Inflammatory responses following direct injection of plasmid DNA into skeletal muscle

Abstract

Transfer of genes by injection of plasmid DNA into skeletal generated widespread inflammation of injected muscle, muscle has a wide variety of applications ranging from with mononuclear infiltrate, comprised largely of macrotreatment of neuromuscular disorders to genetic vacciphages and with both CD4 + and CD8 + T lymphocytes, nation. We examined each component involved in the present. Such inflammation may hamper clinical appliintramuscular injection of plasmid DNA in terms of the cation of this technology and may encourage undesirable induction of inflammatory responses. The insertion of a immune responses in gene therapy trials. Inflammation needle and the injection of a relatively large volume of was not greatly reduced by CD4-or CD8-depleting antisaline caused very little muscle damage except in rare bodies, suggesting this initial inflammation did not involve cases. In contrast, barium chloride-induced regeneration of T cells, but methylation of plasmid DNA before injection muscle, injection of lipopolysaccharide, plasmid backbone substantially lessened the inflammatory response and or plasmid expressing a neo-antigen (␤-galactosidase) all resulted in longer term expression of the transgene.Keywords: gene therapy; regeneration; ␤-galactosidase; muscle; plasmid; inflammation Skeletal muscle has the ability to take up and express naked plasmid DNA following intramuscular injection 1,2 and hence is an attractive platform for (1) providing a systemic source for recombinant therapeutic proteins; 3,4 and (2) as a means of genetic vaccination against either bacteria and viruses, 5-7 or tumour cells. [8][9][10] However, long-term expression of transgenes encoded in the plasmid DNA can be limited by the immune responses (cellular, humoral and innate) evoked during the processes of gene uptake and expression in skeletal muscle fibres. 11-15 These responses, while necessary and desirable for genetic vaccination, can seriously limit the use of direct plasmid injection for gene therapy. Immune responses also play a significant role in determining the success of virus-based gene transfer. [16][17][18][19][20][21][22] In order to start to dissect the relative roles of the responses to vector and transgene, we have concentrated on gene transfer by direct injection of plasmid DNA. This system does not involve injection of any exogenous protein component, allowing the characteristics of the antigen-specific response to be analysed separately. However, plasmid DNA is prepared in E. coli which results both in contamination with lipopolysaccharide (LPS, endotoxin) and the presence of unmethylated CpG motifs. LPS is a known stimulator of immune responses and is frequently used for in vitro stimulation of lymphocyte populations. [23][24][25] Unmethylated CpG motifs are comparatively rare in eukaryotic genomes 26

Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in vivo with reduced tissue damage

“…7,8 Nonviral delivery systems are safe and easy to apply, but suffer from low transduction efficiencies and can also cause local inflammatory reactions and tissue damage which may destroy transduced muscle fibres. 9 New methods have been examined for improving efficiency, using reagents such as cationic lipids and polymers. However, despite their success for gene transfer in vitro, results have been disappointing in muscle in vivo.…”

“…12 Ultrasound is particularly effective for gene delivery when applied in combination with the use of microbubbles, cationic polymers or lipids. [3][4][5][6][7][8][9][10][11][12][13][14][15] Anwer et al 15 reported that application of ultrasound to skin tumours before or after the injection of lipoplexes resulted in a significant increase in gene transfer specifically to the tumour. 15 The magnitude of increased expression ranged from 3-4 to 270-fold.…”

Non-viral gene delivery in skeletal muscle: a protein factory

“…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.…”

Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies

“…Gene Therapy (2001) 8, 1097-1107. duction in vivo. Although transduction yields are sufficient to trigger immune reactions in certain situations, [13][14][15][16] they would not reach the threshold required for the development of a functional benefit. The electrotransfer methodology has been recently adapted to skeletal muscle transduction in vivo with high efficacy and reproducibility, the expression of reporter or secreted proteins being increased by a factor of one to many hundreds.…”