C1 - Base Editing and Prime Editing
97: In Utero and Postnatal Base Editing for a Metabolic Liver Disease in Mice and Nonhuman Primates
Type: Oral Abstract Session
Presentation Details
Session Title: Base Editing and Prime Editing I
A number of genetic diseases amenable to therapeutic gene editing cause pathology before or shortly after birth. In utero gene editing has the potential to mitigate disease before the onset of this pathology. Moreover, normal developmental properties of the fetus including the small size, immature immune system, and abundant, accessible progenitor cell populations are hypothesized to facilitate efficient gene editing. Genetic diseases affecting the liver, including metabolic liver diseases, are some diseases that may benefit from in utero editing. As an example, we have chosen to evaluate a therapeutic base editing strategy for hereditary tyrosinemia type 1 (HT1) applied either before or after birth. HT1 is an autosomal recessive metabolic liver disease in which mutations in the FAH gene, the final step in the tyrosine catabolic pathway, result in accumulation of toxic metabolites and subsequent liver failure with an increased risk of hepatocellular carcinoma. The pathology can begin before birth. Current treatment for HT1 includes daily dosing of nitisinone which inhibits the HPD enzyme upstream in the tyrosine catabolic pathway and prevents the accumulation of toxic metabolites. We hypothesized that adenine base editing to disrupt a splice acceptor or donor site in the HPD gene could inactivate the gene as a therapeutic approach for HT1. To address this hypothesis, we screened a number of gRNAs together with ABE8.8 in HuH-7 cells, a human hepatoma cell line. A lead gRNA, HPD20, was chosen based on on-target editing efficiency and off-target editing profile. To assess the ability of this approach to rescue the disease phenotype, gestational day (E) 16 fetal HT1 mice or 8 week old HT1 mice were intravascularly dosed with an LNP containing ABE8.8 mRNA and a mouse gRNA orthologous to HPD20 gRNA. These studies demonstrated efficient on-target editing with survival, liver function and urine metabolites similar to that seen in HT1 mice maintained on nitisinone and significantly improved compared to HT1 mice in which nitisinone was removed, all of which died by 3 weeks off nitisinone. The target sequence of the human HPD20 gRNA is homologous to the sequence present in Macaca fascicularis. Thus, we next tested the feasibility of in utero base editing in the fetal nonhuman primate (NHP). LNP_ABE8.8_HPD20 was intravascularly delivered into midgestation Macaca fascicularis fetuses. Assessments at short time points after injection demonstrated efficient delivery of the LNP to the fetal liver and moderate editing throughout the liver. To assess a potential benefit of the fetal environment for in vivo editing, we dosed two 3 year old Macaca fascicularis NHPs with LNP_ABE8.8_HPD20 at a comparable dose to that given to the fetuses. On-target liver editing was noted although editing levels were approximately 2- to 4.5-fold lower compared to fetal recipients. This study supports a mutation-agnostic adenine base editing approach for HT1 and demonstrates the feasibility of in utero liver gene editing via LNP delivery in NHPs, highlighting potential advantages to editing during fetal development in this model.
Plain Language Summary
In an ideal world, we would have the ability to identify and fix genes that cause horrible diseases before the diseases have a chance to hurt or kill the affected people. This body of work presents evidence that a one-time injection is enough to fix a broken gene by editing it to correct a disease-causing mutation. We also find that delivering our injection earlier in life matters, and improves how well we can edit the disease gene. With this work, we hope to pave the way to one day offering patients these types of one-time injections to decrease and even prevent the suffering that comes with diseases caused by genetic mutations.
Ana Maria Dumitru1, Marco Carpenter1, Madelynn Whittaker2, Cara Berkowitz1, Valerie L. Luks2, Nicole Kus1, Rosa Choi1, Nicholas F. Archer1, William Johnston3, Arjit Biswas1, Julie Yeo Su Li4, Yiping Fan4, Xiao Wang2, Mohamad Gabriel Alameh1, Jerry Chan5, Kiran Musunuru2, William Peranteau6
1CHOP, Philadelphia, PA,2University of Pennsylvania, Philadelphia, PA,3Children's Hospital of Philadelphia, Philadelphia, PA,4NUS, Singapore, Singapore,5KK Women's and Children's Hospital / Duke NUS Medical School, Singapore, Singapore,6The Children's Hospital of Philadelphia, Philadelphia, PA"
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