Identification of Hematopoietic Stem Cell–Specific miRNAs Enables Gene Therapy of Globoid Cell Leukodystrophy

Hematopoietic stem cell–specific microRNAs allow regulation of therapeutic transgene expression and enable effective gene therapy of globoid cell leukodystrophy. Scratching the Surface of the Holy Grail In Monty Python and the Holy Grail, when King Arthur cuts off one of the arms of the Black Knight, he claims it is only a scratch. Similarly, gene therapy—the insertion of genes into cells to reverse a condition or repair a biological process—has been heralded as a Holy Grail for the treatment of genetic diseases for nearly 40 years. Yet, the complications of gene therapy, including immune responses to the viral vector and cancers that result from insertional mutagenesis, are more comparable to a severed arm than a surface wound. However, researchers with the resiliency of the Black Knight have presided over recent successes, most notably in metastatic melanoma and immune cells, and have reignited the quest for gene therapy solutions to otherwise untreatable diseases. Gentner et al. build on these successes by identifying new microRNAs that can restrict gene therapy vectors to particular immune cell types and thus be used to safely treat globoid cell leukodystrophy (also known as Krabbe disease). Globoid cell leukodystrophy is a rare metabolic disorder caused by a mutation in a lysosomal enzyme called galactocerebrosidase (GALC). In patients who carry the mutation in both copies of the GALC gene, unmetabolized lipids accumulate in myelin-secreting glial cells, rendering them unable to produce the myelin sheath that normally wraps and protects nerves. This aberration results in severe and often fatal degeneration of motor skills. Bone marrow transplantation has been shown to benefit these patients if the disease is caught early enough. Genetic manipulation of the hematopoietic stem and progenitor cells (HSPCs) found in bone marrow may improve this therapy; however, high-level GALC expression in HSPCs, but not in more differentiated immune cells, is toxic. To address this issue, Gentner et al. identified miRNAs—short RNA sequences that often silence gene expression—that were specifically expressed in HSPCs but not in more differentiated cells. They then used these miRNAs in a GALC/HSPC gene therapy system to suppress GALC function in HSPCs upon transfer into a mouse model of globoid cell leukodystrophy. As these cells matured, amounts of HSPC-specific miRNA decreased and GALC expression increased. This approach protected the HSPCs from GALC toxicity, but allowed for successful gene therapy of the disease. In addition, these hematopoietic stem cell–specific miRNAs could be used as simple markers with which to isolate HSPCs for study and transplantation. This work thus provides a basis for improvements in HSPC-mediated gene therapy and may offer globoid cell leukodystrophy patients a new therapeutic option that resembles a scratch more than a chop. Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal storage disorder caused by mutations in the galactocerebrosidase (GALC) gene. Hematopoietic stem cell (HSC)–based gene therapy is being explored for GLD; however, we found that forced GALC expression was toxic to HSCs and early progenitors, highlighting the need for improved regulation of vector expression. We used a genetic reporter strategy based on lentiviral vectors to detect microRNA activity in hematopoietic cells at single-cell resolution. We report that miR-126 and miR-130a were expressed in HSCs and early progenitors from both mice and humans, but not in differentiated progeny. Moreover, repopulating HSCs could be purified solely on the basis of miRNA expression, providing a new method relevant for human HSC isolation. By incorporating miR-126 target sequences into a GALC-expressing vector, we suppressed GALC expression in HSCs while maintaining robust expression in mature hematopoietic cells. This approach protected HSCs from GALC toxicity and allowed successful treatment of a mouse GLD model, providing a rationale to explore HSC-based gene therapy for GLD.