Christopher Vakulskas, director of Enzyme Evolution at Integrated DNA Technologies explains how the company’s novel gene editing tools may one day provide single-treatment cures for diseases caused by single gene mutations, including sickle cell disease.
DHA
“My 12-year-old daughter has sickle thalassemia disease. I as a parent understand how it feels to see your child go through rough times. At first it was so frustrating and overwhelming. She's been in and out the hospital since she was a year old. She's had a lot of blood transfusions, pain, and crises, and at age 5 had a splenectomy… She's so much stronger than I am. I admire her for that.”1
In the US, the Centers for Disease Control and Prevention (CDC) and other medical websites chronicle heart-breaking stories of patients with sickle cell disease (SCD) who experience chronic pain, anaemia, repeated infections, and recurrent hospital stays. The chief symptom of SCD is pain—in fact, sickle cell crises are also referred to as pain crises and are the most common cause for hospitalisation, which may span days or months, and recur throughout life.
Sickle cell disease is a group of recessive genetic disorders of the blood that affects haemoglobin, the molecule in red blood cells that delivers oxygen to cells throughout the body. It is caused by a single nucleotide substitution in the ß-globin gene (HBB), called sickle haemoglobin (HbS), which causes misshapen or “sickled” red blood cells. These sickled erythrocytes, which are rigid and less pliant than their healthy counterparts, clump together to create obstructions in capillaries, which deprives tissues of oxygenated blood. This deprivation can lead to organ damage, particularly of the lungs, kidneys, spleen, and brain. An especially serious complication is pulmonary hypertension, which occurs in about a third of adults with SCD and can lead to heart failure.
In the US, SCD is the most common inherited disorder—globally, 100 million people are carriers of the trait. An estimated 300,000 children are born with SCD every year, and epidemiologists predict that in the next 30 years, 400,000 babies born will be born with SCD annually2. The only true curative treatment is haematopoietic stem cell (HSC) transplantation, which has strong event-free survival (90%) but serious limitation, since 80% of patients lack a human leukocyte antigen-identical sibling donor. And even when matches occur, there may be serious treatment-related risk, including that of graft rejection, infections, prolonged immunosuppression, acute or chronic graft-vs-host disease, and disease relapse.
A promising tool for treating SCD at its root has been gene editing technology, including CRISPR (clustered regularly interspaced short palindromic repeats). Because it is caused by mutation within a single gene, SCD is an excellent candidate for CRISPR gene therapy, as are other single-gene diseases, including Huntington’s disease and cystic fibrosis. In fact, CRISPR is already being evaluated in early-phase clinical trials for several disorders, including SCD.
CRISPR technology is comprised of two components: a nuclease (the best-known is Cas9), which acts like “scissors” in the cleavage of double-stranded DNA at a specific location, and the “guide” that gets it there—generally, a single guide RNA (sgRNA), which forms a complex with the nuclease. After it locates its target, by recognising protospacer adjacent motifs, or PAMs, the Cas9 enzyme cleaves the DNA via a double-stranded break (DSB). The cell’s machinery then seals up the loose ends, either directly, through one of many complex repair pathways including the error-prone nonhomologous end-joining (NHEJ) pathway or, if a homologous donor DNA template is provided during the repair, the homologous recombination (HR) repair pathway. Donor DNA can range from single-nucleotides to large gene cassettes.
As promising as CRISPR technology is, there is of course room for error. One concern is that the nuclease may cut at unintended sites in the genome, since multiple sites can have similar or identical sequences to the ones being targeted. The consequences of these off-target effects (OTEs) may be serious—for instance, the loss of stem cell functions, or the disruption of oncogenes or tumour suppressors, which can set off pathways leading to cancer. Additionally, since no two genomes are the same, OTEs may differ between individuals, making the effects difficult to predict. Finally, since genes often serve multiple purposes, some of which may not yet be fully known, even the effects of on-target editing need to be carefully assessed, particularly when introducing changes that are not simply conversion back to healthy or wild-type sequence. These concerns have understandably limited the testing of CRISPR in humans.
To combat the potential risks associated with the CRISPR-Cas9 system, researchers have tried modifying various parts of the process, to varying degrees of success. A promising method has been the use of recombinant Cas9 enzyme bound to a synthetic guide RNA as a ribonuclear protein complex (RNP)—this can reduce the risk of OTE by limiting the duration of exposure of the CRISPR reagents to the DNA. Use of RNP delivery utilises a ‘fast on, fast off’ strategy, with an initially high level of genome-editing machinery followed by a quick decline. In fact, RNP methods are slated for use in a number of Phase I clinical trials in coming years. However, a remaining issue is that several Cas9 enzyme mutants that have been shown to improve specificity show markedly reduced activity when used in the RNP format, which limits their therapeutic utility.
Our work at Integrated DNA Technologies (IDT) has focused on improving the properties of CRISPR nucleases to be used in tandem with RNP delivery. Using an unbiased bacterial screening system, we evaluated hundreds of novel Cas9 variants, ultimately arriving at a new, high-fidelity Cas9 protein created via a single point mutation (R691A)3. This new high-fidelity, or ‘HiFi,’ Cas9 reduces OTEs, but maintains high on-target activity in living cells when used as an RNP complex. It has outperformed wildtype Cas9 systems for producing highly specific and on-target double-stranded DNA breaks.
We demonstrated the potential of HiFi Cas9 in targeting several important disease-associated loci—in CD34+ hematopoietic stem, progenitor cells (HSPCs), and primary T cells. Importantly, we showed that HiFi Cas9 mediates high-level correction of the SCD-causing p.E6V mutation in HSPCs derived from patients with SCD. Off-target effects were reduced by up to 20-fold compared with wild-type (WT) Cas9. This type of autologous gene editing of HSPCs has the power to be a safer and more effective therapy than allogeneic transplantation, as it reduces the risks and drawbacks mentioned earlier.
Matthew Porteus, professor of paediatrics (stem cell transplantation) at Stanford Medicine, is leading a proposed Phase I SCD trial, evaluating a gene editing therapy to correct the HBB mutation. Preclinical study results are promising: the HiFi Cas9 enzyme has already been used to show that the sickle HBB allele could be efficiently corrected, while simultaneously reducing off-target editing from ~30% to less than 1%.
“We performed an unbiased evaluation of several versions of high fidelity Cas9 enzymes in primary human stem cells. We have been very impressed with the characteristics of this new IDT enzyme. Unlike other versions, this version consistently gives us high on-target editing activity, while having low off-target activity. Because of the retained, excellent on-target activity and improved specificity profile, we are excited to use this version in our future experiments focused on developing novel genome editing-based therapies for several diseases with unmet medical needs,” said Dr Porteus.
We are encouraged by these early findings, and anticipate that HiFi Cas9 will have wide utility across the life sciences, from basic science to therapeutic genome-editing applications. While there is more work to be done, CRISPR gene editing holds enormous promise for personalised medicine, not only for genetic disorders, but also for acquired diseases with genetic mechanisms (e.g., cancers and viral infections, including HIV/AIDS). And while the research is still in early phases, we hope it will encourage patients, their families, and clinicians that there might one day be effective single-treatment cures for some of these most perplexing and devastating diseases.
References
1: Emedicinehealth.com, Sickle cell disease patient comments. https://comments.emedicinehealth.com/sickle_cell_crisis/viewer-comments_em-139.htm. Accessed March 12, 2020.
2: Pleasants S. Epidemiology: a moving target. Nature. 2014;515: S2–3.
3: Vakulskas C., Dever D., et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nature Medicine. 2018;24:1216-1224