TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells

Abstract

CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as β-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because β-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.

Genome editing of HBG1 and HBG2 to induce fetal hemoglobin

“…First, our methods achieved substantially higher rates of target-site editing in HSCs than reported previously after transient expression of Cas9 RNPs 30 or transcription activator-like effector nucleases, 37 which will likely translate to greater therapeutic benefits. The variability of HSC editing that we observed in our studies using Cas9-2xNLS could be due to differences in donors, CD34 1 mobilization methods (ie, G-CSF vs plerixafor), or CD34 1 cell purification.…”

“…The tandem HBG1 and HBG2 genes harbor nearly identical nucleotide sequences, including recognition sites for sgRNA-1. Simultaneous RNP-induced DSBs at both genes can result in the deletion of the intervening 4.9-kb region, 16,30 leaving a single hybrid gene with HBG2 promoter sequences fused to the downstream HBG1 gene ( Figure 4A). We developed 2 assays as proxies for the 4.9-kb deletion.…”

Investigation of the Transcriptional Role of a <em>RUNX1</em> Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

“…On the contrary, the direct RNP-based delivery approach has been established as the preferred method as it is fast and straightforward with improved editing efficiency, selectivity and cell viability. Indeed, a variety of methods such as lipofection 49,50 , electroporation 25,51 , nanoparticles 52 , cell-penetrating peptides 53 , iTOP 54 and TRIAMF 55 have been developed for efficient CRISPR/Cas9 delivery into diverse cell types as well as animal and plant species 24,25,26,56,57,58,59,60,61,62,63 . Since non-coding DNA sequences are hotspots of genetic variations 64 , checking the presence of common SNPs/indels in the target and neighboring PAM sequences is particularly relevant when designing gRNA that targets regulatory elements.…”

Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing

“…Filtroporation is a technique that forces cell suspensions through uniformly sized micropores in a filter membrane to generate mechanical deformation and transient membrane holes just like microfluidics 67 , 125 . Filtroporation was demonstrated to be applicable for RNP-mediated genome editing in HSCs 126 . The filtroporation device consists of a silicone washer, a stainless-steel mesh, a hydrophilic track-etched polycarbonate filter membrane and a polytetrafluoroethylene (PTFE) washer.…”