Juan Carlos Lopez

Andy Marshall

This week, we provide some lightning takes on recent translational papers that caught our eye. We saw several preclinical advances in approaches for pain, neurodegeneration, cardiovascular disease and bone disorders. In the gene-editing arena, several new large DNA insertion technologies and RNA-targeting CRISPR systems came to the fore.

But before we dive in, we want to highlight the New England Journal of Medicine report from the groups of Rebecca Ahrens-Niklas and Lindsey George at the Children’s Hospital of Philadelphia that details a neuroepithelial tumor in a 5-year-old boy with severe mucopolysaccharidosis type I (MPSI, a.k.a. Hurler Syndrome) 4 years after receiving an intracisternal injection of an AAV-9 gene therapy.

Needless to say, approved AAV-based gene therapy products have a long track record of safety, efficacy and long-term transgene expression, but the specter of insertional mutagenesis has always loomed, even though AAV is a predominantly episomal vector. More than five years ago, a paper on hemophilia A dog studies published in Nature Biotechnology reported 1,741 unique AAV integration events in liver and clonal expansions of transduced hepatocytes, with many integrations near growth-related genes. In that case, no tumors were seen. Human liver-biopsy studies after AAV gene therapy have similarly made clear that integration and clonal hepatocyte expansion can happen, while not showing obvious malignant transformation. The NEJM report stands out as providing the first well-documented case of human oncogenesis plausibly linked to AAV vector integration. We can expect it to lead to tighter regulatory and post-marketing oversight of AAV gene therapies, as illustrated by the clinical hold the US Food and Drug Administration (FDA) already placed on Regenxbio’s gene therapy for Hurler, which was reported back in January. The takeaway for the investment community is that this is not entirely unexpected and should be viewed in the context of >6,000 patients receiving AAV gene therapy to date without major long-term toxic effects.

Safety signals have also been a recurring theme for drugs targeting sodium voltage channels (Na_v1.7) in different pain indications. Multiple industry programs have encountered problems with off-target effects and poor clinical translation. Now a team led by Wengsheng Zhang at Sichuan University has identified potent nonopioid analgesics targeting multiple voltage-gated sodium channel isotypes with improved efficacy when tested their efficacy in perioperative rat models (PNAS). We wonder how such a broad approach would mitigate some of the safety flags encountered by previous clinical trials of investigational drugs targeting this pathway. Elsewhere, Xiao-Ming Li and collaborators at Zhejiang University School of Medicine set out to mitigate some of the adverse events of cannabinoid 1 (CB1) agonists, such as reduced locomotion, hypothermia, addiction and analgesic tolerance using so-called biased signaling and targeting downstream signaling cascades mediated predominantly through inhibitory guanine nucleotide binding protein (Gi), rather than beta-arrestin. They show their Gi-biased inhibitors display analgesic properties, but with reduced side effects when tested in mice (Cell). Over recent years, industry has explored cannabinoids to treat a wide range diseases, including chronic kidney disease, glaucoma and even obesity, again with limited clinical success. It will be interesting to see whether drugging a downstream signaling pathway will bring greater reward.

While cannabinoids haven’t exactly set the world of company formation alight, platforms leveraging autophagy biology are another story. In the past five years, Lysoway Therapeutics, Retro Biosciences, Casma Therapeutics, Automera Therapeutics, PAQ Therapeutics and AUTOTAC Bio have all received funding for platforms leveraging auto-phagosomal pathways, such as ATTEC, AUTAC, AUTOTAC, chaperone-mediated autophagy or AUTAB. The latest instantiation of ATTEC is described in a paper by Einar Sigurdsson and researchers from New York University, who develop single-domain antibodies to promote autophagy-mediated tau degradation in patient-derived neurons, improving motor function in tauopathy mice (Science Translational Medicine). Autophagy is also the focus for a collaboration between the Jia-Hong Lu team at the University of Macau and MindRank AI, which developed an AI-based screening platform using a variational autoencoder trained on a library (from MedChemExpress and TSBiochem) of over 1 million compounds to identify brain-penetrant small molecule autophagy enhancers effective in mouse models of Alzheimer’s disease (Nature Biomedical Engineering).

Elsewhere in the neurodegenerative disease field, TDP-43 aggregation is a hallmark of disorders like amyotrophic lateral sclerosis and frontotemporal dementia. Acurastem and Quralis have been tackling these diseases using antisense oligonucleotides (ASOs) to modulate splice-switching of genes affected by mutant TDP-43. But new research from the groups of James Shorter at the University of Pennsylvania, Christopher Donnelly at the University of Pittsburgh, Nicolas Fawzi at Brown University, Brigid Jensen at Thomas Jefferson University and Jeetain Mittal at Texas A&M reveals that short 34-nucleotide RNAs can act as chaperones to inhibit TDP-43 aggregation and prevent neurodegeneration in the mouse. This potentially opens up short RNA chaperones as a new therapeutic modality for protein-folding disorders (Science).

Moving away from the CNS, some intriguing advances in other therapeutic areas popped into our inbox. One of the new frontiers for oligonucleotide therapies is common cardiovascular indications, such as heart failure and atrial fibrillation. For example, Ionis’ transferrin-receptor 1 targeted ASO for downregulating phospholamban in R14-deleted dilated cardiomyopathy just entered phase 1 testing in a development partnership with AstraZeneca. Along these lines, two teams headed by Matthias Nahrendorf and Maarten Hulsman at Harvard Medical School report another target, osteopontin (Spp1), downregulation of which with an antibody–siRNA conjugate targeting TREM2+ cardiac macrophages suppresses atrial fibrillation in mice (Nature Cardiovascular Research).

Another area likely to attract more commercial activity going forward is metabolic bone disease. Last December, the US Food and Drug Administration (FDA) made a landmark regulatory shift, formally qualifying percentage change from baseline at 24 months in total hip bone mineral density (BMD) via imaging as a validated surrogate endpoint (previously, bone disease trial times typically took anywhere from two to five years). Two recent papers discuss new therapeutic approaches to heterotopic bone formation after injury. In the first, two teams led by Benjamin Levi and Michael Dellinger from UT Southwestern show that vascular endothelial growth factor D (VEGF-D)-induced lymphangiogenesis can promote heterotopic bone resorption in mice (PNAS). And across the Atlantic, the groups of Johan Keller and Anke Baranowsky at the University Medical Center Hamburg-Eppendorf target extracellular traps from myeloid cells using an FDA-approved recombinant DNAse 1 Pulmozyme to inhibit traumatic heterotopic ossification in mice (Science Translational Medicine; Roche/Genentech’s Pulmozyme (dornase alpha) is approved only for the pulmonary indication cystic fibrosis).

Moving onto advanced genetic therapeutics, several advances caught our attention in the gene-editing space. While programmable recombinases/integrases capable of introducing genetic cargoes >10 kb have been prominent in journals, momentum in commercializing these approaches has proceeded at a moderate pace, with Brink Therapeutics, Seamless Therapeutics and Stylus Medicine all raising funding in the past three years. The ability of recombinases to introduce large constructs has been touted as a key advantage over prime editing, which traditionally can only achieve desired edits no larger than ~300 bp. In this context, three recent papers disclose alternative prime-editing approaches for the genomic insertion of large sequences, overcoming the sequence size limitation. First, research patented by Ying Zhang’s group at Wuhan University shows that quadruple paired pegRNAs enable prime editing based genomic insertion of sequences as long as 26 kb in vitro (Nature). Second, the teams of Haoyi Wang, Chenxin Wang and Wei Li at the Chinese Academy of Science developed “PRIME-In”, a genome editing platform for the integration of up to 3 kb-long DNA sequences in human T cells independent of double-stranded DNA breaks (Nature Biomedical Engineering). Last, the groups of Erik Sontheimer and Wen Xue at the University of Massachusetts Chan Medical School described a “prime assembly” approach for the insertion of DNA fragments as long as 11 kb (Nature).

Finally, in the area of RNA editing, two recent studies expand the palette of CRISPR–Cas effectors capable of targeting and manipulating cells at the level of transcripts rather than nuclear DNA. A paper from I-Ming Hsing’s group at Hong Kong University of Science and Technology describes the first use of DNA-guided CRISPR–Cas12a effectors for programmable RNA recognition and cleavage (Nature Biotechnology). In a second paper, Yang Liu’s team at the University of Utah, Chase Biesel’s group at University of Würzburg and scientists from Akribion Therapeutics and BRAIN Biotech engineer CRISPR–Cas12a2 for the selective, DNA-triggered killing of virally infected human cells on the basis of their transcriptional profile (Nature).

Conference roundup

Selected startups raising funds in past three years presenting data at the American Society for Cell and Gene Therapy (ASCGT), Boston, May 11–15.

Preclinical financings (from April 21 to May 4)

Preclinical financings (from May 5 to May 11)

Preclinical financings (from May 12 to May 14)

Preclinical deals (from April 16 to April 29)

Preclinical deals (from April 30 to May 13)

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If you’re interested in commercializing your science, get in touch. We can help you figure out the next steps for your startup’s translational research program and connect you with the right investor. Follow us on X, BlueSky and LinkedIn. Please send feedback; we’d love to hear from you (info@haystacksci.com).

Juan Carlos Lopez

Andy Marshall

An old adage in drug development states that any successful program for an advanced medicine must overcome three central challenges: first, delivery; second, delivery, and third … delivery! Lipid nanoparticle (LNP) technology and N-acetyl galactosamine-(GalNAc) conjugates have opened the liver to a wide range of genetic medicines, and transferrin 1 receptor (TfR1) conjugates are beginning to access the CNS via intravenous delivery with brain-shuttle technology. But tissues like the lung, kidney, muscle and heart remain very much a work in progress.

In the pulmonary space, a small cadre of companies are pursuing inhaled LNP delivery technologies. Recode Therapeutics, Vertex Pharmaceuticals and Arcturus are the main players, while other firms such as 4DMT and Krystal Biotech are focusing on viral gene therapies for lung delivery.

Just a few days ago, one of these LNP programs got the chop. The Vertex/Moderna phase 1/2 study of VX-522, an aerosolized LNP to deliver mRNA encoding full-length cystic fibrosis transmembrane conductance regulator (CFTR) to the lungs of cystic fibrosis patients, which had been paused due to tolerability issues, is now permanently discontinued. According to reports, the Moderna LNP was the culprit, leading to lung inflammation. That leaves Recode and Arcturus as the frontrunners, a rather small field, given the entire market opportunity for a pulmonary delivery solution. All told, in 2023, there were 569.2 million cases of chronic respiratory diseases and 4.2 million deaths from respiratory disease.

Recode now is enrolling patients into the phase 2 trial of its Selective Organ Targeting (SORT), LNP platform (RCT2100) that delivers an mRNA encoding CFTR in combination with the small-molecule CFTR potentiator ivacaftor (the SORT technology was originally licensed out of Daniel Siegwart’s group at UT Southwestern). The other LNP platform, Arcturus’ LUNAR LNP technology, also has encouraging interim data from its phase 2 trial in cystic fibrosis patients and from its program delivering ornithine transcarbamylase mRNA.

These LNPs (and most other LNP delivery platforms) are built around the same four common components: an amino ionizable lipid, a helper lipid, a polyethylene glycol lipid and cholesterol. The formulations follow this scheme but with different combinations of proprietary lipid forms; thus, in Arcturus’ LUNAR LNP, distearoylphosphatidylcholine (DSPC) performs the helper lipid function, whereas in Recode’s SORT LNP, it is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Overall, however, just a handful of novel lipid components have gone into humans so far.

According to Siegwart, the field is in dire need of developing a broader palette of cationic lipids that are both efficient and non-toxic for the pulmonary epithelium; ultimately, the goal would be a delivery technology capable of targeting specific cell types in the lung (with many new cell subtypes continuing to be identified).

In a recent article in Nature Biomedical Engineering, Siegwart and his group at UT Southwestern introduce the design and evaluation of a new class of lung-targeting (LuT) lipids that enable the highly efficient and selective delivery of mRNA and CRISPR–Cas9 gene-editing systems to the lungs.

They synthesized and screened a library of 444 lipids using a combinatorial approach, systematically varying amine head groups and hydrophobic tails. Through in vivo testing and structure–activity relationship analysis, they identified key features in the lipids that most effectively targeted the lung: a distinctive ‘tripod-like’ structure, consisting of a quaternary amine head, three long alkyl chains and a short fourth chain.

Compared to benchmark formulations, the best-performing LuT-containing LNPs achieved up to a 25.5-fold increase in mRNA delivery and a 9.2-fold improvement in gene-editing efficiency, with >90% of delivery localized to the lungs. These LuT-LNPs successfully transfected multiple lung cell types, including endothelial, epithelial and immune cells, with some formulations showing preferences for specific cell populations.

Mechanistically, the improved performance was attributable to two main factors. First, the tripod-like structure of lipids promoted endosomal escape by facilitating membrane fusion and LNP disassembly, allowing efficient release of genetic cargo into cells. Second, LuT LNPs formed distinct protein coronas in the bloodstream, particularly enriching for vitronectin, a protein that enhances targeting to lung cells via receptor-mediated uptake.

Siegwart and his team went on to show the therapeutic potential of LuT LNPs. The lead formulation, 1A7B13, enabled effective delivery of IL-10 mRNA in a mouse model of acute lung injury and achieved robust CRISPR–Cas9 gene editing in lung tissue. The LNPs showed minimal toxicity and no significant adverse effects in vivo.

This research establishes clear design principles for lung-targeting LNPs and markedly expands the available toolkit for pulmonary gene delivery. It is just the beginning of the translational path, however.

The Siegwart LuT-LNPs must home through the vasculature to the lungs after being delivered intravenously. This is very different from the aerosolized LNP delivery approaches of Recode and Arcturus currently in clinical testing. There may be a case to be made that some pulmonary vascular disease, lung endothelial targets, lung fibrosis, immune-cell or vascular-compartment targets might warrant the intravenous route, but aerosolized LNP delivery provides lower systemic exposure (and thus higher therapeutic index), is more patient-friendly, and rapidly/directly reaches the airway lumen.

Regardless of the route of administration, the translational challenges associated with targeting the lung remain very difficult. In terms of testing formulations in different models, anatomical differences between mouse, ferret and human airways, including physiological size and branching complexity, impact LNP design and aerosol physics.The formulations used for mice may simply not work for people because of differences in cell composition, and lung epithelial and endothelial membranes and “surfaceomes”. As humans age and develop disease, cell protein and lipid composition may also change, requiring further optimization of LNP formulations. Mice have more narrow airways and faster breathing rates than humans, requiring smaller diameter aerosol droplets (often <2 µm) to ensure particles bypass the upper respiratory tract and reach the alveolar regions.

Moreover, humans have ~23 branches in their airways, whereas mice have only 13, meaning an aerosol optimized for a ‘deep’ reach in a mouse might only reach mid-level bronchi in a human. Furthermore, ferrets are not a widely available model system to study the biodistribution and efficacy of LNPs. Indeed, there are just a few labs in the United States that upkeep ferret colonies.

Last, a human lung’s surface area (~70 m²) is nearly 8.500 times larger than a mouse’s (~82 cm²), and human tidal volume is roughly 6,000 times greater. This requires significant dose scaling and affects how ‘diluted’ the LNPs become once they deposit.

Designing in vitro and in vivo systems representative of human biology and capable of predicting LNP biodistribution is also a tall order (especially with such a small cadre of companies working on the problem). For small molecules, the measurement of efficacy in human basal epithelium-derived patient cells carrying a mutation of interest by and large will translate into what you see in the clinic. The pharmaceutical industry has amassed a lot of data to bolster pharmacology.

Unfortunately, that correlation doesn’t necessarily hold for genetic modalities like mRNA or CRISPR/Cas9 constructs. For these medicines, it is very hard to figure out PK/PD. And so, the translation from preclinical work to the clinic can be tricky for an inhaled LNP technology delivering mRNA. It is difficult to really know the degree of protein expression from an inhaled LNP genetic medicine intracellularly without doing a bronchial biopsy (which is of course highly intrusive). And if you need to test your LNP in patients via biopsy, clinicians historically have been very resistant to carrying out such procedures, particularly in very sick patients like some of people with cystic fibrosis who carry nonsense mutations in CFTR. Thus, there is a need for alternative approaches. Certainly, there is an opportunity for more work on organoids or simpler patient cell-derived assays: 2D or 3D alternatives to large animal models like the ferret.

What is clear is that there are enough patients worldwide living with lung disease that further research in this area needs to be encouraged. In this respect, the findings from Siegwart’s group are a step in the right direction, with broad implications for treating lung diseases by enabling safer and more precise delivery of RNA-based therapeutics and genome-editing technologies.