Korro Bio, Graphite Bio and more biotech startups funded in Sept 2020

By Jonathan Algoo and Richard Murphey

Forty biotech startups raised $2.7B in September 2020. In this post, we dig into the tech of two of these companies: Korro Bio and Graphite Bio.

If you are interested in other biotech startups that raised in September 2020, below is a spreadsheet with details on many of the companies that raised money, including their pipeline (targets, stage and indications), team, patents and publications.

You can view the list in-line, or contact us if you'd like a copy of the excel.

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Startup highlight: Korro Bio, base editing for mRNA

Korro Bio, a company that aims to treat genetic diseases by editing mRNA, raised $92M in its most recent Series A round. Wu Capital led the round, with participation from several crossover investors as well as founding investors Atlas Venture and New Enterprise Associates. The company, led by serial biotech entrepreneur Nessan Bermingham, builds on science developed by Joshua Rosenthal of the University of Chicago’s Marine Biological Laboratory.

Korro Bio’s RNA editing approach relies on directing enzymes of the ADAR (Adenosine Deaminase Acting on RNA) family to target genes with a synthetic oligonucleotide. This family, which is naturally found in all multicellular organisms, catalyzes the conversion of the nucleoside Adenosine to Inosine at particular spots on an mRNA strand. Inosine is structurally similar to Guanosine, and translation enzymes recognize Inosine as a Guanosine nucleoside. Thus ADAR effectively converts A to G on mRNA. Combining a guide oligonucleotide with a nucleotide editor enables targeted mRNA modification, which could be useful to treat diseases caused by G-to-A mutations.

In humans, the ADAR2 variant is composed of three domains: two, called double-stranded RNA binding motifs (DSRBMs), are responsible for identifying locations on an mRNA strand to bind to, and a third catalytic domain performs the chemical transformation of Adenosine. The RNA binding motifs normally locate binding sites by identifying surrounding secondary structures (such as loops or helices) on RNA strands.

There are two schools of thought surrounding the use of ADAR enzymes for RNA editing. Both approaches use a guide RNA that contains a complementary strand which binds to the mRNA sequence of interest. The first is to attach motifs to the guide RNA which resemble the secondary structures that the ADAR enzyme would normally bind to. This RNA would then recruit endogenous ADAR enzymes to the location of interest, where it would catalyze the Adenosine to Inosine conversion. This is, from what I can tell, Korro Bio’s mechanism. This approach can benefit from some of the lessons learned through the development of other antisense oligonucleotide therapies, such as deliverability, manufacturability, and reducing tox ¹.

Image source: Korro Bio

The other approach is to re-engineer the ADAR2 enzyme to avoid using the DSRBM motifs entirely. Joshua Rosenthal, the Scientific Founder of Korro Bio, was heavily involved in developing this approach, so it’s possible Korro or another startup he is involved with could use it in the future. The method Rosenthal developed uses a different targeting mechanism to recruit the catalytic domain to an mRNA sequence, the λ-phage N protein-boxB interaction. In this interaction, the N protein recognizes and binds to the boxB secondary structure, an RNA hairpin. Rosenthal’s group designed their targeting system in two parts: a recombinant protein composed of the N protein connected to the catalytic domain of the ADAR2 enzyme, and a guide RNA which is attached to the boxB hairpin structure. After the guide RNA binds to the sequence of interest, the boxB hairpin recruits the N-protein/catalytic domain complex there. The catalytic domain then transforms nearby Adenosine nucleosides that are bound to the guide RNA strand into Inosine nucleosides².

Image source: Rosenthal lab website.

Korro’s approach is similar to "base editing" technology developed by David Liu’s lab and licensed to Beam Therapeutics. Base editing enables A-to-G edits as well as other edits. A key difference between base editing and Korro's mRNA editing approach is that base editing edits DNA (Korro edits RNA) using CRISPR technology. Advantages of DNA base editing vs. Korro’s approach the include ability to perform more types of edits than A to G (Korro currently only does A to G) and the ability to make permanent edits (this can also be a disadvantage depending on the application). Disadvantages of base editing include a more complex payload (a base editing therapy includes a guide RNA, a deactivated CRISPR enzyme, and a deaminase), which would make base editing therapies more complex to manufacture and deliver. Both base editing and Korro’s tech enjoy advantages over traditional CRISPR editing as they do not require double-stranded DNA breaks and homology directed repair (HDR) to edit DNA. This enables higher editing efficiency and avoids unwanted consequences of HDR, including insertions and deletions or larger-scale genomic rearrangements.

The fact that RNA edits are temporary, while DNA editing is permanent, can be an advantage or disadvantage. A temporary approach is advantageous for certain conditions that aren’t permanent in nature, such as pain relief. Any off-target effects are temporary as well, which can contribute to a better safety profile for potential therapeutics.

At the time of funding, the company had studied the technology in several in vitro as well as in vivo models. The company states that it will use this funding to advance its first candidate to IND filing and expand its product pipeline.

For all oligonucleotide therapies (including gene therapy and gene editing), delivery is a challenge. Korro is targeting the liver, eye and central nervous system, three areas where deliverability of these medicines is better.

Given Korro's crossover syndicate and hot IPO market, Korro could be an attractive IPO candidate in the next year or so. Beam is arguably the closest comp. Beam went public in February 2020 and had yet to initiate IND-enabling studies at the time of IPO. Beam is up 115% over its IPO price of $17 / share and trades at a $2B valuation as of early November 2020.

Startup highlight: Graphite Bio, gene editing for sickle cell disease

Graphite Biosciences raised $45M in its recent Series A round from Versant Ventures. The company aims to develop therapies using its gene-editing platform based on tech developed in the Porteus lab at Stanford University, and is targeting sickle cell disease for its first therapeutic.

CRISPR/Cas9 is a protein complex which, when led by a guide RNA, cuts DNA at a particular location. This leaves a double stranded break at the target location. One of the mechanisms the cell uses to fix this is homologous recombination, a form of homology directed repair (HDR), in which enzymes identify the base located at the break in a nearby DNA strand, incorporate it into the gap, and recombine the strands. This mechanism can be exploited to insert genes of interest at a specific site.

One issue with HDR is low efficiency. The Porteus lab identified and created a number of different methods to make this process more effective at integrating DNA into human cells with high efficiency. These include methods of delivery: delivering a guide RNA as part of a ribonucleoprotein complex instead of as mRNA, and delivering donor DNA using a virus called rAAV6. The group also developed an analytical method using FACS (fluorescence activated cell sorting) and a magnetic bead technology in which they were able to separate cells that had been edited from those that haven’t been with a high effectiveness. Achieving a high separation is particularly important for therapies that would use stem cell transplantation, as edited cells will be competing with unedited ones after transplantation ³. In addition, the group was able to identify a Cas9 variant which naturally has lower off-target effects, which occurs when the enzyme creates a double stranded break at an undesired location ⁴.

These innovations, which lead to a higher integration efficiency (greater than 50%)³ coupled with lower off-target effects, collectively form the basis for a promising value proposition. They allow for the precise editing of nucleotides within individual genes, which would open the door to a number of potential therapeutics. For sickle cell anemia, Graphite Bio’s gene therapy platform permits editing of the abnormal sickle-cell gene itself, as opposed to other CRISPR-based therapies currently in clinical trials which knock out the regulatory gene that decreases fetal hemoglobin production. These other therapies restore a working version of hemoglobin, but do not correct the gene which results in sickle cell in the first place.

Sources

¹ Montiel-Gonzalez, M. F., Diaz Quiroz, J. F., & Rosenthal, J. J. C. (2019). Current strategies for Site-Directed RNA Editing using ADARs. Methods, 156, 16–24. https://doi.org/10.1016/j.ymeth.2018.11.016

² Montiel-González, M. F., Vallecillo-Viejo, I. C., & Rosenthal, J. J. C. (2016). An efficient system for selectively altering genetic information within mRNAs. Nucleic Acids Research, 44(21), e157. https://doi.org/10.1093/nar/gkw738

³ Dever, D. P. et al. (2016). CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature, 539(7629), 384–389. https://doi.org/10.1038/nature20134

⁴ Vakulskas, C. et al. (2018). A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nature Medicine, 24(8), 1216–1224. https://doi.org/10.1038/s41591-018-0137-0