Precision genome editing with tandem autologous transplantation as a therapy for multiple severe immune-mediated diseases

Autologous haemopoietic stem cell transplantation (ASCT) is emerging as an important therapy for patients with severe immune-meditated diseases (IMDs). Recent studies have shown that it is safe and effective for treating severe IMDs such as multiple sclerosis (MS) and scleroderma. This involves collecting bone marrow derived stem cells from a patient and then eliciting severe but transient immunosuppression using a combination of chemotherapy and therapeutic antibodies. This eradicates a large portion of the immune system including the autoreactive cells. Bone marrow derived stem cells are then reinfused and the immune system is reconstituted afresh with minimal long-term side effects.

European Bone Marrow Transplant registry data show that more than 2500 patients have had autologous transplants for IMDs. Only one death has occurred since 2005 following autologous transplantation for MS. ASCT is superior to all other therapies in MS for inducing long-term remissions and it is the only treatment that has been found to reduce disability. In scleroderma, ASCT results in a very marked improvement in survival (86% v 51% at 6 years). Although ASCT can halt disease progression for many patients, 30-50% eventually re-develop their original IMD and 5-10% develop a different IMD. There is therefore significant scope to improve this therapeutic strategy.

In recent years there has been a revolution in molecular biology due to programmable nucleases such as CRISPR-Cas9 because they allow us to make precise changes to the sequence of the genome. They are likely to become hugely important therapeutic tools within the next decade. We are using this technology to modify the genome sequence in bone marrow derived stem cells for curing inherited disorders of blood cell production and the immune system. The next step is to use this approach to treat acquired disorders of the immune system such as IMDs.

Large-scale genetic analyses have revealed a target gene that is broadly protective across 20 different IMDs including multiple sclerosis (MS), scleroderma, rheumatoid arthritis and Crohn's disease. For these diseases there is around a 10- fold risk reduction; meaning that 9 in 10 patients would not have developed the disease had they had two copies of the protective variant. The protective variant does not result in increased risk for malignancy and does not lead to immunodeficiency.

We will develop a strategy that allows us to change the genetic sequence of bone marrow-derived stem cells to mimic the protective genetic variants identified by large-scale genetic studies. These edited cells could then be used to repopulate the immune system with genetically modified cells with a stem cell transplant. This approach would potentially eradicate the harmful immune cells and significantly and permanently reduce the chance of relapse. This is a completely novel approach for treating IMDs and it is potentially applicable to a broad range of different diseases.

Through our editing strategy we will change the DNA sequence that defines the structure of the protein, to mimic the naturally occurring protective variants, which attenuate its function. This protein plays a critical role in immune cell activation and it will cleanly and precisely decrease the immune response.
The editing strategy will be tested in an IMD mouse model, which mimics the pathology of MS, to determine if the edit protects against IMD development. We will also define what proportion of edited cells required for the protective effect by performing transplants in mice using mixtures of edited and normal cells.

Autologous stem cell transplant (ASCT) is an effective treatment in immune-mediated disease (IMDs) because the combination of high dose chemotherapy and anti-thymocyte globulin transiently induces severe immunosuppression, which eradicates a large proportion of pathogenic cells. The stem cell transplant repopulates the immune system without these clones. Our approach is to combine ASCT with genome editing of haemopoietic stem cells (HSCs) to precisely change the genotype of bone marrow-derived immune cells to sequences that are highly protective against developing IMDs. This would represent a novel treatment modality that would potentially be applicable to a broad range of IMDs.

GWAS and UK Biobank analyses have revealed naturally occurring protein coding variants in a target gene that are highly protective against developing a broad range of IMDs (odds ratios of 0.1-0.2). We plan to replicate the effects of these variants using the novel CRISPR-cas9 base editors to make precise edits in the protein coding sequence of the gene.

Significant recent advances (particularly the adoption of modified guide RNAs and purified Cas9 protein) mean that we can now achieve >95% editing in primary human HSC without cell selection. In preliminary experiments we have also been able to achieve >85% editing with Cas9 base editors in primary human haemopoietic stem cells using plasmids. We anticipate it will be possible to achieve >95% editing in human HSCs in the near future using base editor ribonuclearprotein. This will open up new avenues of treatment for IMDs as it will allow us to make precise long-term changes to the immune system.

It is likely that within the next decade genome editing of haemopoietic stem cells will result in long-term cure of many of the inherited disorders of haemopoiesis. Once this technology is established for inherited defects of haemopoiesis it is likely that it will be rapidly adapted for treatment of other disease processes including immune-mediated disease (IMDs), malignancies and possibly infectious diseases such as HIV.

IMDs represent one of the most important classes of diseases: they affect around 10% of the population, their incidence is rising, and they are one of the leading causes of chronic disability and death in the UK and USA, costing billions of GBP/USD annually. At present treatment of IMDs generally consists of drugs to manage symptoms and immunomodulation to suppress active disease, although available therapies often have variable efficacy and can be associated with severe side effects. Generally, it is not possible to cure these diseases indefinitely with current approaches. As a consequence, patients often have successive relapses and suffer a general decline in function over many years. This creates a huge burden of disease and economic impact, which is born by patients, their families and carers, national health services, as well as society more broadly. If it were possible to halt the aberrant immune attack and prevent further disease progression indefinitely through precise, long-term manipulation of the immune system using genome editing, it would potentially have a huge impact.

The proposed work also represents a proof-of-principle for a novel way of treating human disease, which has the potential to be used much more generally. The combination of large-scale genomics projects with genome editing potentially allows protective polymorphisms to be identified and precisely replicated for hundreds of different diseases. The work therefore has the potential to have considerable economic and societal impact.

In our experimental plan we will study the effects of editing the amino acid residues that contribute to the catalytic site of our target protein, which will contribute to the understanding of how this protein and other similar tyrosine kinases function. The technology developed in this proposal and the data generated will provide insights into how genome editing can be safely achieved not only in haemopoietic cells but also more generally. The proposed editing strategy will also be one of the first demonstrations that it is possible to use genome editing technology to treat acquired human disease. It will therefore be of great interest to the biomedical and genome editing communities.