Project Grant R01HD110733

CORRECTIVE EDITING TO TREAT PHENYLKETONURIA - PROJECT SUMMARY PHENYLKETONURIA (PKU) IS AN AUTOSOMAL RECESSIVE DISORDER CAUSED BY MUTATIONS IN THE GENE ENCODING PHENYLALANINE HYDROXYLASE (PAH), RESULTING IN THE ACCUMULATION OF PHENYLALANINE (PHE) TO NEUROTOXIC LEVELS. ALTHOUGH THERE ARE TREATMENT OPTIONS, RANGING FROM A STRICT LOW-PHE DIET TO AN ORAL MEDICATION (SAPROPTERIN, A COFACTOR OF PAH) TO AN INJECTABLE ENZYME SUBSTITUTION THERAPY (PEGVALIASE), MANY PKU PATIENTS FIND IT CHALLENGING TO ADHERE TO THE DIETARY INTERVENTION AND HAVE LIMITED RESPONSES TO OR ACCESS TO THE MEDICAL THERAPIES AND, AS A RESULT, HAVE IMPAIRED COGNITIVE DEVELOPMENT AND DEVELOP A RANGE OF NEUROPSYCHIATRIC PROBLEMS. DURABLE AND, IDEALLY, CURATIVE THERAPIES ARE NEEDED TO ADDRESS THE UNMET MEDICAL NEEDS OF PKU PATIENTS. MORE THAN 1,000 PAH VARIANTS HAVE BEEN CATALOGED IN PATIENTS. THESE VARY IN THEIR CONSEQUENCES FOR PAH ACTIVITY, FROM HAVING LITTLE OR NO EFFECT TO ELIMINATING PAH ACTIVITY COMPLETELY. CERTAIN VARIANTS OCCUR MUCH MORE COMMONLY THAN OTHERS IN PKU PATIENTS. THE MOST FREQUENTLY OCCURRING PATHOGENIC PAH VARIANT WORLDWIDE IS THE R408W (C.1222C>T, P.ARG408TRP) VARIANT. PATIENTS HOMOZYGOUS FOR THIS VARIANT DO NOT RESPOND TO SAPROPTERIN, LIMITING THEIR TREATMENT OPTIONS. IN VIVO GENE EDITING IS AN EMERGING THERAPEUTIC APPROACH TO MAKING DNA MODIFICATIONS IN THE BODY OF A PATIENT, SUCH AS IN THE LIVER. GENE-EDITING TOOLS INCLUDE NUCLEASES, CYTOSINE BASE EDITORS, ADENINE BASE EDITORS, AND PRIME EDITORS. CRISPR BASE EDITORS AND PRIME EDITORS ARE ATTRACTIVE BECAUSE THEY CAN FUNCTION EFFICIENTLY FOR INTRODUCING PRECISE TARGETED ALTERATIONS WITHOUT THE NEED FOR DOUBLE-STRAND BREAKS, IN CONTRAST TO CRISPR-CAS9 AND OTHER GENE-EDITING NUCLEASES. WE AND OTHERS HAVE DEMONSTRATED THE ABILITY OF BASE EDITORS TO MAKE SPECIFIC DNA EDITS WITH VERY HIGH EFFICIENCY AND LIMITED OFF-TARGET EFFECTS IN THE LIVER IN MOUSE MODELS AND NON-HUMAN PRIMATES. WE NOW SEEK TO ASSESS WHETHER A ONE-TIME DELIVERY OF ADENINE BASE EDITING OR PRIME EDITING CAN BE USED TO PERMANENTLY CORRECT THE HUMAN PAH R408W VARIANT IN HUMAN HEPATOCYTES IN VITRO AND IN THE LIVER IN HUMANIZED MICE IN VIVO EFFICIENTLY AND SAFELY AND, IF SO, THE OPTIMAL EDITING SYSTEM TO USE FOR THIS PURPOSE. SUCCESS IN COMPLETING THIS TRANSLATIONAL PROJECT WILL PROVIDE CRITICAL INFORMATION ON THE FEASIBILITY OF AN IN VIVO GENOME- EDITING APPROACH THAT COULD ULTIMATELY YIELD A ONE-SHOT, LONG-TERM THERAPY THAT PERMANENTLY CORRECTS THE MOST FREQUENT PAH PATHOGENIC VARIANT AND THUS SERVES AS A POTENTIAL CURE FOR PKU PATIENTS WITH THIS VARIANT. THE PROPOSED WORK, IF SUCCESSFUL, COULD OPEN THE DOOR TO A NEW THERAPEUTIC MODALITY FOR MANY PKU PATIENTS- NOT JUST FOR PATIENTS WITH THE PAH R408W VARIANT, BUT THOSE WITH OTHER EDITABLE PATHOGENIC VARIANTS IN THE PAH GENE-AS WELL AS PATIENTS WITH SUCH VARIANTS IN OTHER LIVER-EXPRESSED, DISEASE-ASSOCIATED GENES.