RARE Daily

Bringing Precision to CRISPR-Based Genome Editing

June 1, 2023

CRISPR genome editing has the potential to revolutionize the treatment of diseases, but the imprecision of its editing abilities has limited its value. Emendo Biotherapeutics argues that rather than trying to make every disease fit into the standards CRISPR model, the answer lies in making CRISPR fit each disease. We spoke to Rafi Emmanuel, executive vice president of research and development for Emendo Biotherapeutics, about the limits of CRISPR today, the company’s experimental program in severe congenital neutropenia, and how the company is engineering CRISPR to optimize it and make it activity precise.

Daniel Levine: Rafi, thanks for joining us.

Rafi Emmanuel: Thank you, Dan, for having me.

Daniel Levine: We’re going to talk about Emendo Bio, it’s platform technologies and its efforts to develop next generation gene editing therapies. There’s a lot of excitement about the potential of gene editing therapies, but where are we in terms of realizing this potential?

Rafi Emmanuel: Well, the potential of gene editing therapies lies in the single administration of the therapy product to cure the disease. This is unlike gene therapy that is not long-lasting. We are now starting to understand the potential through the first clinical trials that are being conducted. So, we have the very promising results from CRISPR Therapeutics—these are ex vivo clinical trials, and the in vivo clinical trials from Intelia that demonstrated very promising results in terms of efficacy and most importantly in terms of safety. So, we need more data like this, and I think that’s starting with rare diseases such like sickle cell, beta thalassemia, aTTR, and Emendo’s leading indication, severe congenital neutropenia, where the genetic cause of the disease is very clear and the effect of the treatment on the patient’s life will be very traumatic after obtaining the treatment. So, it’s very important to have more confidence in gene editing technologies. Eventually we will be able to implement these technologies also on more prevalent diseases and more complex genetics.

Daniel Levine: What are the primary challenges of using this technology today? Are they delivery, is it a matter of precise editing, or is it something else?

Rafi Emmanuel: Yeah, so if I have to rank the challenges, I would say safety is the biggest challenge of gene editing. So, we remember the effect of the first gene therapy trials conducted in the nineties to treat SCID patients that led to the death of three children from cancer. That incident stopped the clinical trials and the development of the field for decades. Therefore, we at Emendo are working on developing therapeutic products with the highest safety profile. So, the second challenge is the lack of flexibility to design therapeutic strategies using the current technologies. And the delivery—sorry, I would rank them first—because eventually they restrict the tissues that can be targeted and the diseases that can be treated.

Daniel Levine: Before we talk about your platform technologies, I thought it would be useful to explain how gene editing therapy works. Can you walk us through that?

Rafi Emmanuel: Sure. Basically Crispr-based nucleases—we can imagine them as molecular scissors, and once you use them you can target genomic DNA sites using two components. So, we have two entities in these Crispr nucleases. We have the protein and the RNA guide molecule, which basically helps the protein to target specific sequences in the genome. And these sequences are complementary to the sequence of the RNA. And once we have this recognition, we will have a cut in the genomic DNA and following this break, the cells utilize the DNA machinery repairs in order to repair these lesions. And the most prevalent machinery is the non-homologous enjoining, which is basically a machinery that ligates the edges. But while doing that, it can add nucleotides, it can delete nucleotides, meaning that it generates differences in the genomic DNA sequence, which might lead to a knockout in the gene and also changing the expression of genes. And if we also add a template, or introduce a template in addition to the nucleases so the cells would use a machinery that is called a homology-directed repair where it uses the template to correct the lesion according to the sequence that you introduced in this template. So, you can either change the mutation and repair it or even introduce a new sequence to the genome to genomic DNA in order to express different genes.

Daniel Levine: When people hear the term Crispr, I imagine they think of Cas9, which is an enzyme used to cleave DNA. It’s a nuclease. How big a world of nucleases are there and how specific is their activity to an application?

Rafi Emmanuel: So, the world of nucleases is big, and from our experience at Emendo, there is a huge demand for novel diverse nucleases as each has its specific characteristics in term of genomic accessibility, activity, specificity, and delivery modalities. Therefore, there isn’t a nuclease that fits all indications. And the reason for that is that you need to start with the best performing composition for your indication and not rely only on improving the activity or the characteristic of the nucleases because there is always a threshold when you start modifying your nuclease, and if you want to increase its specificity, you would basically compromise the activity. So, you need to modify it as little as possible in order to have this perfect nuclease for your indication. That’s why you need to have a large toolbox of different nucleases with different characteristics to choose from them the best performing nuclease for your indication.

Daniel Levine: Emendo describes its gene editing technology as next generation. What makes it next generation? What does it enable these technologies to do that weren’t previously possible?

Rafi Emmanuel: So, we don’t rely on the first limited sets of nucleases that were discovered, but rather we have a panel of proprietary nucleases that we can further engineer using our powerful platform to use them in our different indications. In this way, we can increase the accessibility of the nucleases to the genome as I’ve mentioned, and to increase the specificity and obtain the highest specificity level to have the best safety profile of the nucleases to be used in the clinic.

Daniel Levine: Emendo has developed two platform technologies. The first is a discovery platform. How does it work? What’s the starting point? What are the inputs and what’s the output?

Rafi Emmanuel: We based on algorithm that was trained on dataset generated at Emendo. We use this algorithm to analyze complex bacterial metagenomic databases and following this analysis, we basically obtain putative nucleases and the corresponding guide sequences that are further validated in the laboratory. And actually, part of these nucleases are active from the beginning with a very high specificity profile, which is basically enough even to be implemented in therapeutic compositions.

Daniel Levine: You also have a platform to optimize candidates. Walk me through the process.

Rafi Emmanuel: So, we use a machine learning algorithm to generate a pool of variants that potentially have improved characteristics and we develop high throughput screen, a platform in order to screen for the relevant and the most potent variants in the relevant cell system. And usually, we need more than one cycle or several iterations in order to have this final variant with the optimal characteristic for our indications.

Daniel Levine: Emando has a preclinical pipeline, but as I look through it, your experimental therapies seem to be targeting indications that involve the eye and liver areas that are most accessible to delivering genetic medicines today. Why target these indications?

Rafi Emmanuel: Well, because accessibility is a major factor for choosing an indication, but I want to add also that these tissues enable us to expand our technological tools so we can utilize nucleases from our proprietary pool and use different delivery modalities and also implement unique therapeutic strategies that are also unique to Emendo in these fields.

Daniel Levine: Have you been able to show your technology allows you to effectively target other cells and tissues within the body?

Rafi Emmanuel: So, efficiency of targeting in vivo is based on the delivery modality, so no, besides the liver and the eye, we haven’t demonstrated editing in other tissues, but we did use our nucleases in different primary cells and we obtained high activity in different cell systems using them as plasmid or messenger RNA and either RNP compositions.

Daniel Levine: You’re developing both in vivo and ex vivo therapies. What determines whether you’ll approach a condition with an in vivo or ex vivo approach?

Rafi Emmanuel: Well, the targeted issue and it’s accessibility to the different delivery vehicles. So, I wouldn’t treat the livered indication ex vivo.

Daniel Levine: Your lead indication is a gene editing approach for severe congenital neutropenia. This is a rare immune condition. Can you explain what it is and how it manifests itself and progresses?

Rafi Emmanuel: Yeah, sure. So severe congenital neutropenia is a neutrophil maturation disorder. Neutrophils are white blood cells that fight infections such as bacteria, viruses, and fungal infections. This disease is manifested by low counts of neutrophils in the blood, which lead to severe and recurrent life-threatening infections. And in most cases, this disease is caused by more than 200 mutations, mainly in the gene called ELANE, which calls for neutrophil elastase. And these mutations are autosomal dominant, meaning that you need only one affected copy of the gene in order to have the phenotype of severe congenital neutropenia. And the severity of the disease differs between different patients according to the mutation and some would respond very well to the standard of care treatment and some would need bone marrow transplantation very early. This is a pediatric disease so if the patient needs bone marrow transplant, it’s done very early. And some would develop or acquire further mutation during their life and they would stop responding or reduce responsiveness to the treatment, which might increase the risk for blood cancer. And this patient would also require bone marrow transplantation.

Daniel Levine: What’s the prognosis for someone diagnosed with this condition today?

Rafi Emmanuel: The prognosis today is basically you just see the count of the neutrophils in the blood and once you see that they are very low below 500 neutrophils per µL. So, they start treating with the GCSF, which is a growth factor that basically overcomes the arrest of differentiation and maturation. And if the patient doesn’t respond to the highest dose of GCSF that can be given, this patient undergoes bone marrow transplantation.

Daniel Levine: What’s your experimental therapy, EMD-101 and how does it work?

Rafi Emmanuel: EMD-101 is an autologous product of edited hematopoietic stem cells. What I mean by autologous is that the source of the cells are from the patients and these hematopoietic stem cells are cells that originated from the bone marrow and they are responsible for the generation of the whole blood cell lineages. So, basically what we do in this product, we mobilize the stem cells from the bone marrow to the blood. These cells are being collected and isolated from the other cells, and we use our therapeutic composition to specifically knock out the mutated copy of the gene and keep the healthy copy intact. In this way, we basically eliminate the source of the disease and we enable the differentiation of the hematopoietic stem cells towards functional neutrophils. This product will be frozen—after editing we freeze the cells. We do a very thorough analysis to assess the safety and the potency of the product, and then it’ll be thawed and injected back to and engrafted to the patient. Now as you could understand, here we need our composition to have the capability to discriminate between the two copies of the gene, the healthy and the mutated copy on the base of one mismatch, one single nucleotide, which basically raises the bar for the specificity level needed from the nuclease. And we could achieve this high bar of specificity using our technological platform. And in addition, I mentioned that this mutation is caused by more than 200 mutations in the gene allele, meaning that we couldn’t develop a composition for each mutation and therefore we thought of a more creative strategy where we achieved a little specific editing, not by targeting the mutation, but by targeting single nucleotide polymorphism, what we call SNPs that are located close to the gene and linked to the mutation. And using this strategy, we can basically treat almost all the patient population using few compositions.

Daniel Levine: What’s the development path forward?

Rafi Emmanuel: We aim to have two clinical sites for this program, one in the United States and one in Germany. That’s why we approached the regulatory authorities in the United States, the FDA, and the PI in Germany. And we recently had meetings with both of them, which were very, very positive meetings. Now we are working on finalizing the studies that would enable us to start our first in-human trial next year. We will start, although this is a pediatric disease, we are going to start with adults. And in the first in-human and following, demonstrating the safety and efficacy of our product, we will gradually reduce the age of the patient and the production of the product will be done here in Israel. So, we are also establishing the site, meaning that all samples, the blood samples will be sent here to Israel, we’ll produce the product and freeze it and send it back to the clinical sites.

Daniel Levine: The Japanese genetic medicines company, AnGes acquired EmendoBio in 2020. Emendo is based in New York and Tel Aviv. What did the acquisition do for Emendo?

Rafi Emmanuel: So, when AnGes acquired Emendo, we remained an independent company and we work and coordinate closely with an AnGes the strategics of Emendo.

Daniel Levine: There are differences in company cultures between a startup and a more mature company. I suspect those differences may be amplified by one company being in Japan and the other in the United States and Israel. How do the companies work together and how have the cultures merged or clashed?

Rafi Emmanuel: Wow. Beautiful question Danny. I think it’s a very interesting cultural experience. The Israeli start startup culture is extremely innovative and versatile, making a bold decision fast. This is very beneficial for developing new technologies and being managed from the United States. We are also influenced by the business culture that is market oriented from the early stages with strong expertise with building structures and value generating companies. And the Japanese business culture is generally more traditional, personal, and details oriented, which I believe eventually generates a balanced company. And I think that after all, we are very fortunate to discover that AnGes is open-minded and accepted the merger of the three cultures.

Daniel Levine: Rafi Emmanuel, executive vice president of Research and Development for Emendo Biotherapeutics. Rafi, thanks so much for your time today.

Rafi Emmanuel: Thank you Danny for this opportunity to speak to you.

This transcript has been edited for clarity and readability.


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