This summer I performed my research in the Arruda lab, which studies gene therapy approaches for treatment of hemophilia A and B – X-linked recessive disorders caused by deficiencies in functional coagulation factor VIII (FVIII) and IX (FIX), respectively. My project addresses one persistent issue in hemophilia B gene therapy: the amount of vector that can be administered safely without risk of an immune response is not always high enough to ensure disease reversal. I am seeking to circumvent this problem by creating a single-substitution FIX variant with enhanced specific activity. A hyperactive variant would evade the vector dosage limitation because a smaller amount of protein would suffice to reverse the disease.
In developing my hypothesis, I focused on a few positions at which single amino acid mutations seemed most likely to enhance protein activity. These were determined by a few key factors. First, recent biochemical findings have allowed us to elucidate a “line of communication” in FIX, where a string of amino acids interconnected by a hydrogen-bonding network is thought to carry essential conformational changes within the protein; due to the role these positions play in the rate-determining step of the reaction, we hypothesized that substitution along this line could improve the rate of activity. Second, both sites were highly conserved in the human population, suggesting their importance to proper FIX function. Lastly, none were recorded to have any association with hemophilia. Thus, our hypothesis developed as follows: single amino acid substitutions at selected positions along the “line of communication” may be sufficient to produce a hyperactive variant.
In order to test this hypothesis, I created variant DNA and protein and then assessed each variant for antigen and activity as compared to wild type FIX. My preliminary (screenings designed to represent the broad chemical categories of amino acids) yielded some dramatic changes in FIX antigen and activity, ultimately suggesting that two of the positions held promise for further experimentation. I have since expanded my screening of these to include the entire array of amino acids, finding several hyperactive variants that will be carried forward for testing in vivo and in combination to probe possible synergistic effects.
My time in the Arruda lab has prompted great professional growth. After beginning my original project in June 2016 and spending the academic year learning the ins and outs of experimental design, technical procedure, data analysis, and presentation of results, I have spent this summer progressing my research more efficiently. I have taken the opportunity to expand and direct my project in response to findings, and feel as though my confidence in working independently has increased greatly.