CRISPR gene-editing technology is being used for a multitude of reasons lately, just one example being the detection of SARS-CoV-2, as was mentioned in our June CRISPR Focus Feature. From creating new types of cancer models to CRISPR/Cas3-enhanced bacteriophages being used to target Escherichia coli bacteria, the versatile technology is making a mark in many fields.
But researchers have also branched out into using Cas systems to target RNA instead of DNA, since RNA targeting is temporary and more flexible. Preclinical data from some of these Cas systems were recently presented at the American Society of Cell and Gene Therapy (ASGCT) Virtual Annual Meeting in May.
DDN spoke with Dr. James Burns, CEO of Locana, and Dr. Kathie Bishop, chief scientific officer of Locana, to find out more about the company’s RNA-targeting Cas systems.
DDN Magazine: Can you tell us more about these RNA-targeting Cas systems and how they work?
Dr. Kathie Bishop & Dr. James Burns: Locana’s RNA-targeting systems were originally derived from work conducted at our founder Gene Yeo’s lab at UCSD [University of California, San Diego]. Gene was the first to discover that you could mutate the traditional CRISPR-Cas9 into what we call RCas9, which specifically binds RNA rather than DNA. In a paper published in Cell in 2017, [Yeo] and collaborators showed that this RNA-targeting Cas9 system could be used to modulate RNA and more specifically destroy disease associated repetitive RNA.
More recently at Locana, we have added to our repertoire of RNA-targeting systems beyond this original RCas9 system. We work with another series of Cas proteins called Cas13d that specifically bind RNA, but have the advantage [in] that they are much smaller in size so they are able to fit within a single AAV delivery vector. We have a number of these Cas13d systems that we are currently working with, and we can efficiently screen through them to find the best system for any given target.
In addition to these Cas13d systems, we also work with a very different set of RNA-binding proteins that are human derived and can be programmed to specifically bind to any given sequence of RNA. These modular RNA binding systems can be paired with a number of different effector molecules to modulate RNA—for example, pairing with an endonuclease enables destruction of a disease-causing RNA. So, taken together, we have a broad menu of RNA-targeting systems which include Cas proteins—as well as human derived non-Cas proteins—and we can efficiently deploy these systems to find the best one for any given RNA target.
DDN: How does this platform differ from traditional gene therapies? What are the advantages of this approach?
Bishop & Burns: Locana’s platform differs from traditional gene therapies in that we are marrying the power of AAV gene delivery with our very flexible RNA-targeting systems. Our platform of RNA binding systems allows us to specifically intervene at the RNA level, and these systems also have broad versatility to modulate RNA using a range of different mechanisms. This could include RNA destruction, [which] can be done for RNA repeat diseases, but also non-repeat RNA. It also includes splicing modulation, enhancement of translation and RNA-editing to change specific bases within the RNA sequence.
And finally, because our systems are very small in size and easily fit within the capacity of the AAV gene delivery vehicle, we have the added flexibility to enable simultaneous destruction of a toxic mutated RNA with replacement of the wild type gene. This is very different than traditional gene therapies that just deliver a replacement gene for monogenic diseases.
DDN: What types of diseases/indications might this platform be able to treat?
Bishop & Burns: Our initial programs are focused on what are called RNA repeat diseases, because our systems are extremely well suited to target these diseases. These include trinucleotide repeat diseases such as myotonic dystrophy, where there is a toxic repeat CTG stretch within the DMPK gene, and CAG repeat diseases which include Huntington’s disease, SCA1 [Spinocerebellar ataxia type 1] and other spinocerebellar ataxias.
In addition, we are working on a third set of RNA repeat diseases called hexanucleotide repeat diseases, including C9orf72-related ALS [amyotrophic lateral sclerosis] and frontotemporal dementia. However, our RNA-targeting systems also enable a wider range of RNA modulation mechanisms and we are also focused on earlier stage research programs, especially in the areas of splice modulation indications, translational enhancement indications and diseases of the retina, where a knockdown and replace approach may be warranted.
DDN: Can you tell us about the effects of the RNA-targeting Cas systems on myotonic dystrophy type 1?
Bishop & Burns: Myotonic dystrophy type 1, or DM1, is a multisystemic disease that affects about 40,000 patients in the U.S. alone and has a similar prevalence worldwide. It is caused by a toxic trinucleotide repeat in the DMPK RNA. Downstream of this RNA repeat, a number of splicing factors are bound by the repeat RNA, leading to broad mis-splicing that is directly associated with symptoms of the disease, such as muscle weakness, wasting and myotonia. We have demonstrated safety and efficacy of our RNA-targeting systems in a series of experiments conducted in muscle cells derived from patients with DM1 and an animal model of myotonic dystrophy.
Our RNA-targeting systems effectively destroyed the toxic RNA repeats and reversed the vast majority of the downstream splicing changes. In addition, in a mouse model of DM1 we observed positive effects on myotonia, one of the hallmark symptoms in DM1 patients. Taken together, this data shows that our RNA-targeting systems are able to correct not only the core genetic cause of the disease but also important downstream molecular and functional changes that occur in patients.
DDN: What will the next step be for continuing research with these RNA Cas systems?
Bishop & Burns: At Locana, our research with our RNA-targeting systems is focused on our core pipeline programs in CAG repeat diseases, myotonic dystrophy type 1, and C9orf72-related ALS and FTD [frontotemporal dementia]. We are currently conducting experiments to identify lead candidates, and will be moving programs toward IND-enabling studies that are required prior to conducting clinical trials.
In addition to this work progressing our pipeline towards the clinic, we are also busy working on expanding our platform capabilities. This includes additional work on a number of splicing diseases, translational enhancement proof of concept, and expanding our repertoire for enabling the destroy and replace approach which can be applied to a number of genetic diseases.
DDN: Is there anything else that you would like to tell DDN? What do you find most exciting about this research?
Bishop & Burns: What makes this work so exciting is that we are creating a new class of genetic medicines by combining two validated approaches for treating diseases. We are using gene therapy for delivery to cells and systems that are engineered to target and manipulate dysfunctional RNA (a validated disease target) involved in human disease. There are many diseases for which this is likely the best option for permanent treatment without changing a cell’s DNA.
We believe we have the broadest technology portfolio for developing systems that can modulate RNA through different mechanisms. Ultimately, as we learn more about these new genetic medicine systems, we see expanding their use beyond genetic diseases to non-genetic diseases as well.