Identification of preexisting adaptive immunity to Cas9 proteins in humans

The CRISPR-Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR-Cas9 system to therapeutically correct human genetic diseases1-6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR-Cas9 system moves toward clinical trials

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses

Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells

β-Thalassemia pathology is due not only to loss of β-globin (HBB), but also to erythrotoxic accumulation and aggregation of the β-globin-binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in β-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize β-globin:α-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing β-thalassemia

Reprogramming Immune Cells <i>in situ</i>:A Targeted LNP Delivery Technology Utilizing IVT mRNA

Cancer immunotherapy has become a standard pillar of cancer treatment throughout the last decade. Immune checkpoint inhibitors (ICIs) and adoptive cell therapies (ACTs) such as tumor-infiltrating lymphocytes, tumor-specific T-cell receptor-modified T-cells, and chimeric antigen receptor (CAR)-engineered T-cells have proven successful in initiating an immunologic response against various cancers. Unfortunately, not all patients respond to ICIs and ACTs requires cumbersome and expensive processes.For currently approved CAR-T therapies, T-cells are first extracted from the patient by leukapheresis followed by being transduced with lentivirus that encodes the CAR. CAR-expressing T-cells are then expanded over a two-week period before finally being re-administrated to the patient. Besides the substantial manufacturing costs associated with this therapy, CAR-T cells often cause toxicity-related side effects upon infusion.This thesis aims to develop a strategy that allows for the generation of transient CAR-transfected T-cells in situ to treat hematological malignancies and thus overcome the lim-itations associated with currently approved CAR-T therapies. The technology uses in vitro transcribed synthetic mRNA that is formulated in lipid nanoparticles (LNPs) as a systemically injectable drug. The LNPs are directed to circulating T-cells via an antibody-based targeting moiety to transiently express disease-specific receptors, thereby bypassing the need to extract, culture, and reinfuse lymphocytes into patients. The thesis then aims to demonstrate the broad applicability of the technology beyond hematological malignancies by targeting other immune cell subsets to overcome solid tumors.First, we demonstrate that the technology can be used to deliver reporter mRNA specifi-cally to human T-cells. We then show that the platform enables specific delivery of functional CAR-encoding mRNA to circulating T-cells directly in vivo. Finally, we extend the application of the technology to reprogram T-cells to become bi-specific T-cell engager-secreting factories, thereby reducing solid tumor burden in mice.Overall, this thesis demonstrates the development and potential applications of a novel groundbreaking immunotherapeutic technology. This technology holds strong promise to be employed in the clinic to treat various cancers and thus improve the lives of patients worldwide