Gene editing immunity
Professor Waseem Qasim, Professor of Cell and Gene Therapy, UCL Great Ormond Street Institute of Child Health
Experience from bone marrow transplantation has created a deep understanding of human immunity, and uncovered powerful immune mediated effects against infections and certain types of cancer. T-lymphocytes express a diverse repertoire of antigen-specific T cell receptors (TCRs) that recognise antigenic peptides in the context of specific human leukocyte antigen (HLA) and genetic engineering is being used to circumvent HLA-restriction and augment efficacy while avoiding non-specific, graft-versus-host effects. Recent advances in gene-editing technologies including TALE nucleases and CRISPR/Cas reagents has allowed the production of ‘universal’ anti-leukaemic cells. The cells can be collected, engineered in a specialist laboratory and cryopreserved until required. Early experience suggests these strategies may offer potent therapies for hard-to-treat malignancies and clinical trials are underway to test the approaches in the transplant arena.
Development of a Subretinally-Delivered, CEP290-Specific CRISPR Medicine for the Treatment of Leber Congenital Amaurosis 10 (LCA10)
Dr Charlie F Albright, CSO, Editas Medicine (Cambridge, MA)
LCA10 is an early-onset, inherited retinal disease caused by mutations in the CEP290 gene. A reduction in CEP290 protein leads to a loss of vision due to the absence of outer segments in photoreceptors which renders these cells non-functional but viable. We aim to restore CEP290 expression by CRISPR editing and thereby photoreceptor function and vision.
To correct the common IVS26 c.2991+1655 A>G mutation in the CEP290 gene, a panel of gRNAs with SaCas9 in human cells was screened. This screen identified a pair of gRNAs that efficiently cleaved intronic sequences flanking the mutation, resulting in the removal of the aberrant splice donor site created by the mutation. In fibroblasts derived from patients homozygous for the IVS26 mutation, editing led to significant reduction of mutant CEP290 mRNA and concomitant restoration of wild-type CEP290 mRNA and protein. The lead gRNAs had no detectable off-target cutting in multiple types of human cells as measured using both unbiased GUIDE-Seq and targeted sequencing methods.
Productive CEP290 gene editing at expected therapeutic levels in neural retina was demonstrated in both human CEP290 IVS26 knock-in mice and cynomolgus macaques following subretinal delivery of a single AAV5 vector expressing both gRNAs and SaCas9 under the control of the photoreceptor-specific GRK1 promoter. Safety studies are underway to support development of the lead candidate. Editing of photoreceptors has the potential to address other inherited retinal diseases.
In Vivo applications of CRISPR
Dr Mohammad Bohlooly, Director, Translational Genomics, Discovery Sciences iMED, AstraZeneca
Animal models with specific gene modification are essential tools for studying development and disease. Technologies that can delete insert or modify the gene sequences of organisms to understand and validate the function of specific genes are an essential part of drug discovery research. The development of the genome editing tool CRISPR/Cas9 has already greatly impacted basic research, drug discovery and translational research by enabling such gene edits in a rapid, precise and controlled manner. As genome editing is developed for therapeutic applications, in vivo validation of genome editing becomes an essential requirement to assess the efficiency and safety of the CRISPR/Cas9 technology.
Genome editing using CRISPR/Cas9 technology represents a fast, multiplexable, efficient and simple technique to generate sophisticated gene manipulation that can be inducible, conditional or introduce new functions such as endogenous reporters in any mammalian species.
CRISPR screens for oncology drug target discovery
Dr Mathew Garnett, Group Leader, Wellcome Trust Sanger Institute
Cancer is caused by multiple molecular alterations that lead to changes in the cellular processes necessary for malignancy. Cancer medicines can exploit cellular rewiring in malignant cells to selectively target tumour-specific dependencies, while sparing healthy cells. The histological, genetic and epigenetic heterogeneity of cancer means that identifying these dependencies is challenging, and limits our ability to develop effective therapies. To identify new oncology drug targets, we are performing whole-genome CRISPR-Cas9 drop-out screens across a large number of genomically-annotated human cancer cell lines. I will discuss these datasets and methods we are developing to systematically link gene essentiality with the molecular features of cancer to guide the development of molecularly targeted oncology therapies.