Welcome to Biotechr

Biotechr is written by Dr. Robert Kruse (@RobertLKruse), who holds a PhD and is currently completing his MD. His research work focused on infectious disease and immunology. This blog is focused on analyzing the latest developments in biotechnologies being developed in academia and industry, with a particular focus on biomedical therapeutics. I hope that the posts are interesting and useful, and hope you join in the discussion with guest posts on the site!

Disclaimer: The thoughts on this blog are not intended as any investment advice regarding any companies that might be discussed, and represent my opinion and not the opinions of my employer. This site is not designed to and does not provide medical advice, professional diagnosis, opinion, treatment or services to you or to any other individual.

Saturday, April 30, 2016

Treating TTR Amyloidosis: Introducing CRISPR

by Robert Kruse

Among the many different programs announced by Intellia Therapeutics ($NTLA) during their disclosures to announce their IPO, one particularly caught our eye at Biotechr. It is a favorite of the biotech world, being targeted by many different companies looking for orphan disease rapid approval and high reimbursement rates with little to no competition. Will the introduction of a CRISPR-Cas9 player lead to the demise of these competitors? Let us delve into the disease and the technologies to find out.

The rare disease of interest is Transthyretin-related (TTR) hereditary amyloidosis, also called Familial amyloid polyneuropathy, which is an autosomal dominant disease characterized by a mutation in the TTR gene (most common being V30M). This mutation leads to the formation of protein aggregates (hence amyloidosis), which are deposited in various organs across the body leading to various toxicities (pain, paresthesia, muscular weakness and autonomic dysfunction) and ultimately death from cardiac and kidney toxicities.

The TTR protein is mostly produced in the liver, and the current standard of care is a liver transplant, which can cure the disease by removing the source of the protein being produced. The downside is the morbidity risk from the liver transplant procedure, the limited availability of livers for transplants, and the need for immunosuppression. Notably, is it the complete transplant of the liver that is necessary, since any remaining sub-fraction of mutated cells can continue producing the TTR proteins causing more aggregates to be formed. That is why it's an autosomal dominant disease after all, since one gene and one mutation causes the effect over the presence of the normal gene. One can imagine that only replacing a portion of the liver will lead to a slower disease course, the extent to which biomedical science simply does not know right now.

This last fact is particularly important when considering the current therapeutic approaches in clinical trials right now. Several RNA knockdown companies have candidates in clinical trials to treat TTR amyloidosis. The players include Alnylam ($ALNY) and Ionis Pharma ($IONS). Alnylam is using the LNP delivery technology developed by Arbutus (formerly Tekmira) ($ABUS), with some royalties from the success of the drug flowing the Arbutus. Alnylam has reported efficacy in clinical trials of knocking down TTR up to 86.8% in patients, with the caveat of a significant number of patients developing infusion-related reactions. Ionis Pharma is exploring a similar strategy, but by using antisense oligonucleotides, that bind to mRNA and cause degradation by RNase H mechanisms (DNA-RNA binding in cytoplasm). The product can be delivered subcutaneously as a benefit, and optimized delivery can occur through conjugation to a GalNAc sugar to the oligonucleotide. Ionis is currently in Phase III trials with an unconjugated ASO, with the trial projecting to be concluded in 2017. Previous studies showed a maximum knockdown of 92% in trials. FDA approval seems likely, but there is the unknown of the long term benefit of the current knockdown levels. Alnylam is also exploring the use of GalNAc conjugates to siRNA and subcutaneous delivery to improve liver uptake for its TTR cardiomyopathy indication.

Into this picture enters Intellia and CRISPR technology. CRISPR allows the permanent cleaving of DNA to inactivate the gene, stopping protein production. In this manner, Intellia could pursue an approach where after a single injection, permanent knockdown of TTR mutants could be achieved, versus the lifelong dosing regimens of the RNA knockdown therapies. This represents a significant advantage that can not be underestimated from the perspective of patient ease and use. There have been no academic papers published on the topic, so the discussion here is all speculative. We don't have data on mouse models to know how much CRISPR could truly alleviate the pathology of the disease. That said, we can already imagine the clinical translation of the approach fairly easily. Given the success of LNP technology in targeting the liver for siRNA applications, the tech can also be applied for the delivery of Cas9 mRNA and gRNA, as has been previously published for Type I Tyrosinemia. Indeed, Intellia's SEC documents reveal a plan to use LNP technology for liver delivery. The transient nature of mRNA and gRNA delivery will alleviate concerns about continued off target editing by Cas9, and any potential immune reactions against Cas9 as well. What we don't know is how efficiently can Cas9 cleave the TTR locus within that 2-3 day window of time, and how much serum knockdown will this lead to eventually. Certainly, with efficient delivery to almost every hepatocyte combined with efficient editing, one could achieve a correction of almost every hepatocyte, replicating the liver transplant therapy in efficacy and representing a real cure.

We don't know if that efficiency will be achieved, but at least the upside of Intellia's approach is there. Autosomal dominant disorders appear to be a great fit for CRISPR therapy, given that the toxic gene must be removed in order for a cure to happen. A concern about the CRISPR approach though, is how they will design the guide RNA's to target TTR. The easiest design would remove both the normal WT allele and the mutant TTR allele, but the normal TTR protein serves an important function in carrying thyroid hormone, thyroxine (T4) around the body, so it might not be able to be removed. It was noteworthy that the current RNA knockdown approaches do target the WT transcript as well. Added complexity could be to cut out the mutant sequence with two gRNA's and try to have homologous recombination with the WT allele, although this process would seemingly cut the normal allele as well. These questions still need to be addressed, likely in academic papers to be published in the near future.

Looking at realistic timelines, Intellia hasn't even announced when a prospective TTR trial will begin. It is likely that they are only going through mouse experiments right now. Their competitor Editas Medicine ($EDIT) could certainly also get into the TTR game as well. If approval is 5-6 years away, in the absolute best case scenario, for Intellia in TTR, that should give Ionis and Alnylam some time with approved drugs on the market to recoup money for investors. Longer term data from Ionis and Alnylam will also provide valuable clinical insights into how TTR knockdown can alleviate symptoms in these patients, setting the benchmarks for future efficacy goals for CRISPR modalities that appear to have potential to replace the RNA knockdown technologies.


  1. Great article, thanks. I'm just a layman trying to learn about the latest in biotech and your article helped put some things in perspective. Wikipedia is also a great help.

    Dumb questions, if I may: Does LNP get the payload all the way into the nucleus? I thought that was more difficult than just into the cell soma. How can LNP get big molecules into the nucleus, like mRNA?

    Also, I have read about attempts to modify genes or transcription / translation, whichever is correct, using exons, codons and introns, and reading frames. But most companies and other research doesn't talk in these terms. They talk instead about gene splicing, DNA and various RNAs, etc. Is there a different way to approach gene modification using exons, etc or is that just theoretical?

    I can "see" how guide RNAs might be able to target genes for deletion, but I don't see how new genetic sequences can be added. Does it just grab some nucleic acid floating around? How does it find the right one that is meant to be inserted. Seems like it could insert the wrong stuff.

    Thanks, Kevin

  2. LNP as a delivery vehicle only gets the payload across the endosomal membrane (which is formed from the cell membrane) and into the cytoplasm. It doesn't deliver to the nucleus. The key is that mRNA or siRNA or a protein only needs to work in the cytoplasm to do its function. mRNA can be read by ribosomes in the cytoplasm directly. The protein may have an appended nuclear localization sequence as necessary for delivery into the nucleus. Your point comes up with DNA delivery, since that needs to go to the nucleus, but there are no active or efficient transport processes for that. Thus, non-viral delivery has work to do there.

    For the second question, exons, codons and introns, and reading frames are just more specific terms about the region or function of the gene that you are trying to manipulate by introducing new DNA. It's just more specific terminology.

    New genetic sequences are added via a DNA repair process called Homologous Recombination. The body also has another route called non-homologous end joining. I would suggest looking up wikipedia articles on those topics. You hit on the right point, since this is a major bottleneck in CRISPR therapies.