New companies are exploiting next-generation CRISPR technologies and powerful

CRISPR alternatives to realize new therapeutic approaches

Just eight years after publishing their groundbreaking discovery on gene editing, Emmanuelle Charpentier, PhD, and Jennifer A. Doudna, PhD, have been awarded the Nobel Prize in Chemistry. By showing that the Cas9 endonuclease from Streptococcus pyogenes could be used like a tiny pair of scissors to selectively cut targeted stretches of DNA, Charpentier and Doudna introduced a new kind of gene editing, one that is readily reprogrammable.

The new approach was dubbed CRISPR in a nod to the Clusters of Regularly Interspaced Short Palindromic Repeats, the DNA regions that are part of a primitive immune system in bacteria. Whereas bacteria rely on CRISPR to encode genetic sequences corresponding to phage DNA (that is, sequences that give rise to RNA molecules that complex with Cas9 and guide it to complementary phage DNA), scientists may use guide RNA of their choosing.

This is the power behind our human-engineered CRISPR-based systems. They have been designed for various applications, including “knockouts” and “knock ins,” both of which rely on the cell’s DNA repair mechanisms. (Knock ins, for example, may accomplish DNA insertions if DNA repair mechanisms incorporate donor DNA.)

Commercial CRISPR-based gene editing systems soon appeared, and they continue to be refined by companies such as Editas Medicine, CRISPR Therapeutics, and Intellia Therapeutics. The first forays into the field of CRISPR medicines aimed at relatively soft targets such as monogenetic diseases. Subsequently, CRISPR and other gene editing technologies have been used to launch more ambitious campaigns, reinforced by ranks of companies that are eager to advance into new territory.

New companies are blurring the lines between gene editing, gene therapy, cell therapy, and small-molecule drug development while they tack on new functionality and target new diseases. Five of these companies are discussed in this article. All are adding to the gene editing arsenal. All are deploying novel therapeutics.

Bespoke molecules fit for purpose

Spotlight Therapeutics is focused on tackling gene editing delivery challenges head-on. Currently, gene editing is done either through cell therapy, which can be complex and time consuming and is mostly customized for each patient, or by viral or nanoparticle delivery systems, which struggle to reach many tissues. Other problems include side effects, payload size limitations, and preexisting immunity to adeno-associated viral (AAV) vectors.

“[Delivery challenges] can exclude about half the patients,” says Spotlight president and CEO Mary Haak-Frendscho, PhD. “We’re working to solve those challenges by taking a pure biologics approach.”

Spotlight’s Targeted Active Gene Editing (TAGE) platform, which delivers CRISPR molecules to selected cell types in vivo, is designed to expand gene editing applications, increase safety, and provide broad access to patients.

TAGE ribonucleoproteins have a modular design with a cell-targeting domain linked to a Cas protein loaded with guide RNA. The cell-targeting module includes molecules that are either cell-penetrating peptides (CPPs), antibodies, antibody derivatives, or ligands. Some molecules use a combination of those elements to achieve cell selectivity with biological function.

For example, according to Haak-Frendscho, an antibody can be used to target a specific cell type, such as a hematopoietic stem cell, and then that antibody is linked to a CPP that can facilitate endosomal escape and translocation to the nucleus for the nuclease and guide RNA. And all those components can be swapped with others. “It allows us to make bespoke molecules fit for purpose,” asserts Haak-Frendscho.

Spotlight has products at the preclinical stage in three therapeutic areas—hemoglobinopathies, ocular diseases, and solid tumors. For hemoglobinopathies, the strategy is to bypass the harsh conditioning stage of treatment used in standard cell therapies and to edit the cells in situ. In ocular diseases, the goal is to avoid the safety challenges of viral or nanoparticle delivery vehicles. And in its work against solid tumors, Spotlight is developing a first-in-class candidate.

“We envision reprogramming the tumor microenvironment,” declares Haak-Frendsho. “Our aim is not to directly kill tumor cells. We want to reprogram the immune contexture of solid tumors to make them more immunologically hot and potentiate a systemic antitumor immune response. That will allow them to respond better to what’s now the standard of care—immune checkpoint blockade.”

Screening for synthetic lethal pairs

Synthetic lethal pairs are genetic perturbations that are harmless alone but lethal in combination. They were first described in fruit flies nearly 100 years ago by the geneticist Calvin Bridges, PhD, and have been an important tool for mapping genetic interaction networks. As well, synthetic lethal pairs can be a powerful mechanism for some drug combinations, particularly in the field of cancer.

Poly ADP ribose polymerase (PARP) inhibitors are the first clinically approved drugs that exploit synthetic lethality between the BRCA1/BRCA2 and PARP genes. However, few other synthetic lethal pairs have been discovered or exploited. Tango Therapeutics was founded in 2017 with the goal of targeting previously undruggable cancer by using CRISPR to discover new synthetic lethal pairs.

Unlike many of the startups based on CRISPR, Tango is not developing CRISPR therapeutics. It is using CRISPR as a target discovery tool, and then using standard small-molecule drug discovery methods on those targets.

“We put 5,000 genes at a time into CRISPR vectors and introduce that whole library of 5,000 genes at once into a single cell line containing a genetic mutation profile found in a particular type or subtype of cancer,” says Tango CEO Barbara Weber, MD. Using CRISPR, Tango is able to knock out each gene individually within a pool of cells in culture and determine which gene knockouts cause cell death.

Most of the modified cells will survive, but some will die. In a slain cell, the original mutation and the loss of the gene disabled by CRISPR contribute to a synthetic lethal pair. Tango now has five targets in its drug discovery pipeline and is developing molecules against them, with first-in-human dosing expected in the first half of 2022, according to Weber.

Weber says that several companies are using CRISPR to discover novel oncology targets, but because there are many potential targets that await discovery, it is unlikely that Tango will often find itself competing head to head with other companies. Still, coincidentally, Tango does have a direct competitor for one or two of its targets. “There’s probably in the range of 500 novel synthetic pairs out there to be discovered,” Weber notes.

Deleting viral infectious diseases

Excision BioTherapeutics is using CRISPR to cure viral infectious diseases—a goal that has been attempted before. “[We employ] CRISPR in a way that’s slightly different than the rest of the global community in CRISPR and gene editing more broadly,” says Daniel Dornbusch, the company’s CEO. “The challenge has been that viruses evolve around any previous attempt to deactivate them.”