Compositions comprising kozak sequences selected for enhanced expression

A method of inducing increased expression of a human transgene in a target cell, comprising:

• contacting a target cell with a plurality of rAAV particles comprising a nucleic acid expression construct comprising:
• a human transgene coding sequence;
• a Kozak sequence;
• and
• an enhancer element operably linked to a promoter,
• wherein the Kozak sequence is non-native to one or more of the transgene, the enhancer element and/or the promoter,
• wherein the expression construct is flanked on each side by an inverted terminal repeat sequence, and
• wherein said contacting results in the target cell increasing expression of human transgene as compared to prior to the contacting, thereby increasing the expression of a human transgene.

A method of generating a consensus Kozak sequence, the method comprising:

• assembling a collection of multiple native Kozak sequences associated with genes of interest,
• for each nucleotide position of at least a first native Kozak sequence, comparing a first identity of a first nucleotide within the at least a first native Kozak sequence to a first identity of a first nucleotide within a second native Kozak sequence, wherein the first nucleotide in the at least a first native Kozak sequence and the first nucleotide of the second Kozak sequence occupy the same position relative to the length of the at least a first and the second native Kozak sequence,
• identifying a predominant nucleotide at each position, wherein identifying comprises one or more of:
• nucleotide identity, purine identity, or pyrimidine identity, and assembling a consensus sequence comprising the predominant nucleotide for each position.