Manufacture of CRISPR RNPs for Clinical Use

Shawn Shafer, PhD, genome editing platform lead, Aldevron

Shafer spoke about manufacturing ribonucleoproteins (RNPs) for clustered regularly interspaced short palindromic repeats (CRISPR) for clinical applications. CRISPR RNP consists of a guide RNA (sgRNA) complex with the CRISPR-associated protein 9 (Cas9). The Cas9 protein is complexed with the guide RNA as it associates with the genomic DNA. Once the guide RNA is aligned with the complementary genomic DNA sequence, the catalytic nucleus domains cleave the genomic DNA, creating a double-stranded break — and that is the process researchers use to inactivate (knock out) a gene.

Research shows that inactivating endogenous T-cell genes improves the therapeutic profile for those immune cells, and CRISPR RNPs are the best way to accomplish that. They are immediately active and circumvent the need for transcription and translation, behaving more like small molecules. That makes them easily titratable, and researchers can develop in vitro assays to characterize their performance and composition.

Knocking out T-cell genes has been shown to improve efficacy of therapeutic T-cell profiles. Knocking out endogenous T-cell receptor genes can prevent crossreactivity and result in a singularly targeted T cell to a specific cancer antigen. When we think about making off-the-shelf or universal allogeneic T cells, knocking out the human leukocyte antigen (HLA) genes is an important consideration.

Aldevron was approached by therapeutic partners with a need for characterizing CRISPR RNPs under good manufacturing practice (GMP) conditions. Shafer described project goals to identify the ideal complexing conditions for guide RNA and Cas9 proteins to create an in vivo cutting assay that would be applicable from one RNP to another for determining the best way to identify free Cas9 sgRNA and the RNP composition to create a standard quality control (QC) panel for RNPs. Long-term stability studies were conducted to elucidate storage conditions and longevity of CRISPR RNPs as potential off-the-shelf products.

Shafer reviewed data from the efforts thus far, beginning with development of an in vitro cutting assay to characterize RNPs in a drug-like way, in vitro, and in a replicable method. The goal was to define a linear range of cutting and limit of detection for RNP activity. Once a reliable in vitro cutting assay, was developed, Shafer’s group then moved to assessing ideal ratios of guide RNA to Cas9 — also performed in vitro and substantiated and backed by cellular cutting activity. Slides demonstrated results that showed robust cutting and almost a complete abolition of the target template DNA. Shafer stressed the importance of finding the optimal ratio of guide RNA to protein so as to reduce the cost of reagents in clinical assays.

Shafer spoke about optimizing guide RNA and Cas9 protein ratios in vitro. His group looked at a number of different assays beginning with differential scanning polarimetry and then moving on to size-exclusion chromatography, neither of which provided the results needed to differentiate between guide-RNA bound and unbound Cas9. Finally, however, analytical cation-exchange chromatography achieved reliable separation of elution times between RNP and free Cas9 following optimization efforts. Shafer illustrated how the quality and composition of the guide RNA can have a substantial impact on the reliability of the assay.

To treat CRISPR RNPs as a drug, the group investigated long-term storage conditions at –80 °C, –24 °C, and 30 °C for up to six months, and then at –80 °C for up to 24 months. Results confirmed that storage at colder temperatures preserved RNP activity and composition. The group was encouraged to see a robust ability of the RNP to cut, with few degradation products, when stored at –80 °C and at –20 °C. But at 4 °C and three months, degradation products appeared. At 30 °C the free guide RNA was gone, indicating that it probably was fully degraded. The conclusion was that for up to 12 months, storage of the RNP at –80 °C is a reliable way to contain these products. The group is continuing this work to take the experiments out to two years.

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