The Needle Issue #16


Welcome to The Needle, a newsletter from Haystack Science to help you navigate the latest translational research, with a roundup of the latest news on preclinical biotech startups from around the world.

This issue, we take a look at epigenome editing, which has its first products entering the clinic and continues to bear fruit with some recent compelling preclinical efficacy in metabolic and neurodevelopmental disorders. In our survey of the translational literature, startups Advanced Biodesign, GlyTR Therapeutics, Epigenic Therapeutics, and Regel Therapeutics all disclosed notable advances. In other startup news, Sanofi tops up its venture fund and preclinical financings continue to proceed at a healthy clip, with a bumper crop of 15 financings since our last issue. In contrast, publicly disclosed preclinical licensing deals remain at a trickle. Any financings or collaborations we missed, let us know (info@haystacksci.com).

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Commercial interest in targeted epigenetic therapies — agents that target specific genes without altering bases in their sequence or causing double-strand breaks or even single nicks in the DNA — continues to grow, as underscored by the latest financing announced by Epigenic Therapies. The unique selectivity and specificity of targeted epigenetic therapeutics offers compelling advantages over small-molecule epigenetic drugs, which target a specific epigenetic reader, writer or eraser, but affect genes across the genome and affect many diverse tissues, leading to narrow therapeutic windows that make them difficult to develop for conditions outside of cancer.

Today, Haystack is aware of at least eight private companies (nChroma Bio (resulting from a merger of Chroma and Nvelop), Encoded Therapeutics, Epigenic Therapeutics, Epitor Therapeutics, Moonwalk Bio, Navega Therapeutics, Regel Therapeutics, and Tune Therapeutics), and two public companies (Modalis Therapeutics and Sangamo Therapeutics) that are pursuing the targeted epigenetic approach against disease (let us know if you know of any others). Another company, Flagship Pioneering’s Omega Therapeutics, went out of business in August after filing for bankruptcy in February. A smaller set of companies are also pursuing targeted epigenetic therapies against RNA modifications.

All of these therapies are designed around an alluring set of simple principles: take a gene-specific DNA-binding domain — zinc-finger proteins (ZFPs), ‘dead’ Cas9 (dCas9) with mutations in its RuvC and HNH endonuclease domains, or transcription activator-like effectors (TALEs) — and tether it via an amino acid linker to an enzymatic effector module. This effector is either an enzyme that directly places or removes a specific epigenetic modification (e.g., TET, histone demethylases or the histone acetyltransferase p300) or a transcriptional activator (e.g., VP16) or repressor (e.g., KRAB).

A particularly compelling application for such treatments is genetic disorders of haploinsufficiency (like Dravet’s) or imprinting disorders (like Angelman’s or Prader Willi). There are also many of these diseases where the therapeutic genes would be too large (>4.0 kb) for a traditional AAV gene-therapy approach; in contrast, epigenetic editing machinery can be packaged into an AAV vector.

Currently, the diseases being pursued by companies include hepatitis B, hypercholesterolemia, epilepsies (SCN1A (Dravet syndrome) and SCN2A), chronic pain, and muscular dystrophies. Those with the most advanced programs are Encoded’s AAV-9 intrathecally delivered SCN1A-targeting zinc finger protein linked to a VP16 activation domain in phase 1 testing for Dravet and Sangamo’s AAV- STAC-BBB-delivered SCN9A-targeting zinc finger protein linked to a KRAB repressor domainin a phase 1/2 trial for patients with chronic pain. In this context, two papers published in the past couple of weeks represent important proofs of the efficacy of targeted epigenetic therapies.

In a first paper published in Nature, the groups of Kevin Bender and Nadav Ahituv at UCSF (scientific co-founders of Regel Therapeutics) sought to test a targeted epigenetic therapy in patients with SCN2A mutations that exhibit decreased NaV1.2 function. These individuals have impaired action potentials, synaptic transmission and manifest diverse neurological symptoms and seizures, with few therapeutic options, beyond symptomatic anti-seizure medications that have a dizzying range of debilitating side effects.

The UCSF teams leveraged conditional genetic knock-in technolgoy or CRISPRa technology — an AAV-delivered SCN2A-promoter-targeting dCas9 fused to a VP16 activator domain — to upregulate transcription of the SCN2A gene. Using either approach, they were able to boost transcript levels from the healthy SCN2A allele, ameliorating electrophsiological deficits and chemical-induced seizure activity in Scn2a+/− mouse models. Importantly, these effects were seen in adolescent mice, which conventionally have been thought to be too old to respond to treatment. This suggests that rescue of normal dendritic excitability with epigenetic agents at later stages of life might be capable of restoring neuronal function, with implications for patients.

In a separate set of experiments, the authors showed that their epigenetic approach was able to rescue neurophysiological activity in haploinsufficient neuron-like cells from SCN2A-knockout human embryonic stem cells. This cross-species reproducibility provides further confidence that CRISPRa-mediated upregulation could be translated into human treatments.

In a second paper in Nature Biotechnology, a team from Epigenic Therapeutics (Shanghai, China) describes the design and validation of optimized epigenetic regulators (EpiRegs) to silence genes in a precise, durable way without altering genomic DNA. Epigen’s Shaoshai Mao and his collaborators at the Chinese Academy of Sciences and the First Affiliated Hospital of Anhui Medical University tested combinations of TALE- and dCas9-based systems, systematically optimizing effector domains and fusion architectures, looking for effective regulators of gene expression. The best-performing variant, EpiReg-T (a TALE-based system, which eliminates the need for a guide RNA), achieved 98% silencing of target genes in mice, substantially outperforming dCas9-based versions.

Using lipid nanoparticles (LNPs) for delivery, a single administration of EpiReg-T in macaques induced long-term repression of the PCSK9 gene, which encodes a validated target for the treatment of hypercholesterolemia. EpiReg-T reduced PCSK9 expression by >90% and LDL-cholesterol by about 60%, with effects persisting for nearly a year (343 days).

Mechanistically, the team used whole-genome bisulfite sequencing and cleavage under targets and tagmentation (CUT&Tag) to show that EpiReg-T induced stable DNA methylation and repressive histone marks at the PCSK9 promoter. The silencing persisted even after liver regeneration and could be reversed by targeted epigenetic activation. Multiomic analysis in mice, macaques and human hepatocytes confirmed high specificity of the manipulation and minimal off-target effects. Overall, these finding, as well as similar results reported in April by Chroma Medicine, establish epigenetic editing as a promising therapeutic platform for durable and reversible gene silencing.

Overall, targeted epigenetic therapies offer clear safety advantages over small molecules that indiscriminately target all genes under the control of an epigenetic eraser or writer enzymes. They avoid the potential risks associated with creating single- or double-strand DNA breaks associated with CRISPR/Cas9 gene, base or prime editing therapies. And they avoid the insertional mutagenesis risks associated with traditional viral gene therapies. What’s more, in applications requiring gene upregulation in haploinsufficient disease, these approaches maintain the endogenous regulatory context of the functional allele. This is in stark contrast to traditional gene-therapy replacement approaches, where overexpression of an introduced therapeutic gene can often lead to toxicities and immunogenecity.

Of course, questions still linger around the persistence of the changes elicited by these epigenetic agents. Will they persist in patients for long periods — for years or even decades? If they can, then epigenetic therapy may offer compliance advantages over small molecules, antibodies, ASOs or even siRNAs, which have treatment durations of six months or less.

Like all genetic medicines, though, delivery remains the key headache. Thus far, AAV vectors, lipid nanoparticles or ribonucleoproteins (RNP) have all been explored to deliver epigenetic therapies (with some evidence that RNPs might have advantages because they can result in higher dCas9 dosages within target cells). For AAV vectors, the fact that targeted epigenetic therapy might only need to be given once might be an advantage in terms of immunogenicity/neutralization concerns against the vector.

A broader point is that the safety profile of targeted epigenetic editors may offer advantages if AAV vectors are used as delivery vehicles: if the epigenetic agents themselves can be delivered at high dosage (given their intrinsic favorable safety profile and presumed maximal tolerated dose), perhaps AAV vector dosages could be lower than current practice. With many current gene therapies requiring dosages of 1013 or more viral particles/kg in patients, it is increasingly becoming clear that unacceptable liver toxicities arise from the virus at these levels in clinical studies. It will be interesting to follow this space as more agents enter human testing.

Translational papers: Best of the rest

Target biology

USP10/GSK3B-mediated inhibition of PTEN drives resistance to PI3K inhibitors in breast cancer | JCI

ZDHHC11-mediated palmitoylation is impaired in human osteoarthritis and alleviates chondrocyte senescence in knockout mouse models | Nature Aging

Carboxypeptidase D deficiency identified as defect in hearing-loss patients rescuable with arginine supplementation | JCI

Autoimmune response to C9orf72 protein in amyotrophic lateral sclerosis | Nature

ANTXR1 blockade enhances cardiac function in preclinical models of heart failure with preserved ejection fraction | Nature Cardiovascular Research

S-acyl transferase ZDHHC13 modulates tumor microenvironment interactions to suppress metastasis in melanoma models | JCI

Proof-of-concept studies

Advanced Biodesign identifies small-molecule covalent inhibitor of aldehyde dehydrogenase 1A isoform in xenograft and syngeneic mouse models of triple negative breast cancer | Cell Chemical Biology

Identification of a USP5 small-molecule inhibitor therapy for AML with t(8;21) chromosomal translocation through ubiquitin-mediated AML1-ETO degradation in patient-derived xenografts | Science Translational Medicine

AAV9 encoding glycophagy gene Gabarapl1 reverses diabetic heart disease in human engineered cardiac tissue and type 2 diabetic mice | Nature Cardiovascular Research

Identification of small-molecule histone decrotonylase inhibitor that causes epigenetic upregulation of epithelial transcription factor HNF1A, rescuing EGFR-tyrosine kinase inhibitor resistance in mouse models of lung cancer | PNAS

Small-molecule inhibitor of ATPase VCP, identified by CRISPR screens as a therapeutic vulnerability in patient derived cholangiocarcinoma organoids | PNAS

Oral inhibitor of interaction of S-nitrosoglutathione reductase and transcription factor ETS-related gene (ERG) ameliorates diabetic vascular complications in limbs of rodent models of diabetes | Science Translational Medicine

A TGF-βR/IL-2R immunomodulatory fusion protein transforms immunosuppression into T cell activation to enhance adoptive T cell therapy | PNAS

Ocular delivery of lipid nanoparticles-formulated mRNA encoding lanosterol synthase ameliorates cataract in rats | Nature Communications

Five-year analysis showing efficacy and safety of a bidirectional AAV hexosaminidase A and B gene therapy in Tay-Sachs sheep | JCI

Targeted clearance of extracellular Tau using aptamer-armed monocytes alleviates neuroinflammation in mice with Alzheimer’s disease | Nature Biomedical Engineering

Cancer immunotherapy

GlyTR Therapeutics’ lectin-scFvs fusions mediate velcro-like density-dependent targeting of pantumor-associated carbohydrate antigens and killing in cell lines and xenograft mouse models of triple-negative breast, ovarian or colon cancer | Cell

Small-molecule inhibitor of palmitoyl protein thioesterase1 (PPT1) reactivates immune surveillance in cold tumors by restoring STING signaling in cancer cells | PNAS

In vivo CRISPR screens identify modifiers of CAR T cell function in mouse models of multiple myeloma | Nature

PSCA-targeted CAR-T cells engineered to secrete PD-L1–IL-12 fusions show superior killing of prostate cancer cell lines and xenograft mouse models | Nature Biomedical Engineering

Editors, integrases, transposases galore

Integra Biosciences’ protein language model-guided design of hyperactive PiggyBac transposases or Cas9-directed transposases for T-cell engineering | Nature Biotechnology

Megabase-scale human genome rearrangement with programmable bridge recombinases | Science

Multiplex base editing of BCL11A +58 and +55 enhancers reduces BCL11A mRNA avoids double-strand breaks and boosts fetal hemoglobin production to treat sickle cell disease | Cell

AAV-packaged dCas6 and endoribonuclease fusions silence cytoplasmic and nuclear transcripts in cell culture with reduced off-targets compared to CRISPRi and RNAi | Nature Communications

Platforms

Binding interface mimicry of natural peptides and their cognate receptors by machine learning (PepMimic) generates nanomolar potency de novo peptides | Nature Biomedical Engineering

Single-cell CRISPR knockout technology identifies new CARs for T-cell immunotherapy in a xenograft mouse model of human leukemia | Nature

Dual membrane receptor degradation via folate receptor targeting chimera | Nature Communications

Startup news

Sanofi commits an additional $625 million to Sanofi Ventures to accelerate investment in biotech and digital health startups

Universities continue to spin out startup funds and strengthen ties with venture community:

Trio Impact Invest launched by Karolinska Institutet, KTH, and Stockholm University to spur early-stage ventures

Monash University establishes a ‘Monash Boston hub” to meet surging US and European demand for drug discovery expertise and clinical trial trackrecord.

All of which is good news considering the general frosty financing climate:

In the world of accelerators, two players combine forces:

BioInnovation Institute (BII) biotech and Accelerace deep tech accelerators join forces to accelerate startup formation

And several regenerative medicine startups receive non-dilutive funding:

The Maryland Stem Cell Research Fund handed out million dollar grant awards to Rooster Bio, HOHCells, Theradaptive and Stemora.

Following up on the ‘Endpoints 11’ listed in our last issue, Fierce Biotech has announced its Fierce 15, with three of these, Congruence Therapeutics and Trace Neuroscience and TrimTech Therapeutics still in the preclinical stage (Haystack’s myopic interest). Congratulations to Umoja Biopharma for making both lists!

Finally, various accolades handed out to startups during prize season:

BioAlberta names health and longevity play BioAro as “Company of the Year”, joining previous winner Pacylex Pharmaceuticals, a developer of N-myristoyltransferase inhibitors for antibody–drug conjugates for cancers.

Abbvie Japan and Biolabs announces Neko Pharma, developing an ultrastable antibody-like scaffold, as the winner of the Innovation Award, with the Golden Ticket for one-year use of labs and 10 million yen.

Preclinical financings

Preclinical deals

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Until next issue,

Juan Carlos and Andy

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