Startup Spotlight: Ariya Bio - A Unique CRISPR-Based Approach to Treat Haemoglobinopathies

A single ex vivo-edited therapy for sickle cell disease and beta thalassemia

- Which gene-editing reagents are you using and what is your chosen delivery strategy?

Jan: While our therapeutic strategy is novel, our gene-editing approach is similar to what others are doing from a protocol point of view. We are using Cas9 ribonucleoprotein and single guide RNAs to target the locus of interest (in this case the HBD promoter), which are delivered to cells via electroporation. We add single-stranded oligodeoxynucleotides (ssODNs) as the donor template; this allows us to insert the motifs mentioned earlier. In a therapeutic setting, all those reagents would be delivered to a patient’s own blood stem cells ex vivo via electroporation, and the edited cells would then be transplanted back into the patient’s bone marrow.

Mandy: Although we have chosen to focus on ex vivo patient-derived stem cell editing at present, our therapeutic mechanism is not restricted to an ex vivo approach, and we may explore in vivo promoting-editing strategies in the future.

- There are some ongoing attempts to develop therapies that correct the single disease-causing mutation in SCD. Such treatments should eliminate defective HbA1 from patient blood. What happens to the defective HbA1, in SCD, when you upregulate HbA2?

Mandy: Based on our data so far, we don’t believe that our approach to upregulate HbA2 leads to downregulation of β-globin, so HbA1 would still be present even with increased HbA2, but this shouldn’t be a major concern. The SCD mutation causes the defective haemoglobin molecules to polymerise, but studies have shown that expression of beneficial haemoglobins (such as HbA2) should prevent this, if there is enough HbA2 present.

As a side note, we are aware that others are developing therapies that target the underlying mutation in SCD. This is obviously an attractive approach for SCD, but since beta-thalassemia is caused by any of several hundred mutations in the HBB gene, any therapy to fix the root cause of disease would require a separate development programme for every type of mutation. With our approach, we can address both patient groups and potentially treat many more patients with a single therapy.

The attraction with haemoglobin A2 as a therapeutic target

- Do you believe that HbA2 is superior to foetal haemoglobin from a therapeutic point of view?

Mandy: Yes, we believe that HbA2 is a better target for several reasons. Firstly, it is more similar in structure and function to adult haemoglobin, with 93 % sequence homology between the two, compared to foetal haemoglobin, which only shares 76 % homology with HbA1. In addition, from a functional standpoint, foetal haemoglobin has elevated oxygen affinity compared to the adult haemoglobins, and the potential implications of persistently high levels of foetal haemoglobin on human physiology, in particular during pregnancy, are not yet clear.

Jan: The high levels of foetal haemoglobin that are achieved in the ongoing development programmes are not widely observed in nature. In contrast, HbA2 is already expressed in all adult red blood cells, albeit in small amounts, which suggests that it should be safe to express. Another interesting fact here is that HbA2 is actually the predominant form of haemoglobin in some species, such as dogs, which indicates that high levels of HbA2 are safe in mammals.

- So how and when did Ariya Bio get started?

Mandy: It really started with a fundamental question about how HbA2 was regulated. We were not thinking about developing a therapy at all in the beginning, but rather looking at genetic regulation of HbA2 and trying to figure out why two types of haemoglobin exist in adults, and why one of them is so lowly expressed.

Although we only started working on Ariya Bio quite recently, I began my PhD in 2015 and my dissertation was on the genetic regulation of haemoglobins. Jan and I started working together in 2021. During my PhD, I was looking closely at the promoters of the various haemoglobin genes and noticed that quite a few transcription factor elements were missing in HBD, whose protein product is required for the δ-globin subunits in HbA2. Other groups have reported transgenic approaches to replace these elements by inserting a KLF1 motif in the HBD promoter, but we were the first to show that CRISPR-mediated insertion of KLF1, β-DRF, and TFIIB motifs into the endogenous HBD promoter can lead to a 10-fold upregulation in HbA2 expression in various cell types. These results were very exciting and highlighted to us the therapeutic potential of upregulating HbA2 to treat haemoglobinopathies. No one else was doing it, so we started Ariya Bio to translate our scientific discoveries into a new therapy.

Lead programmes in the pre-clinical stage

- I know it’s early days, but what can you share about Ariya’s pipeline so far?

Jan: Our lead programmes are focused on the HbA2-upregulating strategy we’ve discussed here, for two indications: severe SCD and transfusion-dependent BT.

We are currently at the pre-clinical stage with both programmes, with a plan to advance ARY001 and ARY002 to the clinic for SCD and BT, respectively. We are still optimising the gene-editing reagents, and we plan to start in vivo engraftment studies before the end of 2023. These studies are crucial, as they will test how effectively CRISPR-edited patient-derived cells can engraft and express HbA2. They will likely run for a few months, and hopefully after that point we can begin to compile the data needed to submit an investigational new drug (IND) or clinical trial application (CTA) towards the middle or end of 2024.

Jan: Regarding clinical development, we are open for several scenarios including co-development with a partner, and we have already initiated talks with suitable industry partners. We are also in the very early stages of exploring whether we can adapt our promoter-editing strategy to other indications. We can’t say more about which diseases we are looking at, but we are very excited about how far we can go with our novel approach.

- Thanks to you both for speaking with me. We look forward to future developments from Ariya Bio!

Related articles from CMN

Disease Roundup: Four Gene-Editing Candidates in the Clinic for Sickle Cell Disease

Clinical Roundup: Gene-Edited Therapies for Beta Thalassemia