Linear DNA fragments for gene expression

A method for increasing levels of a polypeptide in a subject comprising the steps of:

• (a) delivering a linear double stranded nucleic acid expression construct into a selected tissue;
• and
• (b) applying a cell-transfecting pulse to the selected tissue;
• wherein:
• the construct is substantially free from a viral backbone;
• the polypeptide is encoded by a gene sequence on the linear double-stranded nucleic acid expression construct;
• and
• the linear double-stranded nucleic acid expression construct is delivered in an area comprising the cell-transfecting pulse.

A method for increasing levels of a polypeptide in a subject comprising the steps of:

• (a) placing a plurality of electrodes in the selected tissue,
• (b) delivering the linear double stranded nucleic acid expression construct into the selected tissue;
• and
• (c) applying an electrical pulse to the plurality of electrodes;
• wherein:
• the construct is substantially free from a viral backbone;
• the polypeptide is encoded by a gene sequence on the linear double-stranded nucleic acid expression construct;
• and
• the linear double stranded nucleic acid expression construct is delivered to the selected tissue in an area that interposes the plurality of electrodes.

A construct for plasmid mediated gene supplementation, the construct being a linear double-stranded nucleic acid expression plasmid comprising:

• (a) a promoter;
• (b) a nucleotide sequence of interest;
• and
• (c) a 3′ untranslated region;
• wherein:
• the construct is substantially free from a viral backbone;
• the promoter, the nucleotide sequence of interest, and the 3′ untranslated region are operably linked;
• and
• the nucleotide sequence of interest comprises a growth hormone releasing hormone;
• the promoter comprises a tissue-specific promoter;
• the 3′ untranslated region comprises a human growth hormone 3′ UTR;
• in vivo expression of the nucleotide sequence of interest is regulated by the promoter.

A method for increasing levels of a polypeptide in a subject comprising the steps of:

• (a) placing a plurality of electrodes in the selected tissue,
• (b) delivering the linear double stranded nucleic acid expression construct into the selected tissue;
• and
• (c) applying an electrical pulse to the plurality of electrodes;
• wherein the polypeptide is encoded by a gene sequence on the linear double-stranded nucleic acid expression construct;
• the linear double-stranded nucleic acid expression construct comprising:
• a promoter;
• a nucleotide sequence of interest;
• a 3′ untranslated region;
• and
• the promoter, the nucleotide sequence of interest, and the 3′ untranslated region are operably linked;
• the nucleotide sequence of interest comprises a growth hormone releasing hormone;
• the promoter comprises a tissue-specific promoter;
• the 3′ untranslated region comprises a human growth hormone 3′ UTR;
• and in vivo expression of the nucleotide sequence of interest is regulated by the promoter;
• the construct being substantially free from a viral backbone;
• the linear double stranded nucleic acid expression construct is delivered to the selected tissue in an area that interposes the plurality of electrodes;
• the delivering step comprises injection;
• and
• the tissue comprises muscle.