MacColl, Gavin Stuart; (2000) Functional and immunological aspects of growth hormone gene transfer into muscle cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Several practical aspects of gene transfer into muscle using plasmid vectors were studied in this thesis. First, the influence of promoter sequences on growth hormone expression by muscle cells was studied following transfection with various plasmid constructs containing a rat growth hormone complementary DNA (GH cDNA). Second, the plasmid vector which produced the highest levels of recombinant GH in vitro was delivered by intramuscular injection into GH deficient dwarf rats, and the effects were studied. Third, the immunogenicity of plasmid vectors were studied in immunologically normal Balb/C mice, and in MRL/MpJ mice which had a genetic background for a mild autoimmunity. The findings included: 1. The combination of cytomegalovirus immediate early promoter (CMV), myosin light chain enhancer and GH cDNA (pcDNA3E-GH), generated significantly more biologically active GH in differentiated C2C12 mouse myotubes than either CMV promoter, or muscle specific promoter used individually (p<0.05). 2. Intramuscular transfer of a plasmid reporter vector was more effective in 4 week old male rats, than in older animals. Subsequent injections of pcDNA3E-GH into dwarf rats did not significantly increase weight gain, or circulating insulin like growth factor 1 (IGF-1) levels. However, immunoreactive IGF-1 was detected in the colons of 4 of 5 rats compared with only 1 of the controls, suggesting that this methodology could be used to modulate local IGF-1 production in the gastrointestinal tract. 3. MRL/MpJ mice given i.m. injections of pcDNA3E at 4, 6, 8 and 10 weeks, generated significantly higher serum anti-double stranded DNA (dsDNA) immunoglobulins, than saline injected controls (p<0.05). Additionally, sera from 6 of the 7 of the plasmid injected MRL/MpJ mice contained anti-cell nuclear immunoglobulins, compared with 2 control sera. No increase in dsDNA antibodies was observed in Balb/C mice injected with pcDNA3E, or with plasmids with fewer eukaryotic sequences, suggesting that plasmid DNA injection into muscle may only alter circulating levels of anti-dsDNA antibodies in circumstances where individuals are susceptible to specific types of autoimmunity.

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