CRISPR/Cas-based gene therapy in Adult-Onset Leukoencephalopathy with axonal spheroids and pigmented glia: designing the optimal guide RNA

Abstract

Adult-onset leukoencephalopathy with spheroids and pigmented glia (ALSP) is a rare, early-onset neurodegenerative disease. The disease is rapidly pro-gressive and leads to death on average six years after the manifestation of the first symptoms such as cognitive decline, psychiatric and motor disturbances. The disease is caused by mutations in colony-stimulating-factor-1 receptor (CSF1R). This receptor is expressed on cells of the myelogenous line, such as microglia, that is supposed to play a central role in the pathophysiology of ALSP. However, the exact pathophysiological mechanisms are still largely un-known. This is one of the reasons why treatment options are currently limited to symptomatic therapy. Recently, allogenic hematopoietic stem cell transplan-tation has been reported to have a stabilizing effect on the patients’ condition in several cases. Disadvantages of allogenous transplantation include the need for a suitable donor and the necessary immunosuppression. In order to avoid these, an alternative approach might be to perform autologous stem cell transplantation combined with ex vivo gene therapy. For this purpose, the pa-tients’ CSF1R could be knocked out using the CRISPR/Cas system, a highly efficient gene editing tool that has already been applied in clinical trials for other diseases. For an efficient CRISPR/Cas-based gene knockout, appropri-ate single guide RNAs (sgRNAs) need to be established. One of the objectives of this project was therefore to identify an optimal sgRNA for efficient CSF1R knockout. For this purpose, five sgRNAs were designed and tested in in the THP-1 cell line, which is known to express CSF1R at a high level. Their efficiency was evaluated on DNA level by Sanger sequencing and on protein level by Western Blotting. Two suitable sgRNAs with a high ef-ficiency on DNA and protein level were identified as candidates for a thera-peutic knockout strategy. Further experiments will be needed to evaluate their efficiency in hematopoietic stem cells as well as their specificity, as accidental off-target effects might lead to side effects. Furthermore, two of the tested sgRNAs were chosen for the generation of ho-mozygous and heterozygous CSF1R knockout lines in induced pluripotent stem cells (iPSC). For this approach, human iPSC of a healthy donor were treated with two guides at the same time, so that a part of the gene was cut out. Clones were then screened by PCR and two obviously homozygous and het-erozygous clones each were sequenced in order to confirm the knockout state. The five most probable off-targets for each of the sgRNAs were also se-quenced in the clones and compared to the untreated iPSC control. No off-target effects were detected. Subsequently, the clones’ stem cell characteris-tics were assessed. The cells showed the characteristic high expression of alkaline phosphatase and were stained positive for pluripotency markers. On RNA level, the expression of pluripotency-related genes was confirmed by RT-PCR. The clones were able to spontaneously differentiate into cells of all three germ layers which was verified by immunocytochemistry. Whole genome sin-gle-nucleotide polymorphism (SNP) genotyping performed by Life & Brain Ge-nomics did not reveal any chromosomal aberrations or copy number variations and confirmed the genetic identity of the generated knockout iPSC clones with the original iPSC and fibroblasts they were derived from. The generated knockout line is ready be used in future experiments to improve the under-standing of disease mechanism in ALSP.