Human stem cell-based model of MYCN-driven neuroblastoma

Neuroblastoma (NB), a disease of neural crest (NC) origin, is the most common extracranial solid tumor in childhood. High-risk NB patients represent the subgroup with the worst prognosis and frequently harbor amplification of MYCN. Due to its biochemical structure as a transcription factor, MYCN has been difficult to target directly with small molecules. Alternatively, identification of cooperating partners of MYCN-induced tumorigenesis can reveal a more therapeutically viable target, such as anaplastic lymphoma kinase (ALK). While genetically engineered mouse models (GEMMs) of NB driven by MYCN and ALK exist, recent studies have found significant differences between mouse models of disease and the human tumors they are intended to represent. In support of this, mouse and human NC cells differ in development and marker expression, and the genetic requirement for transformation of human cells has been shown to be more complex than mouse cells, suggesting that a human cell-based NB model would be more relevant. To develop a human cell-based model of NB, we started with a normal human induced pluripotent stem (iPS) cell line derived integration-free from a healthy adult and transduced empty vector, ALK F1174L (active mutant), doxycycline-inducible MYCN (DOX-MYCN), or ALK F1174L/DOX-MYCN. These iPS cells were differentiated towards NC cells and subsequently implanted orthotopically into renal capsules of mice fed on dox chow. Within 3 months, 60% of mice developed tumors with ALK F1174L/DOX-MYCN, 10% with DOX-MYCN, and 0% with both empty vector and ALK F1174L alone. Tumors were transplantable and demonstrated histologic characteristics consistent with NB, including morphology and expression markers. Thus, we are demonstrating the first, to our knowledge, human stem cell-based model of NB. We are using this system to further investigate similarities and differences with GEMMs of NB, test and establish novel candidate drivers of NB, and evaluate potential therapeutic options

Correction of Fanconi Anemia Mutations Using Digital Genome Engineering

Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using ‘digital’ genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy