Pre-existing immunity does not impair the engraftment of CRISPR-Cas9-edited cells in rhesus macaques conditioned with busulfan or radiation.

CRISPR-Cas9-based therapeutic genome editing approaches hold promise to cure a variety of human diseases. Recent findings demonstrate pre-existing immunity for the commonly used Cas orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans, which threatens the success of this powerful tool in clinical use. Thus, a comprehensive investigation and potential risk assessment are required to exploit the full potential of the system. Here, we investigated existence of immunity to SpCas9 and SaCas9 in control rhesus macaques (Macaca mulatta) alongside monkeys transplanted with either lentiviral transduced or CRISPR-SpCas9 ribonucleoprotein (RNP)-edited cells. We observed significant levels of Cas9 antibodies in the peripheral blood of all transplanted and non-transplanted control animals. Transplantation of ex vivo transduced or SpCas9-mediated BCL11A enhancer-edited cells did not alter the levels of Cas9 antibodies in rhesus monkeys. Following stimulation of peripheral blood cells with SpCas9 or SaCas9, neither Cas9-specific T cells nor cytokine induction were detected. Robust and durable editing frequencies and expression of high levels of fetal hemoglobin in BCL11A enhancer-edited rhesus monkeys with no evidence of an immune response (>3 years) provide an optimistic outlook for the use of ex vivo CRISPR-SpCas9 (RNP)-edited cells.

High-throughput imaging method for direct assessment of GM1 ganglioside levels in mammalian cells.

GM1-gangliosidosis is an inherited autosomal recessive disorder caused by mutations in the gene GLB1, which encodes acid ß-galactosidase (ß-gal). The lack of activity in this lysosomal enzyme leads to accumulation of GM1 gangliosides (GM1) in cells. We have developed a high-content-imaging method to assess GM1 levels in fibroblasts that can be used to evaluate substrate reduction in treated GLB1(-/-) cells [1]. This assay allows fluorescent quantification in a multi-well system which generates unbiased and statistically significant data. Fluorescently labeled Cholera Toxin B subunit (CTXB), which specifically binds to GM1 gangliosides, was used to detect in situ GM1 levels in a fixed monolayer of fibroblasts. This sensitive, rapid, and inexpensive method facilitates in vitro drug screening in a format that allows a high number of replicates using low working volumes.

Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel ß-galactosidase:RTB lectin fusion.

New enzyme delivery technologies are required for treatment of lysosomal storage disorders with significant pathologies associated with the so-called "hard-to-treat" tissues and organs. Genetic deficiencies in the GLB1 gene encoding acid ß-galactosidase lead to GM1-gangliosidosis or Morquio B, lysosomal diseases with predominant disease manifestation associated with the central nervous system or skeletal system, respectively. Current lysosomal ERTs are delivered into cells based on receptor-mediated endocytosis and do not effectively address several hard-to-treat organs including those critical for GM1-gangliosidosis patients. Lectins provide alternative cell-uptake mechanisms based on adsorptive-mediated endocytosis and thus may provide unique biodistribution for lysosomal disease therapeutics. In the current study, genetic fusions of the plant galactose/galactosamine-binding lectin, RTB, and the human acid ß-galactosidase enzyme were produced using a plant-based bioproduction platform. ß-gal:RTB and RTB:ß-gal fusion products retained both lectin activity and ß-galactosidase activity. Purified proteins representing both fusion orientations were efficiently taken up into GM1 patient fibroblasts and mediated the reduction of GM1 ganglioside substrate with activities matching mammalian cell-derived ß-galactosidase. In contrast, plant-derived ß-gal alone was enzymatically active but did not mediate uptake or correction indicating the need for either lectin-based (plant product) or mannose-6-phosphate-based (mammalian product) delivery. Native ß-galactosidase undergoes catalytic activation (cleavage within the C-terminal region) in lysosomes and is stabilized by association with protective protein/cathepsin A. Enzymatic activity and lysosomal protein processing of the RTB fusions were assessed following internalization into GM1 fibroblasts. Within 1-4h, both ß-gal:RTB and RTB:ß-gal were processed to the ~64kDa "activated" ß-gal form; the RTB lectin was cleaved and rapidly degraded. The activated ß-gal was still detected at 48h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of ß-gal:RTB supported significantly greater accumulation of ß-galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived ß-gal. These data demonstrate that plant-made ß-gal:RTB functions as an effective replacement enzyme for GM1-gangliosidosis - delivering enzyme into cells, enabling essential lysosomal processing, and mediating disease substrate clearance at the cellular level. RTB provides novel uptake behaviors and thus may provide new receptor-independent strategies that could broadly impact lysosomal disease treatments.

Detection of respiratory viruses with a multiplex polymerase chain reaction assay (MultiCode-PLx Respiratory Virus Panel) in patients with hematologic malignancies.

Respiratory viral pathogens are a common cause of morbidity in patients with hematologic malignancies. Sensitive molecular assays have increased the detection of common respiratory viruses and expanded the panel of detectable viruses. Both a rapid viral culture with direct fluorescence antibody (DFA) staining and a PCR-based assay (MultiCode-PLx Respiratory Virus Panel) were performed on patients with hematologic malignancies, who underwent collection of a nasopharyngeal swab or bronchoalveolar lavage from October 2006 to April 2007. Eighty-two samples from 70 patients were obtained; all patients had upper respiratory tract symptoms. Respiratory viruses were detected in 10 samples (12%) by conventional virological methods and in 31 samples (38%) by the MultiCode-PLx assay. This increased diagnostic yield resulted from better sensitivity for those viruses detectable by both methods and detection of viruses not covered by the antigen detection/rapid culture method (human metapneumovirus, coronaviruses and rhinoviruses). The MultiCode-PLx assay frequently identified respiratory viral infections which are not detected by rapid viral culture/DFA; 40% of these patients had pneumonia in addition to the upper respiratory tract symptoms. Improved diagnostics for respiratory viruses may lead to more effective management and better outcomes in this patient population.