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.

Targeted Active Gene Editing (TAGE)
Spotlight Therapeutics is developing a new class of biologics called Targeted Active Gene Editing (TAGE) agents. Each TAGE agent consists of a cell-targeting domain, a cell-penetrating peptide (CPP), and a Cas protein loaded with a guide RNA (gRNA). In this image, a TAGE agent targets immune cells in vivo to perform gene editing, reprogram the tumor microenvironment, and potentiate a systemic antitumor response.

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.”

Excision BioTherapeutics developed a novel approach to CRISPR-based therapies
To fight viral infectious diseases, Excision BioTherapeutics has developed a novel approach to CRISPR-based therapies. The company notes that with standard gene editing approaches (left panel), which execute single base cuts or small cleavages, viral escape may occur, precluding cures. To prevent viral escape and realize cures, Excision has developed an alternative approach (right panel), one that uses two or more guide RNAs (gRNAs) to excise large sections of viral DNA.

To meet this challenge, Excision is applying CRISPR in a different way, using technology licensed from Temple, as well as technology licensed from the University of California, Berkeley. (The former technology originated in the laboratory of Kamel Khalili, PhD; the latter, Doudna’s laboratory.) Essentially, Excision is using CRISPR editors to remove viral DNA to return the human genome to normal or nearly normal.

The twist is that Excision makes more than one cut within the viral genome, using two or more guide RNAs. That allows the removal of some or all of the viral sequence so that there’s not enough left to replicate. One of the advantages of this approach is that targeting unique viral sequences minimizes the chances of off-target effects.

The company’s approach may be used on any virus that enters the host cell’s nucleus. Excision’s pipeline includes therapeutics against HIV, hepatitis B, John Cunningham virus (JCV), herpes viruses—all preclinical. A therapeutic against SARS-CoV-2 is also in development. Excision published a study in Nature Communications in 2019 describing an antiviral therapeutic that eliminated HIV from the genomes of living animals, achieving functional cures.

The company expects that its HIV program will enter Phase I/II human clinical trials in 2021. “For the most part,” Dornbusch observes, “the difference between our programs is the sequence of guide RNAs.”

Harnessing homology-directed repair

CRISPR systems that execute double-strand breaks in DNA often do so with great specificity. But even perfectly placed double-strand breaks must be repaired. To date, most CRISPR systems accomplish repairs via non-homologous end joining (NHEJ), an error-prone pathway that is useful when gene knockouts are desired. In general, however, knockouts don’t achieve medical ends. According to Josh Lehrer, MD, CEO of Graphite Bio, knockouts are better at disrupting genes than correcting them. Just cutting DNA to knockout a gene, he says, won’t “correct the underlying mutation causing disease.”

Using technology developed at Stanford University by Matthew Porteus, MD, PhD, GraphiteBio is harnessing a natural repair process of cells called homology-directed repair. In homology-directed repair, when there is DNA damage, the cellular machinery “basically grabs the sister chromosome and lines up the similar sequences,” explains Lehrer, “and then it pastes in any missing nucleotides by copying them over from the sister chromosome, which serves as a template.”

Graphite Bio’s innovation is to use a CRISPR system to trick the cell. The system provides a repair template to correct the disease-causing mutation. The company’s most advanced program is for sickle-cell disease, which is caused by a mutation that occurs in the β-globin gene and leads to abnormal hemoglobin.

Starting with stem cells harvested from the patient, Graphite Bio’s CRISPR technology cuts the β-globin gene near the point mutation that causes sickle-cell disease. Then a template is delivered using the virus AAV6. The cell copies over the change and corrects the point mutation, allowing the production of normal hemoglobin. In the patient, that transforms sickle-cell disease to something like sickle-cell trait—which is a normal condition of having one sickle-cell allele and one normal allele.

The clamshell nuclease

Not every pioneering gene editing company relies on CRISPR technology. For example, Precision BioSciences is harnessing a nuclease derived from I-CreI. In nature, I-CreI is encoded by a parasitic gene found in algae. There, the gene’s sole purpose is to insert itself into the genome of algae and propagate its existence.

In Precision BioSciences’ ARCUS gene editing platform, I-CreI is no longer selfish, but selfless. The genomic interloper has been tamed by Precision’s co-founders, Derek Jantz, PhD, and Jeff Smith, PhD, who are now the company’s chief scientific officer and chief technology officer, respectively. “It was a technology that had already evolved for the specific purpose that they were looking to achieve,” observes Chris Heery, MD, Precision’s chief medical officer.

Heery says that the nuclease is like a clamshell. “When the gene sequence is bound on either side of the clamshell, that’s when the clamshell clamps down, and that’s when the cutting occurs.” That binding is necessary for the protein to cut the DNA, differentiating ARCUS from other gene editing technologies.

Using directed evolution and in silico design, Precision’s founding scientists have engineered I-CreI to bind to a section of 20–22 base pairs with high specificity without touching any other part of the genome. On top of that, the small size of ARCUS means it can be bundled into a very small delivery vector more efficiently than CRISPR-Cas9 or most other gene editing tools.

Precision’s lead chimeric antigen receptor T-cell product is targeted at CD19. According to a clinical study presented by the company in 2019, the product was able to knock out the T-cell receptor to avoid graft vs. host disease. The study also showed that the cells could expand against a target antigen. The company is now working on getting the cells to last longer in patients. It also has other programs in development with a similar platform, but different targets—CD20 and B-cell maturation antigen.

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