a disulphfide linkage, raises the transfection efficiency of the 1:1 complex by 2–3 orders of magnitude.⁸⁵ This augmentation of transfection efficiency is likely due to the combination of the DNA condensing and the pH buffering effect in the endosomal compartment by polyamidoleamine together with the destabilizing effect of the peptide on cell membranes. Again the effect of such types of peptide on gene delivery in muscle is untested.

As a development of this idea, Wyman and colleagues designed a polypeptide called KALA (WEAKLAKALAKALAKHLAKALAKALKACEA), which differs from GALA principally in the replacement of the seven uncharged A residues by positively charged K residues.⁸⁶ KALA too is an amphipathic peptide: when in alpha-helical conformation, one face displays hydrophobic leucine residues, while the opposite face displays hydrophilic lysine residues. The KALA peptide was reported to cause membrane disruption as well as binding and condensing DNA. Transfection efficiency of non-myogenic cells with KALA/DNA complexes in vitro is over 100 times greater than that with the optimal poly-L-lysine/DNA complex delivery system. Relatively high transfection efficiency was also achieved in C2C12 myoblasts and myotubes. However, the effect of KALA on gene transfer in vivo in muscle has yet to be explored.⁸⁷

So far we have discussed enhancement of plasmid entry into cells and their release from endosomes into the cytoplasm. Another objective in the development of non-viral gene delivery systems is to improve the efficiency of transport of plasmid DNA from the cytoplasm into the nucleus. This may be particularly important in muscle fibres, where, although plasmid entry may be relatively easier due to its vast surface area of T-tubule system, translocation of naked plasmid to the nucleus is likely to be more problematic. This is mainly because of the non-dividing nature of the myonuclei which excludes the possibility of plasmid entry during mitosis. Thus plasmid DNA can gain access to myonuclei only through the nuclear pore complex. This route of entry is standardly used by nuclear proteins such as transcription factors which are actively imported by mediation of NLS within the cargo proteins. It is conceivable that a combination of NLS with plasmid DNA might improve efficiency of gene delivery (see reviews^78,88,89). Attempts have been made to either add nucleoproteins or NLS into cocktails of plasmid solution or to covalently link NLS to plasmid DNA, either directly⁹⁰ or indirectly through DNA-binding agents such as cationic polymers and peptides.⁹¹ Collas and Alestrom in experiments involving micro-injection of plasmid into the cytoplasm of zebrafish eggs, reported that use of NLS increased nuclear accumulation of plasmid DNA and expression of the transgene.⁹² Beneficial effects were also obtained in liver gene delivery experiments in vivo⁹³ and in different cell lines⁹⁴ when NLS was used in combination with a liposome-mediated delivery system. However, little or no benefit over plasmid alone was reported when NLS was used in combination with adenovirus/PEI for gene transfer into myoblasts.⁷²

The limited benefit of simply adding NLS to a plasmid DNA delivery system suggests the need for a more stable linkage of NLS to plasmid DNA either directly or indirectly through DNA-condensing and membrane-destabilizing polymers. Again the success of this type of approach has, so far, been limited (see review by Bremener et al⁷⁸). This ineffectiveness may be attributable to modifications of DNA and increase in size of plasmid/polymer complexes, hampering the entry of DNA into cells. Clearly this approach to gene delivery is still in the early stages of development and exhaustive testing of the variety of NLS sequences in combination with different agents may lead to identification of optimal conditions for gene delivery into the nuclei of specific cell types.

Electroporation

Electropermeabilization, commonly referred to as electroporation, is a physical process which exposes cells to a brief, high-intensity electric field that induces temporary damage to the plasma membrane, allowing the influx of large molecules to which it is normally impermeable. This technique has been used for nearly two decades for transfection of cells in vitro.⁹⁵ Its initial application in vivo was to transfer DNA into skin and liver cells, and was first reported for gene transfer into skeletal muscle in vivo by Aihara and Miyazaki in 1998.⁹⁶ Since then, progressive improvements have made it the most efficient approach among non-viral gene delivery systems in skeletal muscle. Applications of this technique have been extended from treating muscular dystrophy to using muscle for systemic delivery of therapeutic proteins.^{97,98,99,100,101} The mechanisms by which this method enhances transgene expression appear to be relatively simple. Electroporation is effective only when plasmid DNA is injected into the muscle prior to, not after, electroporation. This suggests that the electric pulse acts directly on the charged DNA molecules and forces them to migrate through ECM and cell membranes and enter muscle fibres, although membrane permeabilization may also play a role.⁹⁷ The entry of plasmid directly into cytoplasm may bypass the endosome–lysosome pathway, reducing the degree of DNA degradation (Figure 4).

Figure 4.

Schematic diagram of the proposed routes by which naked plasmid DNA is delivered to muscle fibres and transgene products are secreted into circulation. The route of endocytosis (a) is exemplified by lipoplex-mediated plasmid delivery whereas the route of passive entry (b) is by electroporation. The efficiency of plasmid delivery into myonuclei through the route of endocytosis can be improved by modulation of plasmid, membrane targeting, endosome destabilization and nuclear targeting. Mechanisms which regulate transcription, translation of transgene and secretion of transgene products are largely unknown and have not been explored as a means of improvement in transgene expression.

Full figure and legend (346K)

The effectiveness of gene transfer in muscle relies on the widest possible dissemination of DNA or virus within the area to be electrically pulsed. However, the abundant ECM in the perimysium and endomyosium of muscle restricts severely the diffusion of injected plasmid, thus limiting the region over which fibres can be transduced. The pore size of basal lamina has also been shown to selectively limit the penetration of larger viruses, such as adenovirus, by comparison with the smaller ones, such as AAV.¹⁰² Such size constraints may also be limiting factors for efficient delivery of large plasmids. To improve dissemination of both viral and non-viral vectors, some proteases have been tested for their ability to break down the barriers. To facilitate the diffusion of AAV in muscles, Favre et al used hyaluronidase which hydrolyses hyaluronic acid, a major constituent of the muscle ECM, reporting up to a three fold increase in infection efficiency.¹⁰³ Likewise, Wells and colleagues recently reported that pretreatment of muscle with hyaluronidase improves the penetration of naked plasmid within the tissue and expression. To their surprise, hyaluronidase pretreatment also reduced muscle damage.²²

Although electroporation has shown high efficiency of gene transfer in primates as well as in rodents, several aspects need to be improved and the efficacy in human is yet to be tested. The voltage gradient required to achieve efficient gene transfer, about 200 V/cm, has detrimental effects on muscle fibres, provoking degeneration particularly in the centre of electric field. One can envisage the practical difficulties of adapting such a technique to the larger scales encountered in the human subject, particularly for gene therapy of genetic diseases, such as the muscular dystrophies, which require body-wide delivery of therapeutic plasmids. Extensive tissue damage may also act as a 'Danger Signal', promoting the development of immune responses to transgene products.¹⁰⁴ Efficiency of gene transfer with electroporation also appears to be greatly dependent on the size of the plasmid DNA; a 5 kb plasmid expressing -gal can transduce more than half the fibres (about 2000) of the tibialis anterior muscle in a mouse while far fewer were transduced with a 12.5 kb vector expressing human dystrophin.¹⁰⁵ This is clearly a major impediment to application of this technique to Duchenne's muscular dystrophy.

Other approaches

Several other developments have emerged recently, which have the potential to improve the efficiency of naked plasmid DNA delivery systems both locally and systemically in muscles, and are worthy therefore of discussion in brief.

Microbubbles and ultrasound

Transfection of mammalian cells with plasmid DNA by sonication was reported by Fechheimer et al as early as 1987.¹⁰⁶ This technique has some advantages for gene delivery. Firstly, the energy of ultrasound (US) can be focused on a relatively small area wherever gene transduction is required. This is especially important for gene delivery into deeply located tissues. Hence the current use of this technology is in targeting tumours. Plasmid DNA or microspheres (such as microbubbles) bearing plasmids (MSP) can be injected intravenously and US energy applied to the target region. Secondly, the sonication needed to achieve enhanced gene expression by either plasmid alone or coupled to MSP is within the range emitted by diagnostic transducers.^107,108,109 Thus, if efficient gene transfer could be achieved by this technique, it would have great advantages in clinical use over other physical methods, such as electroporation. Thirdly, enhanced transgene expression can be achieved by US alone or in combination with other non-viral delivery systems, such as lipoplex-mediated gene delivery. Lipoplexes in combination with US can enhance transgene expression by several orders of magnitude.^110,111 Manome et al reported that US of 20 W/cm², at continuous 1 MHz, increased -galactosidase expression by 3–270-fold, in vitro and in vivo.¹¹² In the presence of microbubbles, US enhanced gene expression up to 300-fold over plasmid alone. The main mechanism by which US enhances gene transfer is thought to be acoustic cavitation, which can effect transient non-lethal perforations in the plasmalemma and possibly the nuclear membranes.¹⁰⁷ Microbubbles, by acting as cavitation nuclei, may potentiate pore formation in cell membranes, thus facilitating the entry of plasmids into cells and their release from endosomes.¹¹³ Our preliminary results using US and microbubbles for gene delivery in muscle also showed a significant increase in the number of fibres transduced with a GFP marker gene (unpublished observation).

Local ischemia, vasodilation and high-pressure injection

As discussed earlier, one common hurdle for gene delivery in muscle, by either naked plasmid, electroporation, microbubbles or virus infection, is the dispersion of the vehicles carrying transgenes within muscles. Several groups have tried to overcome this problem by intravenous administration of the vehicles with local ischemia, vasodilation and high-pressure, or using high-pressure injection. Takeshita et al reported that gene expression in skeletal muscle transfection is significantly augmented by administration of transgenes under ischemic conditions created by ligation of femoral artery of rat limb.¹¹⁴ Similarly, Wolff's group reported widespread transgene expression when naked plasmid in a large volume was delivered by rapid intra-arterial injection in combination with occlusion of large draining blood vessels.^115,116 They also found moderate increases in transgene expression when collagenase, papaverine and ischemia were used. These results suggest that local ischemia, and more particularly the high pressure induced by vessel obstruction and high volume of transfection reagent, probably create enough force to break ECM and membrane barriers and facilitate entry of plasmid into target cells. Similar protocols have also been reported to be effective for gene delivery into the diaphragm¹¹⁷ and for virus-mediated gene delivery. Greelish et al reported efficient delivery of human delta-sarcoglycan to distal hindlimb muscles through infusion of recombinant AAV into the femoral artery in conjunction with histamine-induced endothelial permeabilization.¹¹⁸

Particle bombardment is well known to be effective for immunization by gene therapy and is an alternative means of achieving forceful delivery of transgenes into muscle. Stable and long-term expression of a marker gene in fish muscle was reported with naked plasmid delivered by gene gun.¹¹⁹ Walther et al used a low-volume, high-speed hand-held jet injector for direct gene transfer of naked plasmid into tumours, and reported that expression of the transgene was more efficient and more widespread within tumour tissues than the control procedure of direct injection from a syringe.¹²⁰ The advantages of forceful delivery of transgenes in muscle with these methods remain to be widely tested.

Enhancing transgene expression at transcription or post-transcription levels

Entry of transgenes into the nucleus does not guarantee efficient expression. It is well documented that plasmid DNA may persist within target cells with no detectable expression.³³ It is not well understood how transgene expression is regulated in muscle. Studies of transgene expression with viral infection systems suggest that there is room for improvement of transgene expression at both the transcriptional and post-transcriptional levels. For instance, the post-transcriptional regulatory element (PRE) present in hepatitis B virus (HBV) can stimulate the cytoplasmic accumulation of transgene mRNA.¹²¹ Zufferey et al reported a substantial increase in levels of expression in a transgene-, promoter- and vector-independent manner when a PRE of woodchuck hepatitis virus (WPRE) was inserted in the 3' untranslated region of coding sequences carried by either oncoretroviral or lentiviral vectors.¹²²

Taking a different approach to this problem in AdV-mediated gene transfer, it has been shown that transcriptionally active drugs such as retinoic acid (RA) in combination with histone deacetylase inhibitor trichostatin A (TSA) can enhance and prolong transgene expression in cell lines and skeletal muscle up to seven-fold over controls.¹²³ This effect may be mediated, in part, by the direct activation of RA receptors on the CMV promoter which contains repeated RA responsive element (RARA). Perhaps more importantly, these drugs may reactivate expression of the transgene via remodelling of chromatin. This enhancing effect is unlikely to be limited to the genes delivered by AdV. Indeed, expression of a transgene, in vitro, delivered by polyoma virus pseudocapsids can also be enhanced by these drugs (N Krauzewicz, personal communicaton, MRC clinical Science Centre, London, UK). We too have observed an increase in the number of myoblasts expressing a GFP transgene when TSA and RA are added after PEI-mediated gene transfer with naked plasmid DNA (unpublished observation). The inclusion of PRE in vector constructs and use of drugs that activate transcription might represent an important ancillary strategy for vector design and efficient gene expression.

Conclusion

The efficiency of transgene delivery and expression by non-viral delivery systems in muscle has been improved significantly. This is mainly due to the construction of new vectors, the use of gene delivery enhancing reagents and the development of novel delivery systems. As illustrated in Figure 4, nearly all of the measures taken so far focus on tackling obstacles which prevent the efficient entry of plasmid into cytosols and nuclei, or which cause DNA degradation. However, although it is known that plasmid DNA can persist in myonuclei for long periods, little is known about how the expression of transgenes in episomal DNA is controlled by cellular transcriptional and translational machineries. Nor do we know how the protein products of transgenes are transported from muscle fibre cytoplasm into the circulation. The ultimate efficiency of transgene expression and delivery of the gene products could therefore be interfered with or improved at each of a number of stages. For example, recent evidence suggests that active RNA polymerases are concentrated in discrete 'factories' where they work together on many different templates. The evidence that such factories specialize in the transcription of particular groups of genes prompts the speculation that these factories could be targeted for efficient transcription.¹²⁴

Although gene therapies have not lived up to hopes voiced for them 19 years ago,¹²⁵ research in this topic has nonetheless made a number of useful advances. Principal among these is the accurate definition of the biological hurdles that limit what is achievable and the identification of what appear to be realistic targets for our current technologies. In this regard, skeletal muscle has been shown repeatedly to be a robust target organ. Gradual refinement of our understanding of how muscle fibres take up plasmid DNA and of the mechanisms that mediate transcriptional and post-transcriptional controls of transgene expression look likely to lead to further improvements in efficiency of transgene expression to the point where naked plasmid delivery would be a genuine option for therapeutic application. This approach, avoiding viral vectors, has the virtue of minimizing contentiousness in the passage from animal experiments to human trials.

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