A first-in-human gene therapy trial using a recombinant adeno-associated viral (rAAV) vector for acute intermittent porphyria (AIP) reveals that higher doses would be required to reach therapeutic levels of the porphobilinogen deaminase (PBGD) transgene. We developed a hyperfunctional PBGD protein to improve the therapeutic index without increasing vector dose. A consensus protein sequence from 12 mammal species was compared to the human PBGD sequence, and eight amino acids were selected. I291M and N340S variants showed the highest increase in enzymatic activity when expressed in prokaryotic and eukaryotic systems. In silico analysis indicates that isoleucine 291 to methionine and asparagine 340 to serine variants did not affect the active site of the enzyme. In vitro analysis indicated a synergistic interaction between these two substitutions that improve kinetic stability. Finally, full protection against a phenobarbital-induced attack was achieved in AIP mice after the administration of 1 x 10(11) gc/kg of rAAV2/8-PBGD-I291M/N340S vector. // three times lower than the dose required to achieve full protection with the control rAAV2/8-hPBGD vector. In conclusion, we have developed and 3 characterized a hyperfunctional PBGD protein. The inclusion of this variant sequence in a rAAV2/8 vector allows the effective dose to be lowered in AIP mice.