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Notable Recent Events in Gene Therapy Translation January 2023 to January 2026

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Notable Recent Events in Gene Therapy Translation January 2023 to January 2026

PDF version: Notable Recent Events in Gene Therapy Translation January 2023 to January 2026

Clinical gene therapy has seen a lot of big wins and a lot of big setbacks over the past few years. To help myself keep track of recent important events in the field, I decided to write up this catalogue of key happenings. Though the landscape is ever-evolving, this resource should nonetheless be useful in the relatively near future and perhaps serve as a historical record later on. It has been fascinating to read up on the industry’s dynamics! I hope any readers out there who encounter this page will find my catalogue similarly interesting and valuable.

BioMarin and Roctavian (AAV)

  • June 2023: The FDA approved BioMarin’s Roctavian, an AAV5 gene therapy for hemophilia A. It is the first AAV gene therapy for hemophilia [16].

Sarepta Therapeutics and Elevidys (AAV)

  • June 2023: the FDA approved (Accelerated Approval pathway) Sarepta’s AAVrh74 gene therapy for Duchenne’s muscular dystrophy (DMD) in ambulatory of male patients 4-5 years old [1]. This treatment is known as Elevidys. It treats DMD by expressing a microdystrophin to replace the role of the defective endogenous dystrophin. The per patient cost of the one-time Elevidys treatment was listed at $3.2M [2]. It used a high AAV dose of 1.33×1014 vg/kg [3].
  • October 2023: Sarepta’s Elevidys failed to reach statistical significance for its primary endpoint (functional mobility) in a phase 3 clinical trial, though substantial evidence of secondary endpoint effects was reported by the company. A p value of 0.24 for the main functional mobility test was reported [4].
  • February 2024: Despite its setbacks, the FDA still accepted an efficacy supplement to the Biologics License Application (BLA) of Elevidys, which removed age and ambulation restrictions from the treatment.
  • March 2025: Sarepta reported a patient death due to acute liver failure after Elevidys treatment [5]. A recent cytomegalovirus infection was identified by the reporting physician as a possible contributing factor, but the main reason was probably the high AAV dose.
  • June 2025: Sarepta reported another patient death due to acute liver failure after Elevidys treatment [6]. Both of the deaths were in non-ambulatory teenage boys.
  • June 2025: Sarepta paused shipments of Elevidys to non-ambulatory patients. The FDA began investigating the Elevidys deaths. Sarepta also reported a third death (which had actually occurred in June), this time of a 51-year-old non-ambulatory patient in one of their clinical trials for limb-girdle muscular dystrophy (LGMD) [7]. This patient had been treated with an investigational gene therapy which leveraged the same AAVrh74 capsid as Elevidys [8].
  • July 2025: The FDA issued a major action bundle which placed Sarepta’s muscular dystrophy gene therapy clinical trials on hold, revoked AAVrh74’s platform technology designation, and made a voluntary request for Sarepta to immediately cease all Elevidys distribution [9]. Roche then paused shipments of Elevidys for non-ambulatory patients outside of the USA [10].
  • July 2025: Sarepta initially resisted the request to stop distributing Elevidys. But after further consideration, Sarepta agreed to the FDA’s request [7]. To mitigate financial losses, Sarepta performed major restructuring, laying off about 36% of their workforce (~500 employees) [11].
  • July 2025: The Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) issued an official negative opinion on the conditional marketing authorization (CMA) of Elevidys for ambulatory DMD patients of 3-7 years [12]. Roche noted that over 900 DMD patients (760 ambulatory) had received Elevidys, demonstrating a manageable safety profile.
  • July 2025: The FDA began investigating the death of an 8-year-old boy who died in June after receiving Elevidys [13]. However, the treating physician deemed this death unrelated to the Elevidys treatment [14].
  • November 2025: The FDA approved a new label for Elevidys which included a Boxed Warning, limited the indication to ambulatory DMD patients 4 years of age and older, and added new recommendations for safety and monitoring [15].

Vertex Pharmaceuticals, CRISPR Therapeutics, and Casgevy (ex vivo electroporation)

  • November 2023: The United Kingdom MHRA (Medicines and Healthcare products Regulatory Agency) approved Casgevy (developed by Vertex Pharmaceuticals and CRISPR Therapeutics through a strategic partnership) for the treatment of sickle cell disease and transfusion-dependent beta thalassemia. Casgevy is the first CRISPR-Cas9 gene therapy applied in humans. It performs gene editing ex vivo on blood stem cells derived from the patient (via electroporation of Cas9) to knock out the erythroid-specific enhancer region of the BCL11A gene, disinhibiting production of fetal hemoglobin [17].
  • December 2023: The FDA approved Casgevy to treat sickle cell disease [18]. Both the FDA and MHRA approvals represented the first regulatory authorization of a CRISPR therapy in the world.
  • January 2024: The FDA approved Casgevy to also treat transfusion-dependent beta thalassemia [19].

Bluebird Bio and Lyfgenia (ex vivo lentivirus)

  • December 2023: The FDA approved Bluebird Bio’s Lyfgenia to treat sickle cell disease [18]. Lyfgenia is an ex vivo lentiviral gene therapy which delivers a copy of HbAT87Q into blood stem cells derived from the patient. This hemoglobin behaves similarly to normal hemoglobin and thus treats the sickle cell condition. The one-time Lyfgenia treatment price is $3.1M.

Orchard Therapeutics and Lenmeldy (ex vivo lentivirus)

  • March 2024: The FDA approved the Orchard Therapeutics gene therapy Lenmeldy to treat children with metachromatic leukodystrophy (MLD) [20], [21]. Lenmeldy is an ex vivo gene therapy which uses a lentiviral vector to modify a patient’s stem cells to add a functional copy of the ARSA (arylsulfatase A) gene. This gene encodes the ARSA enzyme and degrades harmful sulfatides. MLD patients possess mutations in their endogenous ARSA enzyme gene. Lenmeldy is the most expensive therapy in the world at $4.2M [21].

Krystal Biotech and Vyjuvek (topical HSV-1)

  • May 2023: The FDA approved Krystal Biotech’s Vyjuvek to treat dystrophic epidermolysis bullosa (DEB). It uses a herpes simplex virus 1 (HSV-1) vector to deliver normal copies of the COL7A1 (collagen type VII alpha 1 chain) gene into wounds caused by the disease [22]. A healthcare professional applies the vector topically to the patient’s wounds once per week. The treatment costs $630,500 per year [21].

Abeona Therapeutics and Zevaskyn (ex vivo RVV + topical cell sheets)

  • April 2025: The FDA approved Zevaskyn, a unique autologous cell sheet gene therapy, for treatment of recessive dystrophic epidermolysis bullosa (RDEB) [23]. Cells derived from the patient are collected and transduced with a replication-incompetent retroviral vector (RVV) carrying a copy of the COL7A1 gene [24]. These cells are used to grow sheets of skin which are grafted onto the patient’s wounds. The treatment costs $3.1M and provides up to 12 cell sheets.

Pfizer and Beqvez (AAV)

  • April 2024: The FDA approved Pfizer’s Beqvez, an AAVRh74var vector encoding factor IX, for treatment of hemophilia B [25]. It was priced at $3.5M per patient [26].
  • May 2025: Pfizer discontinued Beqvez due to lack of patients adopting the therapy (no patients had received the treatment outside of clinical trials) [26]. After discontinuing Beqvez, Pfizer was left with no gene therapies on the market or even in development, a setback for the gene therapy field.

Rocket Pharmaceuticals and RP-A501 (AAV)

  • May 2025: During a phase II clinical trial for treatment of Danon disease with an AAV9 vector (6.7×1013 vg/kg), a patient died [27]. This led the FDA to put a clinical hold on the trial.
  • August 2025: The FDA lifted its clinical hold on RP-A501 less than 3 months later [28]. They confirmed that Rocket Pharmaceuticals had addressed the issues relating to the clinical hold by changing the dose to 3.8×1013 vg/kg and modifying their immunomodulatory drug regime. Rocket Pharmaceuticals thus resumed the clinical trial.

Intellia Therapeutics and NTLA-2001 (LNP)

  • August 2024: In a phase I clinical trial, Intellia Therapeutics demonstrated the first redosing data of an in vivo CRISPR therapy in humans [29]. This therapy treats transthyretin amyloidosis (ATTR) by knocking out the TTR gene in hepatocytes. It uses a lipid nanoparticle (LNP) delivery platform. It should be noted that further redosing was not planned for NTLA-2001 specifically, but the data may be useful for future therapies from Intellia.
  • October 2025: During the MAGNITUDE-2 phase III clinical trial for NTLA-2001, a patient experienced a grade 4 liver adverse event and was hospitalized. As a result, the FDA placed the MAGNITUDE and MAGNITUDE-2 trials on clinical hold [30]. (The MAGNITUDE trial is for treating ATTR with cardiomyopathy and the MAGNITUDE-2 trial is for treating ATTR with polyneuropathy).
  • January 2026: The FDA lifted its clinical hold on MAGNITUDE-2 [31].

UniQure and AMT-130 (AAV)

  • December 2024: UniQure and the FDA reached an agreement for Huntington’s disease gene therapy AMT-130 to enter the accelerated approval pathway [32]. AMT-130 uses an AAV5 vector encoding a miRNA which inhibits expression of the mutant Huntingtin protein [33]. The AAV5 is delivered directly to the brain via a micro-catheter [34].
  • September 2025: UniQure showed a statistically significant 75% slowing of Huntington’s disease progression in a phase I/II clinical trial for AMT-130, a major breakthrough [35].
  • November 2025: Although UniQure had previously reached an agreement with the FDA for AMT-130 to enter the accelerated approval pathway (and had later presented the breakthrough data on slowing Huntington’s disease progression by 75%), the FDA stated the data were insufficient to support approval due to the use of an external control group [36]. UniQure continues to work with the FDA in an effort to move AMT-130 forward despite this setback.

Capsida Biotherapeutics and CAP-002 (AAV)

  • September 2025: In a phase I/II clinical trial (CAP-002 SYNRGY) intended to treat syntaxin-binding protein 1 (STXBP1) encephalopathy, Capsida Biotherapeutics used an AAV capsid which had been engineered to efficiently cross the blood-brain-barrier while exhibiting liver-detargeting. The first patient (a child) dosed with this treatment died [37]. The trial was placed on clinical hold.
  • January 2026: After investigating, Capsida found that the patient’s death was triggered by cerebral edema, but the root cause remained undetermined (i.e. no one knows whether the treatment had been directly responsible for the death) [38]. All of this has resulted in increased scrutiny on the field of blood-brain-barrier crossing AAVs.

Replimune and RP1 (HSV-1)

  • July 2025: The FDA issued a Complete Response Letter (CRL) to Replimune’s biologics license application (BLA) for treatment of advanced melanoma with RP1 [39], an oncolytic engineered herpes simplex virus 1 (HSV-1) equipped with a fusogenic protein that encodes GM-CSF (granulocyte-macrophage colony-stimulating factor). The CRL voiced concerns about the RP1 IGNYTE trial’s design, interpretability, and inadequate controls, and the heterogeneity of the patient population. This represented a major (and highly publicized) setback for the company.
  • October 2025: After presenting a revised trial design during a type A meeting with the FDA, Replimune’s BLA resubmission was accepted [40]. This means that RP1 is cleared for further FDA evaluation towards possible approval for marketing in the U.S.

Beam Therapeutics, BEAM-302 (LNP), and BEAM-101 (ex vivo electroporation)

  • March 2025: In a phase I/II clinical trial of its base editor formulation BEAM-302, Beam Therapeutics showed successful human proof-of-principle for treating alpha-1 antitrypsin deficiency (AATD) [41]. The formulation consists of a lipid nanoparticle (LNP) carrying guide RNA and an mRNA (the latter encodes the base editor).
  • March 2025: The FDA cleared Beam’s investigational new drug application (IND) for BEAM-302 to treat AATD [42].
  • December 2025: Beam announced strong safety and efficacy data from its phase I/II clinical trial BEACON for their BEAM-101 ex vivo base editor therapy sickle cell disease therapy [43]. In BEAM-101, stem cells from a patient are electroporated with base editors which modify the promoter regions of HBG1/2 genes, preventing binding of the transcriptional repressor BCL11A. After reintroduction of the cells into the patient’s bone marrow, this increases expression of fetal hemoglobin, combating the sickle cell disease.

References:

[1]     “FDA Approves First Gene Therapy for Treatment of Certain Patients with Duchenne Muscular Dystrophy,” 2023. https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapy-treatment-certain-patients-duchenne-muscular-dystrophy

[2]     “After delays, Sarepta’s DMD gene therapy Elevidys finally crosses FDA finish line at $3.2M,” 2023. https://www.fiercepharma.com/pharma/sareptas-dmd-gene-therapy-finally-makes-it-accelerated-approval-finish-line-restricted

[3]     “Sarepta Therapeutics Announces Topline Results from EMBARK, a Global Pivotal Study of ELEVIDYS Gene Therapy for Duchenne Muscular Dystrophy,” 2023. https://investorrelations.sarepta.com/news-releases/news-release-details/sarepta-therapeutics-announces-topline-results-embark-global-0

[4]     “Sarepta Fails Confirmatory Trial for DMD Therapy, Still Eyes Label Expansion,” 2023. https://www.biospace.com/sarepta-fails-confirmatory-trial-for-dmd-therapy-still-eyes-label-expansion

[5]     “Community Letter: ELEVIDYS Safety Update,” 2025. https://www.sarepta.com/community-letter-elevidys-safety-update

[6]     “FDA investigates patient deaths after treatment with Sarepta’s gene therapy,” 2025. https://www.reuters.com/business/healthcare-pharmaceuticals/fda-investigates-patient-deaths-after-treatment-with-sareptas-gene-therapy-2025-06-24/

[7]     “Sarepta, bowing to FDA pressure, pauses shipments of Duchenne gene therapy Elevidys,” 2025. https://www.fiercepharma.com/pharma/sarepta-getting-back-fdas-good-side-pauses-shipments-duchenne-gene-therapy-elevidys

[8]     “Sarepta Therapeutics Announces Pipeline Progress for Multiple Limb-Girdle Muscular Dystrophy Programs,” 2025. https://investorrelations.sarepta.com/news-releases/news-release-details/sarepta-therapeutics-announces-pipeline-progress-multiple-limb

[9]     “FDA Requests Sarepta Therapeutics Suspend Distribution of Elevidys and Places Clinical Trials on Hold for Multiple Gene Therapy Products Following 3 Deaths,” 2025. https://www.fda.gov/news-events/press-announcements/fda-requests-sarepta-therapeutics-suspend-distribution-elevidys-and-places-clinical-trials-hold

[10]   “Roche pauses shipments of Elevidys gene therapy outside US,” 2025. https://www.reuters.com/sustainability/boards-policy-regulation/roche-pauses-shipments-elevidys-gene-therapy-outside-us-2025-07-22/

[11]   “Sarepta to lay off about 500 employees after Duchenne gene therapy setbacks,” 2025. https://www.biopharmadive.com/news/sarepta-layoffs-restructuring-elevidys-duchenne-research-cuts/753256/

[12]   “Roche provides regulatory update on ElevidysTM gene therapy for Duchenne muscular dystrophy in the EU,” 2025.

[13]   “FDA Investigating Death of 8-Year-Old Boy Who Received Elevidys,” 2025. https://www.fda.gov/news-events/press-announcements/fda-investigating-death-8-year-old-boy-who-received-elevidys

[14]   “Sarepta Therapeutics Provides Clarifying Statement on ELEVIDYS,” 2025. https://investorrelations.sarepta.com/news-releases/news-release-details/sarepta-therapeutics-provides-clarifying-statement-elevidys

[15]   “FDA Approves New Safety Warning and Revised Indication that Limits Use for Elevidys Following Reports of Fatal Liver Injury,” 2025. https://www.fda.gov/news-events/press-announcements/fda-approves-new-safety-warning-and-revised-indication-limits-use-elevidys-following-reports-fatal

[16]   A. Philippidis, “BioMarin’s ROCTAVIAN Wins Food and Drug Administration Approval As First Gene Therapy for Severe Hemophilia A,” Hum. Gene Ther., vol. 34, no. 15–16, pp. 665–668, Aug. 2023, doi: 10.1089/hum.2023.29251.bfs.

[17]   “Vertex and CRISPR Therapeutics Announce Authorization of the First CRISPR/Cas9 Gene-Edited Therapy, CASGEVYTM (exagamglogene autotemcel), by the United Kingdom MHRA for the Treatment of Sickle Cell Disease and Transfusion-Dependent Beta Thalassemia,” 2023. https://investors.vrtx.com/news-releases/news-release-details/vertex-and-crispr-therapeutics-announce-authorization-first

[18]   “FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease,” 2023, [Online]. Available: https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapies-treat-patients-sickle-cell-disease

[19]   “Vertex Announces US FDA Approval of CASGEVYTM (exagamglogene autotemcel) for the Treatment of Transfusion-Dependent Beta Thalassemia,” 2024. https://investors.vrtx.com/news-releases/news-release-details/vertex-announces-us-fda-approval-casgevytm-exagamglogene

[20]   “FDA Approves First Gene Therapy for Children with Metachromatic Leukodystrophy,” 2024. https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapy-children-metachromatic-leukodystrophy

[21]   A. Mullard, “FDA approves gene therapy for metachromatic leukodystrophy, the tenth for a genetic disease and the priciest yet,” Nature Reviews Drug Discovery, 2024.

[22]   “FDA Approves First Topical Gene Therapy for Treatment of Wounds in Patients with Dystrophic Epidermolysis Bullosa,” 2023, [Online]. Available: https://www.fda.gov/news-events/press-announcements/fda-approves-first-topical-gene-therapy-treatment-wounds-patients-dystrophic-epidermolysis-bullosa

[23]   “ZEVASKYN,” 2025. https://www.fda.gov/vaccines-blood-biologics/zevaskyn

[24]   “FDA approves cell-sheet-based gene therapy for severe skin disease,” 2025. https://www.nature.com/articles/d41573-025-00082-2

[25]   “U.S. FDA Approves Pfizer’s BEQVEZTM (fidanacogene elaparvovec-dzkt), a One-Time Gene Therapy for Adults with Hemophilia B,” 2024. https://www.pfizer.com/news/press-release/press-release-detail/us-fda-approves-pfizers-beqveztm-fidanacogene-elaparvovec

[26]   “Pfizer Jettisons FDA-Approved Hemophilia B Gene Therapy Beqvez,” 2025, [Online]. Available: https://www.cgtlive.com/view/pfizer-jettisons-fda-approved-hemophilia-b-gene-therapy-beqvez

[27]   “Patient Dies After Treatment With Rocket Pharmaceuticals’ Danon Disease Gene Therapy RP-A501 in Phase 2 Trial,” 2025. https://www.cgtlive.com/view/patient-dies-after-treatment-rocket-pharmaceuticals-danon-disease-gene-therapy-rp-a501-phase-2-trial

[28]   “Rocket Pharmaceuticals Announces FDA Has Lifted the Clinical Hold on the Pivotal Phase 2 Trial of RP-A501 for the Treatment of Danon Disease,” 2025. https://ir.rocketpharma.com/news-releases/news-release-details/rocket-pharmaceuticals-announces-fda-has-lifted-clinical-hold

[29]   “Intellia Announces Positive Clinical Proof-of-Concept Data for Redosing a CRISPR-Based Therapy with its Proprietary LNP-Based Delivery Platform,” 2024. https://ir.intelliatx.com/news-releases/news-release-details/intellia-announces-positive-clinical-proof-concept-data-redosing

[30]   “Intellia Puts Phase 3 Trials for Transthyretin Amyloidosis Gene Editing Therapy Nex-Z on Hold Following Grade 4 Liver AE,” 2025. https://www.cgtlive.com/view/intellia-phase-3-trials-transthyretin-amyloidosis-gene-editing-therapy-nex-z-hold-grade-4-liver-ae

[31]   “Intellia Therapeutics Announces FDA Lift of Clinical Hold on MAGNITUDE-2 Phase 3 Clinical Trial in ATTRv-PN,” 2026. https://ir.intelliatx.com/news-releases/news-release-details/intellia-therapeutics-announces-fda-lift-clinical-hold-magnitude

[32]   “uniQure Announces Alignment with FDA on Key Elements of Accelerated Approval Pathway for AMT-130 in Huntington’s Disease,” 2024. https://uniqure.gcs-web.com/news-releases/news-release-details/uniqure-announces-alignment-fda-key-elements-accelerated

[33]   “Silencing the mutant huntingtin gene.” https://www.uniqure.com/programs-pipeline/huntingtons-disease

[34]   “Phase I/II Clinical Trial of AMT-130,” 2025. https://www.uniqure.com/programs-pipeline/phase-1-2-clinical-trial-of-amt-130

[35]   “uniQure hopes to launch 1st Huntington’s gene therapy next year following phase 1/2 success,” 2025. https://www.fiercebiotech.com/biotech/uniqure-hopes-launch-1st-huntingtons-gene-therapy-next-year-following-phase-12-success

[36]   “uniQure’s ballyhooed gene therapy for Huntington’s hits FDA roadblock,” 2025. https://www.fiercebiotech.com/biotech/fierce-biotech-layoff-tracker-2026

[37]   “Capsida pauses phase 1 gene therapy trial after child dies,” 2025. https://www.fiercebiotech.com/biotech/capsida-pauses-phase-1-gene-therapy-trial-after-child-dies

[38]   “An Important Update Regarding Our CAP-002 Program: A Letter to the STXBP1 Community,” 2026. https://capsida.com/an-important-update-regarding-our-cap-002-program-a-letter-to-the-stxbp1-community/

[39]   “Replimune Receives Complete Response Letter from FDA for RP1 Biologics License Application for the Treatment of Advanced Melanoma,” 2025. https://ir.replimune.com/news-releases/news-release-details/replimune-receives-complete-response-letter-fda-rp1-biologics

[40]   “Replimune Announces FDA Acceptance of BLA Resubmission of RP1 for the Treatment of Advanced Melanoma,” 2025.

[41]   “Beam Therapeutics Announces Positive Initial Data for BEAM-302 in the Phase 1/2 Trial in Alpha-1 Antitrypsin Deficiency (AATD), Demonstrating First Ever Clinical Genetic Correction of a Disease-causing Mutation,” 2025. https://investors.beamtx.com/news-releases/news-release-details/beam-therapeutics-announces-positive-initial-data-beam-302-phase

[42]   “Beam Therapeutics Announces Clearance of Investigational New Drug Application for BEAM-302 for the Treatment of Alpha-1 Antitrypsin Deficiency (AATD) by the United States (U.S.) Food and Drug Administration,” 2025. https://investors.beamtx.com/news-releases/news-release-details/beam-therapeutics-announces-clearance-investigational-new-drug

[43]   “Beam Therapeutics Reports Updated Data from BEACON Phase 1/2 Trial of ristoglogene autogetemcel (risto-cel) Highlighting Durable, Differentiated Profile in Sickle Cell Disease (SCD) at American Society of Hematology (ASH) Annual Meeting,” 2025. https://investors.beamtx.com/news-releases/news-release-details/beam-therapeutics-reports-updated-data-beacon-phase-12-trial

The Path to Scalable Psychiatric Gene Therapy and a Future of Cures for Widespread Mental Illnesses — Restoring Joy to a Billion Lives

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The Path to Scalable Psychiatric Gene Therapy and a Future of Cures for Widespread Mental Illnesses — Restoring Joy to a Billion Lives

PDF version: The Path to Scalable Psychiatric Gene Therapy and a Future of Cures for Widespread Mental Illnesses – By Logan Thrasher Collins

The Promise of Psychiatric Gene Therapy

Current psychiatric interventions remain insufficient to address the highly prevalent mental illnesses which plague more than a billion people (1 in 7) across the world.1 Widespread and debilitating diseases like major depressive disorder (MDD),2 anxiety disorders,3 schizophrenia,4 bipolar disorders,5 post-traumatic stress disorder (PTSD),6 substance abuse disorders,7,8 and personality disorders9,10 pose an enormous global health burden and are one of the most central causes of human suffering. Patients frequently do not respond to pharmacological interventions for these conditions, resulting in vast numbers of people struggling through life without options for proper management. As a result, at least 800K people die by suicide annually.11 Today’s neuropharmacology industry employs small molecule drugs which modulate neurochemical states, utilizing strategies like neurotransmitter reuptake inhibition and receptor agonism. Mechanistic underpinnings of many neuropharmacological treatments are not well understood.2,12 Furthermore, small molecules suffer from extensive off-target binding13 and frequently come with side effects, many of which can be extremely debilitating and/or dangerous. Though it has been cemented into place by partial successes,12 the way we currently treat mental illnesses is woefully inadequate.

Why might gene therapy solutions eventually offer better treatment options for common psychiatric disorders compared to traditional small molecule pharmaceuticals? Gene therapies possess a number of intrinsic advantages like cell-type-specific targetability, direct in situ expression of therapeutic proteins or RNAs, and the capacity to dynamically respond to environmental conditions. They also have potential for spatiotemporal programmability via emerging sonogenetics14–17 and chemogenetics18 approaches. (Sonogenetics has particular promise, which will be discussed in more detail later). In addition, gene therapies may be engineered to downregulate (RNAi or CRISPRi)19 or even ablate (CRISPR knockout)20 expression of almost any gene in the genome, offering an unprecedented array of new therapeutic targets. CRISPRa might also be employed to upregulate target genes without altering the genome.19 Engineering gene therapies which express multiple proteins or RNAs at once may synergistically improve efficacy.21,22 Importantly, gene therapies can be engineered to persist for long periods of time23,24 or to only provide a burst of short term expression. Depending on the genetic payload, one or the other of these durations may represent the most optimal choice. Gene therapies altogether provide a much larger space of possibilities for precision alteration of brain states than has been possible for small molecule treatments. I would argue that this space’s capabilities remain severely underexplored primarily because of a lack of delivery system capabilities.

Better Delivery Systems are Needed

Gene therapy promises to open a new world of precision psychiatric treatments, yet its potential has gone unrealized. This makes sense as there are a number of obstacles which render psychiatric gene therapy a difficult target. Among these, the challenges of brain delivery, safety, and manufacturing scalability represent particularly recalcitrant bottlenecks. Adeno-associated virus (AAV) gene therapies have progressed furthest in the brain delivery field. Industry players like 4DMT, Dyno Therapeutics, Apertura Therapeutics, and Capsida Biotherapeutics have made efforts via directed evolution, rational design, and machine learning towards optimizing AAV capsids for blood-brain-barrier (BBB) crossing efficiency. However, safety concerns stemming from several patient deaths in systemically administered AAV therapies over the past few years have slowed this progress. Additionally, limitations in AAV manufacturing capacity pose a problem for scaling the vector to populations of 1M+ patients.25 This will be discussed in more detail further on. It should be noted as well that AAVs are limited by their small DNA packaging capacity of 4.7 kb. To unlock the potential of psychiatric gene therapy, we need safer and more scalable delivery systems.

Although there exist multiple obstacles to overcome before gene therapy can realize its potential as a psychiatric modality, I propose a lack of delivery systems represents a foundational missing piece. Without strong delivery vehicles to feasibilize solutions, the field of psychiatric gene therapy will not be credible enough to receive substantial investment. It is a “tools problem”. As mentioned earlier, there is a particular need for vectors which at once possess high safety, scalability, and efficacy. I strongly suspect that the emergence of vectors with these qualities would seed an explosion of efforts towards gene therapies for brain diseases, which would eventually allow us to tackle psychiatric ailments. While psychiatric diseases are unlikely to represent the initial targets of brain gene therapies, opening the door to brain delivery will in my view be necessary to take steps in the direction of modernizing psychiatry through precision genetic medicines.

Examination of the Current Landscape

As mentioned earlier, AAV gene therapies are the current frontrunner for brain delivery yet possess both scalability and safety limitations. I will explain the scalability issue using publicly available information on AAV manufacturing: Final yields (after purification) of AAVs have been reported or modeled in scientific literature with values ranging from around 7.5×1012 vg/L to 7.5×1013 vg/L.26–28 I will thus assume here that 5×1013 vg/L is a typical yield. For this rough calculation, I will also assume that the yield scales linearly with bioreactor volume. As such, a 2,000 L bioreactor would make batches of around 1017 vg and a 200 L bioreactor would make batches of around 1016 vg. According to a 2024 report, a 200 L AAV production run at cGMP quality costs about $2M (including analytics).29A 2022 meta-analysis study of clinical AAV doses states that per patient systemic delivery amounts range from 3.5×1013 vg total to 1.5×1017 vg total.30 Huang et al.’s highly promising AAV BIhTFR1 capsid (the basis for Apertura Therapeutics) was originally administered to mice at a higher dose of 1014 vg/kg and a lower dose of 5×1012 vg/kg.31 Generously (perhaps too generously) assuming that the lower dose is sufficient, this would equate to about 4×1014 vg total in an average 80.7 kg North American adult human.32 Dividing 1016 vg from a $2M (200 L) batch by 4×1014 vg per dose, this means each batch would provide 25 doses for about $80,000 each. Even if substantial improvements in manufacturing yield and in lowering required dosage happen, I am skeptical that systemic AAV approaches will scale to disease indications with 1M+ patients. Despite this, AAVs remain still a central point in the gene therapy industry for a reason and paradigm-shifting approaches to manufacturing25 and/or efficacy might still change the current scalability challenges.

Another existing modality is transient focused ultrasound BBB opening (BBBO). I would argue that BBBO is extremely promising for some applications but not a universal solution. Treatment of many brain diseases necessitates brain-wide delivery. By contrast, BBBO is generally a localized delivery technique.33 Although some common psychiatric diseases fit these parameters, most common ailments with clean clinical endpoints do not. Some work has been done to extend BBBO ultrasound to larger-volume delivery through multiple sonication34,35 or raster scanning,36 but this remains much less well-developed by comparison to localized BBBO approaches and may exhibit greater safety concerns. Indeed, while single-site BBBO possesses a fairly strong safety profile, there is still evidence it can cause problematic inflammatory responses and occasional microhemorrhages.37–39 Also, the level of risk may increase if delivery of vectors with large diameters (e.g. 100 nm) is needed.40,41 As BBBO involves a device, an injection of microbubbles, a procedure, and its own set of safety concerns, it adds complexity which can increase regulatory burden. But I do not think BBBO should be discounted. In some situations, it possesses enormous advantages. These situations may indeed include potential treatments for certain psychiatric disorders. Though BBBO does not universally solve the problem of safe and scalable delivery, I expect it may still play a major role in the field of brain gene therapy.

Intranasal delivery represents a highly promising alternative to intravenous injections which maintains minimal invasiveness. It circumvents the BBB by allowing delivery vectors to migrate through the olfactory (and to a lesser degree trigeminal) nerves into the brain.42,43 This minimizes toxicity by vastly reducing exposure of peripheral organs to the delivery vector. Additionally, much lower doses of delivery vector can be used for intranasal delivery, which might bring AAVs back into the equation as a potentially scalable option. The main drawback of the intranasal route is that the vast majority of delivered DNA accumulates in the olfactory bulb and adjacent brain regions.42,44 Roughly, as the distance from these regions increases, the amount of DNA delivered decreases.44 In a study by Chukwu et al., intranasal delivery of AAV9 was shown to achieve 15% transduction efficiency and 9% gene expression efficiency on average across the brain compared to intravenous delivery.44 Remarkably, this intranasal delivery decreased exposure of peripheral organs by a factor of 13,400 compared to intravenous injection. It should be noted that AAV9 has limited BBB crossing efficiency compared to optimized capsids like AAV BIhTFR1.31 Indeed, intravenous AAV BIhTFR1 transduces brain 40-50 times more efficiently than intravenous AAV9 in humanized TfR1 mice. Yet overall, I would speculate that novel intranasal delivery systems have strong potential for safer and more scalable gene therapies. The intranasal route deserves serious consideration.

Translational Strategies

The path to psychiatric gene therapy may require a detour focusing on “easier” high-prevalence brain disease indications with more clearly defined clinical endpoints. This detour will allow the field to consolidate, cultivating enough successes to justify the financial risk of pursuing psychiatric diseases. Additionally, manufacturing, regulatory, and clinical infrastructure for brain gene therapy in large patient populations may establish itself in this way. That said, I do think it would be beneficial for companies to pursue psychiatric indications in parallel. Even if these initial attempts do not pan out, they may help strengthen the field’s knowledge base and infrastructure. What are some high-prevalence brain indications with clear clinical endpoints which represent strong potential targets for early brain delivery? I will start by nominating stroke, Parkinson’s disease (PD), and epilepsy (open to suggestions here). Though it will by no means be easy to develop efficacious gene therapies for such conditions, I remain optimistic that this foundation of successes in brain treatment is attainable.

In my view, sonogenetic genes have immense potential as payloads for psychiatric gene therapy. Sonogenetics broadly speaking involves delivery of genes encoding mechanosensitive proteins, often ion channels.45 The mechanosensitive proteins change state (e.g. channel opening) in response to ultrasound waves, allowing neuromodulation through transcranial focused ultrasound (tFUS). Sonogenetic gene therapy should thus enable both millimeter-scale spatial resolution and cell-type-specific targeting for neurostimulation, offering unprecedented possibilities for treatment of psychiatric diseases.14–17 This technological convergence could radically transform how mental illness is treated. But delivery nonetheless remains among the most central challenges which must be overcome before sonogenetics reaches clinical feasibility. Neuromedicine cannot explore sonogenetic therapies without a strong foundation of enabling delivery systems.

From a translational perspective, one of the greatest strengths of sonogenetics is that the different effects of stimulating a chosen neuronal cell type across numerous human brain regions may rapidly be tested. While this could have benefits at the preclinical level as well, the most important outcomes will likely occur during clinical stage testing. As an example, consider an anxiety disorder patient who has received a gene therapy which expresses mechanosensitive ion channels in a brain-wide fashion across GABAergic neurons. A clinician might leverage tFUS stimulation in the patient’s lateral amygdala for a few weeks.46,47 If that strategy did not improve the patient’s symptoms, the clinician may easily switch the tFUS stimulation to the central amygdala region46 or the bed nucleus of the stria terminalis (BNST)48 or the lateral septum.49 Many distinct brain regions could be explored without needing to develop a new therapeutic. This would allow fast clinical screening of strategies for modulating neural circuits towards better mental health. Since the challenges of clinical trials represent a massive limiting factor for therapeutics in general, the ability to quickly explore this space of possibilities could dramatically accelerate discovery. Combining the incredible precision of sonogenetic tFUS with such a rapid screening strategy may reveal superior therapeutic targets for combating mental illness.

Conclusion

As I have discussed in this essay, a lack of scalable delivery systems is a central roadblock to the promise of psychiatric gene therapy. Because of this, I have begun developing strategies for overcoming the scalable delivery problem through a number of ideas. I have done early-stage experiments towards a couple of these ideas while others remain in the ideation stage. (I cannot publicly describe the details of my ideas on the internet because public disclosure of IP precludes patentability). I should note that I am currently a graduate student and expect to complete my PhD in around six months. This article represents part of my efforts to lay the groundwork for my upcoming scalable brain delivery plans.

Mental illness represents one of the most profound challenges facing humanity. It affects how we live our lives and interact with the world. It affects people we love. It takes away precious time from people who otherwise could have been experiencing the extraordinary beauty of life and the universe. I believe that positive emotional experiences represent the most fundamental form of value in the cosmos. Mental illness blocks people off from experiencing joy, which is in my view an immeasurable tragedy that needs to be righted. It is time to do the science needed to reset our brains to live life to the fullest.

If you are interested in discussing anything related to this space, please reach out to: logan (dot) phospholipid (at) gmail (dot) com!

If you would like to read more about me and my scientific and entrepreneurial background, please check out my bio at: https://logancollinsblog.com/

References:

1.        Mental disorders (World Health Organization). https://www.who.int/news-room/fact-sheets/detail/mental-disorders (2025).

2.        Marx, W. et al. Major depressive disorder. Nat. Rev. Dis. Prim. 9, 44 (2023).

3.        Craske, M. G. et al. Anxiety disorders. Nat. Rev. Dis. Prim. 3, 17024 (2017).

4.        Leucht, S. et al. Schizophrenia. Nat. Rev. Dis. Prim. 11, 83 (2025).

5.        Vieta, E. et al. Bipolar disorders. Nat. Rev. Dis. Prim. 4, 18008 (2018).

6.        Yehuda, R. et al. Post-traumatic stress disorder. Nat. Rev. Dis. Prim. 1, 15057 (2015).

7.        Strang, J. et al. Opioid use disorder. Nat. Rev. Dis. Prim. 6, 3 (2020).

8.        MacKillop, J. et al. Hazardous drinking and alcohol use disorders. Nat. Rev. Dis. Prim. 8, 80 (2022).

9.        De Brito, S. A. et al. Psychopathy. Nat. Rev. Dis. Prim. 7, 49 (2021).

10.      Gunderson, J. G., Herpertz, S. C., Skodol, A. E., Torgersen, S. & Zanarini, M. C. Borderline personality disorder. Nat. Rev. Dis. Prim. 4, 18029 (2018).

11.      An, S. et al. Global prevalence of suicide by latitude: A systematic review and meta-analysis. Asian J. Psychiatr. 81, 103454 (2023).

12.      Hyman, S. E. Revitalizing Psychiatric Therapeutics. Neuropsychopharmacology 39, 220–229 (2014).

13.      Roth, B. L. Molecular pharmacology of metabotropic receptors targeted by neuropsychiatric drugs. Nat. Struct. Mol. Biol. 26, 535–544 (2019).

14.      Liu, T. et al. Sonogenetics: Recent advances and future directions. Brain Stimul. 15, 1308–1317 (2022).

15.      Xian, Q. et al. Modulation of deep neural circuits with sonogenetics. Proc. Natl. Acad. Sci. 120, e2220575120 (2023).

16.      Hahmann, J., Ishaqat, A., Lammers, T. & Herrmann, A. Sonogenetics for Monitoring and Modulating Biomolecular Function by Ultrasound. Angew. Chemie Int. Ed. n/a, e202317112 (2024).

17.      Tang, J., Feng, M., Wang, D., Zhang, L. & Yang, K. Recent advancement of sonogenetics: A promising noninvasive cellular manipulation by ultrasound. Genes Dis. 11, 101112 (2024).

18.      Miyakawa, N. et al. Chemogenetic attenuation of cortical seizures in nonhuman primates. Nat. Commun. 14, 971 (2023).

19.      Bendixen, L., Jensen, T. I. & Bak, R. O. CRISPR-Cas-mediated transcriptional modulation: The therapeutic promises of CRISPRa and CRISPRi. Mol. Ther. 31, 1920–1937 (2023).

20.      Doudna, J. A. The promise and challenge of therapeutic genome editing. Nature 578, 229–236 (2020).

21.      Zhao, M., Zhao, Z., Koh, J.-T., Jin, T. & Franceschi, R. T. Combinatorial gene therapy for bone regeneration: Cooperative interactions between adenovirus vectors expressing bone morphogenetic proteins 2, 4, and 7. J. Cell. Biochem. 95, 1–16 (2005).

22.      Won, Y.-W. et al. Synergistically Combined Gene Delivery for Enhanced VEGF Secretion and Antiapoptosis. Mol. Pharm. 10, 3676–3683 (2013).

23.      Wang, J. & Vos, J.-M. H. Infectious Epstein-Barr virus vectors for episomal gene therapy. in Gene Therapy Methods (ed. Phillips, M. I. B. T.-M. in E.) vol. 346 649–660 (Academic Press, 2002).

24.      Alba, R., Bosch, A. & Chillon, M. Gutless adenovirus: last-generation adenovirus for gene therapy. Gene Ther. 12, S18–S27 (2005).

25.      Collins, L. T., Ponnazhagan, S. & Curiel, D. T. Synthetic Biology Design as a Paradigm Shift toward Manufacturing Affordable Adeno-Associated Virus Gene Therapies. ACS Synth. Biol. 12, 17–26 (2023).

26.      Reid, C. A., Hörer, M. & Mandegar, M. A. Advancing AAV production with high-throughput screening and transcriptomics. Cell Gene Ther. Insights (2024).

27.      Cameau, E., Glover, C. & Pedregal, A. Cost modelling comparison of adherent multi-trays with suspension and fixed-bed bioreactors for the manufacturing of gene therapy products. Cell Gene Ther. Insights (2020).

28.      Smith, J., Grieger, J. & Samulski, J. Overcoming bottlenecks in AAV manufacturing for gene therapy. Immuno-oncology Insights (2018).

29.      Gangurde, R. & Winitsky, S. Gene therapy: are high costs and manufacturing complexities impeding progress? https://www.parexel.com/insights/blog/gene-therapy-are-high-costs-and-manufacturing-complexities-impeding-progress (2024).

30.      Au, H. K. E., Isalan, M. & Mielcarek, M. Gene Therapy Advances: A Meta-Analysis of AAV Usage in Clinical Settings. Front. Med. Volume 8-, (2022).

31.      Huang, Q. et al. An AAV capsid reprogrammed to bind human transferrin receptor mediates brain-wide gene delivery. Science (80-. ). 384, 1220–1227 (2024).

32.      Walpole, S. C. et al. The weight of nations: an estimation of adult human biomass. BMC Public Health 12, 439 (2012).

33.      Gorick, C. M. et al. Applications of focused ultrasound-mediated blood-brain barrier opening. Adv. Drug Deliv. Rev. 191, 114583 (2022).

34.      Batts, A. J. et al. A multifunctional theranostic ultrasound platform for remote magnetogenetics and expanded blood-brain barrier opening. Brain Stimul. Basic, Transl. Clin. Res. Neuromodulation 18, 1939–1951 (2025).

35.      Nouraein, S. et al. Acoustically targeted noninvasive gene therapy in large brain volumes. Gene Ther. 31, 85–94 (2024).

36.      Felix, M.-S. et al. Ultrasound-Mediated Blood-Brain Barrier Opening Improves Whole Brain Gene Delivery in Mice. Pharmaceutics vol. 13 1245 at https://doi.org/10.3390/pharmaceutics13081245 (2021).

37.      McMahon, D. & Hynynen, K. Acute Inflammatory Response Following Increased Blood-Brain Barrier Permeability Induced by Focused Ultrasound is Dependent on Microbubble Dose. Theranostics 7, 3989–4000 (2017).

38.      Kovacs, Z. I. et al. Disrupting the blood–brain barrier by focused ultrasound induces sterile inflammation. Proc. Natl. Acad. Sci. 114, E75–E84 (2017).

39.      Patwardhan, A. et al. Safety, Efficacy and Clinical Applications of Focused Ultrasound-Mediated Blood Brain Barrier Opening in Alzheimer’s Disease: A Systematic Review. J. Prev. Alzheimer’s Dis. 11, 975–982 (2024).

40.      Chen,  Hong & Konofagou,  Elisa E. The Size of Blood–Brain Barrier Opening Induced by Focused Ultrasound is Dictated by the Acoustic Pressure. J. Cereb. Blood Flow Metab. 34, 1197–1204 (2014).

41.      Shumer-Elbaz, M. et al. Low-frequency ultrasound-mediated blood-brain barrier opening enables non-invasive lipid nanoparticle RNA delivery to glioblastoma. J. Control. Release 385, 114018 (2025).

42.      Patel, D., Patel, B. & Wairkar, S. Intranasal delivery of biotechnology-based therapeutics. Drug Discov. Today 27, 103371 (2022).

43.      Crowe, T. P., Greenlee, M. H. W., Kanthasamy, A. G. & Hsu, W. H. Mechanism of intranasal drug delivery directly to the brain. Life Sci. 195, 44–52 (2018).

44.      Chukwu, C., Yuan, J. & Chen, H. Intranasal versus intravenous AAV delivery: A comparative analysis of brain-targeting efficiency and peripheral exposure in mice. Gene Ther. (2025) doi:10.1038/s41434-025-00585-y.

45.      Legon, W. & Strohman, A. Low-intensity focused ultrasound for human neuromodulation. Nat. Rev. Methods Prim. 4, 91 (2024).

46.      Babaev, O., Piletti Chatain, C. & Krueger-Burg, D. Inhibition in the amygdala anxiety circuitry. Exp. Mol. Med. 50, 1–16 (2018).

47.      Benarroch, E. E. The amygdala: Functional organization and involvement in neurologic disorders. Neurology 84, 313–324 (2015).

48.      Giardino, W. J. & Pomrenze, M. B. Extended Amygdala Neuropeptide Circuitry of Emotional Arousal: Waking Up on the Wrong Side of the Bed Nuclei of Stria Terminalis. Front. Behav. Neurosci. Volume 15–2021, (2021).

49.      Wang, D. et al. Lateral septum-lateral hypothalamus circuit dysfunction in comorbid pain and anxiety. Mol. Psychiatry 28, 1090–1100 (2023).

Amygdala Structure, Function, and Clinically Relevant Pathways

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Amygdala Structure, Function, and Clinically Relevant Pathways

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Amygdala Structure, Function, and Clinically Relevant Pathways – by Logan Thrasher Collins

Anatomy

The amygdala consists of nuclei which can be grouped into (i) the basolateral nuclear group (BLA), (ii) the superficial cortex-like laminated region (sCLR) which contains the cortical nuclei (Co), and (iii) the centromedial nuclear group.1 The BLA consists of the lateral nucleus (LA) and basal nucleus (BA). In turn, the BA consists of the basolateral nucleus and the basomedial nucleus. The centromedial nuclear group consists of the central nucleus (Ce), medial nucleus (Me), and intercalate cell mass (IC). In turn, Ce consists of a lateral (CeL) subdivision and a medial (CeM) subdivision. The centromedial nuclear group (Ce, Me, and IC) along with the bed nucleus of the stria terminalis (BNST) and sublenticular substantia innominata together comprise the centromedial extended amygdala.

The cellular composition of the BLA nuclei and the sCLR’s Co nuclei resembles that of the cerebral cortex in that the majority of the neurons are pyramidal-like glutamatergic cells while the rest are local GABAergic inhibitory interneurons.1 The inhibitory interneurons include parvalbumin-containing neurons which mainly synapse on the soma and proximal dendrites of the pyramidal cells and somatostatin-containing neurons which mainly synapse on the distal dendrites of the pyramidal neurons. By contrast, the composition of the Ce and Me nuclei resembles the striatum in that many of the neurons are similar to GABAergic medium spiny neurons.

Overview of Amygdala Connectivity

Signals flow into the amygdala primarily via synapses in the BLA. Inputs from cortical sensory areas and from the thalamus (relaying subcortical signals) synapse on neurons in the LA.1 These inputs in the LA facilitate coincidence detection and associative learning tying together the sensory cortical representations of the world with subcortical information coming in via the thalamus. The LA pyramidal-like neurons send excitatory signals to the BA’s projection neurons and to the BA’s interneurons. (It should be noted that the subnuclei of the BA are also interconnected with the prefrontal cortex, hippocampus, and striatum). Next, the BA projects glutamatergic inputs to the CeM’s GABAergic projection neurons. These BA glutamatergic projections additionally synapse on the inhibitory interneurons of the IC and the CeL, both of which regulate the CeM neurons. (An additional layer of complexity comes from further inhibitory interneuron circuits within the LA, BA, IC, CeL, and CeM). Finally, the CeM’s GABAergic projection neurons send output signals to the hypothalamus and brainstem.

Sensory inputs to the amygdala’s LA come from several sources.1 Sensory association areas of the temporal cortex carry visual and auditory information. These areas are part of the ventral stream of sensory processing, which encodes analyses of complex features to facilitate face recognition and auditory recognition. The insular cortex, which encodes somatosensory and visceral sensations, also sends inputs to the LA. Subcortical sensory inputs to the LA come via the thalamus. In addition to LA inputs, the CeM receives visceral and nociceptive inputs directly from the pons. The sCLR receives olfactory input from the olfactory bulb as well as from higher olfactory areas.

Interestingly, the amygdala sends outputs back to cortical sensory association areas as well as primary sensory areas.1 These modulate the valence of specific sensory stimuli, which can be thought of as a way to assign emotional value to particular stimuli.

The amygdala has strong bidirectional interactions with the orbitofrontal cortex (OFC).1 In particular, the OFC receives strong inputs from the BA and targets the IC’s GABAergic neurons. The amygdala also interacts with the dorsal anterior cingulate cortex (dACC) and ventral anterior cingulate cortex (vAAC). The BA sends outputs to the dACC while the vACC projects back to the BA. The BA projects to the entorhinal cortex and receives inputs from the hippocampus as well, which may help tie emotional significance of particular events undergoing processing to associated memories. Finally, the amygdala receives subcortical inputs from arousal systems, including basal forebrain cholinergic inputs, ventral tegmental area (VTA) dopaminergic inputs, noradrenergic locus coeruleus inputs, and rostral raphe serotonergic inputs. The amygdala also projects back to all of these neuromodulatory regions and can influence the arousal systems.

CeM outputs to the hypothalamus and brainstem facilitate visceral behavioral responses to fear. These projections trigger various endocrine and autonomic peripheral nervous system responses such as secretion of adrenocorticotropic hormone (ACTH) into the blood and increased activation of the sympathetic nervous system.

Fear Learning

As mentioned earlier, the convergence of cortical inputs and subcortical inputs onto LA neurons facilitates associative learning between neutral stimuli and unpleasant stimuli. The neutral stimulus is often referred to as the “conditioned stimulus” (CS) while the unpleasant stimulus is referred to as the “unconditioned stimulus” (UC). In classical animal studies, the CS might take the form of a neutral sound (e.g. a tone) while the UC is often an electrical shock to the feet. It is variable as to which input pathways carry information about the CS and which input pathways carry information about the UC.2

In auditory fear learning, both the subcortical thalamic pathway afferents and the auditory cortex afferents have been shown to carry sensory CS information into the LA.2 When an animal must discriminate between two distinct CS sounds to learn which sound is associated with a foot shock UC, the auditory cortical pathway is thought to be necessary because plasticity in the auditory cortex facilitates the discriminative learning. Interestingly, the primary auditory cortex has been shown to carry information about complex multifrequency sounds into the LA while the more ventral associative areas of the auditory cortex bring information about simpler tone sounds.

Both the cortical and subcortical pathways have also been shown to carry parts of the UC. In particular, the parabrachial nucleus of the brainstem has been shown to encode nociceptive UC information. To transfer this information, the parabrachial nucleus projects to the CeM and CeL nuclei of the amygdala.2–4 However, the parabrachial nucleus does not project to the LA.2 It remains unknown if there is a separate (probably glutamatergic) input to the LA which carries aversive information for associative fear learning.

It should also be noted that evidence implicates neuromodulatory systems as carrying part of the UC signal during fear learning. Locus coeruleus noradrenergic projections have been shown to contribute about half of the strength of the fear learning signal.5 That is, when silenced during fear learning in rats, a 50% decrease in learned fear occurred. Additionally, a subpopulation of dopaminergic neurons from the VTA which projects to the BA has been shown to contribute about 30% of the strength of the fear learning signal.6 That is, when silenced during fear learning in mice, a 30% decrease in learned fear occurred. Acetylcholine inputs from the basal forebrain into the BLA have also been demonstrated to be necessary for efficient fear learning.7 These neuromodulators may also be released, though to a lesser degree, during fear memory recall. Finally, serotoninergic neurons (especially from the raphe nuclei) have been implicated to sometimes act on the 5-HT1A receptors of GABAergic interneurons of the LA to inhibit fear learning in LA pyramidal cells.1 That said, serotonin can have other effects in the BLA and its influence is not fully understood.8

Amygdala, Emotion, and Anxiety

The amygdala represents a central part of circuits relating to fear and anxiety as well as of circuits of general emotional valence. Elevated amygdala activity with decreased top-down regulation from the vmPFC has been shown in people with higher anxiety.1,9 It is important to note that the vmPFC overlaps with the ACC and OFC, which were discussed earlier. The vmPFC can facilitate the process of fear extinction: the decline of a learned fear via repeated exposure of a neutral CS without the associated aversive UC. As such, decreased functional connectivity between the amygdala and vmPFC is common in people with anxiety disorders.

The vmPFC facilitates fear extinction by sending excitatory input from the OFC to GABAergic neurons in the IC, which then inhibit the BA’s inputs to the CeM. The BA itself also sends excitatory projections up to the vmPFC which can induce the vmPFC’s fear extinction circuits. A distinct group of excitatory neurons in the BA target the dACC, which then sends excitatory projections back to the amygdala’s BLA to facilitate fear learning (in contrast to the vmPFC projections).10,11 Indeed, LTP occurring via this circuit within the dACC contributes to the formation and maintenance of fear memory. In this way, the dACC is a direct part of the learning network which creates fear memories.

Inhibitory interneurons within the amygdala act as important regulators of anxiety responses.12 In the BLA, inhibitory interneurons can suppress the magnitude of anxiety by releasing GABA onto the pyramidal projection neurons. Inhibitory interneurons in the CeL can also constrain the activity of amygdala output projection neurons of the CeM, which leads to decreased fear behavior. But it should be noted that the BLA can receive sensory input associated with either threatening or rewarding stimuli. Because of this, its projection neurons trigger different behavioral responses (threat or reward behaviors) depending on the nature of the stimulus.

There exist non-overlapping populations of putative projection neurons in the BLA which are thought to fire in response to threat and reward stimuli separately.12,13 These populations are thought to develop via the Hebbian associative learning described previously, which leads to formation of fear pathways for some stimuli, but can also promote association of rewarding stimuli with neutral stimuli and thus form learned emotional pathways of positive valence.13 Additionally, inhibitory interneurons of the BLA suppress threat-related projection neurons when reward-related projection neurons are active and vice versa. With anxiety disorders, these interneuron circuits are frequently dysregulated in that negative valence is assigned to neutral or reward stimuli, leading to activation of only the threat-related projection pathway.

Extended Networks of the Amygdala and Anxiety

As has been discussed to some degree so far, the amygdala does not function in isolation. It makes numerous reciprocal connections with other brain areas to facilitate its operation. Some of the most important of these include cortical regions like the vmPFC, OFC, and ACC, which were discussed earlier. But extended subcortical structures like the BNST and hippocampus (which have so far only been mentioned briefly) also play major roles.

The BNST is a collection of nuclei nearby to the amygdala which is recruited during sustained fear and anxiety responses.13 It is thought that the BNST specifically activates during prolonged stressful periods of greater than 10 minutes in duration.14 The BLA sends glutamatergic projections into the BNST’s anterodorsal (ad) nucleus. Interestingly, these excitatory inputs to the BNST ad nucleus promote anxiolytic outcomes. Additionally, local inhibition of the ad nucleus from the BNST’s oval (ov) nucleus promotes anxiogenic outcomes. The BNST’s ad nucleus facilitates anxiolytic states by sending its own (predominantly) GABAergic projections to the VTA to increase positive emotional valence, to the lateral hypothalamus (LH) to decrease risk avoidance, and to the parabrachial nucleus of the brainstem to decrease respiration rate.15

BNST ad projections to the VTA are mostly GABAergic neurons synapsing onto VTA inhibitory interneurons.16 It should be noted that there are also ventral BNST (vBNST) GABAergic and glutamatergic projections which synapse onto different populations of VTA inhibitory interneurons, triggering anxiogenic phenotypes and anxiolytic phenotypes respectively.17 A major population of BNST ad projections to the lateral hypothalamus are GABAergic neurons preferentially synapsing onto GABAergic target neurons. Among these is a subpopulation of GABAergic projection neurons targeting GABAergic lateral hypothalamus neurons which also produce orexin (a neuropeptide which stimulates food intake behaviors and promotes wakefulness).16 BNST ad GABAergic projections to the parabrachial nucleus probably inhibit glutamatergic neurons which themselves would otherwise signal for increased respiratory rate.18

The amygdala also interacts with the hippocampus. As mentioned earlier, the BLA sends excitatory inputs to the hippocampal formation by first synapsing at the entorhinal cortex (EC), which then sends its own excitatory inputs to the hippocampus.13 These inputs are necessary for acquisition of contextual fear memories, likely mediated by the BLA amygdala’s fear learning mechanism in combination with hippocampal memory representations.

In addition, the BLA sends glutamatergic synapses directly onto pyramidal cells in the ventral hippocampus (vHPC) CA1 region, increasing anxiety-like behavior when these BLA projections are active. In part, the vHPC mediates its effects on anxiety through glutamatergic projections to the lateral septum, which sends its own projections onwards to the hypothalamus. The vHPC glutamatergic projections stimulate activation of corticotropin releasing factor receptor 2 (CRFR2) expressing GABAergic projection neurons in the lateral septum through a mechanism which is not fully understood.13,19 These GABAergic projection neurons inhibit the anterior hypothalamic area (AHA), which itself inhibits the paraventricular nucleus (PVN) of the hypothalamus as well as the periaqueductal gray (PAG). In this way, the lateral septum disinhibits the paraventricular nucleus and the periaqueductal gray, which leads to neuroendocrine and behavioral outcomes associated with persistent anxiety.

Conclusion

While this writeup serves as an initial primer on the amygdala, there remain a plethora of relevant neural circuits to explore beyond what has been described here. Nonetheless, I hope that the information provided will offer a useful starting point for learning about the amygdala’s structure, function, and effects on mammalian emotions. As further reading, I specifically recommend references #1, #9, #12, and #13.

References

1.      Benarroch, E. E. The amygdala: Functional organization and involvement in neurologic disorders. Neurology 84, 313–324 (2015).

2.      Palchaudhuri,  Shriya, Osypenko,  Denys & Schneggenburger,  Ralf. Fear Learning: An Evolving Picture for Plasticity at Synaptic Afferents to the Amygdala. Neurosci. 30, 87–104 (2022).

3.      Han, S., Soleiman, M. T., Soden, M. E., Zweifel, L. S. & Palmiter, R. D. Elucidating an Affective Pain Circuit that Creates a Threat Memory. Cell 162, 363–374 (2015).

4.      Herry, C. & Johansen, J. P. Encoding of fear learning and memory in distributed neuronal circuits. Nat. Neurosci. 17, 1644–1654 (2014).

5.      Uematsu, A. et al. Modular organization of the brainstem noradrenaline system coordinates opposing learning states. Nat. Neurosci. 20, 1602–1611 (2017).

6.      Tang, W., Kochubey, O., Kintscher, M. & Schneggenburger, R. A VTA to Basal Amygdala Dopamine Projection Contributes to Signal Salient Somatosensory Events during Fear Learning. J. Neurosci. 40, 3969 LP – 3980 (2020).

7.      Jiang, L. et al. Cholinergic Signaling Controls Conditioned Fear Behaviors and Enhances Plasticity of Cortical-Amygdala Circuits. Neuron 90, 1057–1070 (2016).

8.      Bocchio, M., McHugh, S. B., Bannerman, D. M., Sharp, T. & Capogna, M. Serotonin, Amygdala and Fear: Assembling the Puzzle. Front. Neural Circuits Volume 10–2016, (2016).

9.      Zhang, W.-H., Zhang, J.-Y., Holmes, A. & Pan, B.-X. Amygdala Circuit Substrates for Stress Adaptation and Adversity. Biol. Psychiatry 89, 847–856 (2021).

10.    Toyoda, H. et al. Interplay of Amygdala and Cingulate Plasticity in Emotional Fear. Neural Plast. 2011, 813749 (2011).

11.    Jhang, J. et al. Anterior cingulate cortex and its input to the basolateral amygdala control innate fear response. Nat. Commun. 9, 2744 (2018).

12.    Babaev, O., Piletti Chatain, C. & Krueger-Burg, D. Inhibition in the amygdala anxiety circuitry. Exp. Mol. Med. 50, 1–16 (2018).

13.    Calhoon, G. G. & Tye, K. M. Resolving the neural circuits of anxiety. Nat. Neurosci. 18, 1394–1404 (2015).

14.    Hammack, S. E., Todd, T. P., Kocho-Schellenberg, M. & Bouton, M. E. Role of the bed nucleus of the stria terminalis in the acquisition of contextual fear at long or short context-shock intervals. Behavioral Neuroscience vol. 129 673–678 at https://doi.org/10.1037/bne0000088 (2015).

15.    Kim, S.-Y. et al. Diverging neural pathways assemble a behavioural state from separable features in anxiety. Nature 496, 219–223 (2013).

16.    Giardino, W. J. & Pomrenze, M. B. Extended Amygdala Neuropeptide Circuitry of Emotional Arousal: Waking Up on the Wrong Side of the Bed Nuclei of Stria Terminalis. Front. Behav. Neurosci. Volume 15–2021, (2021).

17.    Jennings, J. H. et al. Distinct extended amygdala circuits for divergent motivational states. Nature 496, 224–228 (2013).

18.    Kaur, S. et al. Glutamatergic Signaling from the Parabrachial Nucleus Plays a Critical Role in Hypercapnic Arousal. J. Neurosci. 33, 7627 LP – 7640 (2013).

19.    Anthony, T. E. et al. Control of Stress-Induced Persistent Anxiety by an Extra-Amygdala Septohypothalamic Circuit. Cell 156, 522–536 (2014).

An Introduction to Ebolavirus Biology

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An Introduction to Ebolavirus Biology

PDF version: An Introduction to Ebolavirus Biology – Logan Thrasher Collins 

I wrote this educational primer as a fun exploration of a topic not related to my current research. While such knowledge may be useful in the event of some future ebolavirus epidemic, it is mostly just an exercise in curiosity and intellectual enrichment. I hope that you too enjoy learning about this fascinating (but scary!) virus as you browse my writeup. Also, if you’re an ebolavirus expert with concepts, edits, and/or ideas to offer, feel free to reach out with your additional insights! Shoutout: I’d like to give a special shoutout/thanks to Jain et al. (reference 4) and Bodmer et al. (reference 2). I used their papers extensively throughout the creation of writeup!

Genome

The ebolavirus genome consists of an 18.9 kb negative-sense single-stranded RNA (ssRNA) which encodes seven genes.1,2 Each gene is flanked by a 3’ and 5’ untranslated region (3’UTR and 5’UTR) which contain start and end signals. The start signals have the consensus sequence of 3’-CUNCUUCUAAUU-5’ and the end signals have the consensus sequence 3’-UAAUUC(U)5/6-5’. Since 3’UAAUU-5’ is found in both the start and end signals, they can overlap and (for most types of ebolavirus) do so at the junctions between the VP35-VP40, GP-VP30, and VP24-L genes. The rest of the genes have intergenic regions with non-overlapping start and end signals between them.

The 5’ and 3’ ends of the genome contain elements called the 5’ trailer and 3’ leader. The 5’ trailer contains parts of the antigenomic replication promoter and the 3’ leader contains parts of the genomic replication promoter. There is also a second genomic replication promoter in the NP untranslated region. Genomic replication promoters initiate RNA-dependent RNA polymerase (RdRP) replication of the negative-sense ssRNA genome while antigenomic replication promoters initiate replication of the positive-sense copy version of the ssRNA genome.

In total, the ebolavirus genome encodes seven proteins.1 The seven proteins encoded by the ebolavirus genome include NP (nucleoprotein), VP24 (membrane-associated protein interfering with interferon signaling), VP30 and VP35 (polymerase matrix protein acting as interferon antagonist), L (the RdRP for replication), VP40 (matrix protein), and GP (glycoprotein).1,2 The proteins will be discussed with more detail in the next section.

The GP RNA itself undergoes mRNA editing, so the GP can take three different forms.2,3 The unedited GP mRNA (~80% of transcripts) encodes a precursor of soluble glycoprotein or sGP. The edited GP0 mRNA (~20% of transcripts)4,5 arises from viral polymerase stuttering at a slippage region sequence of seven consecutive uridines, which leads to addition of an adenosine and a frameshift allowing expression of GP1,2. VP30 may help facilitate resolution of a stem loop involved in the stuttering of the viral polymerase.6 Finally, sometimes either two adenosines are added or one adenosine is omitted from the mRNA (5% of transcripts), leading instead to expression of a small soluble GP precursor protein (ssGP).2,3

Structure

At a glance, ebolavirus consists of its ssRNA genome, a nucleocapsid and accessory proteins, and an envelope bearing its glycoproteins. The NP adopts a helical structure when complexed with the ssRNA genome, forming the nucleocapsid.7 VP35 and VP24 associate with the surface of the NP-RNA complex. VP40 forms the matrix between the envelope and the nucleocapsid. VP30 also binds the nucleocapsid and is important for transcription initiation.4 GP is a transmembrane protein which plays roles in cellular attachment and transduction.

NP

NP’s main function is to encapsidate the ssRNA genome, forming a helical ssRNA-NP complex (the nucleocapsid).2,8 The NPs form a left-handed helix with 24 subunits per turn. Each NP subunit binds six nucleotides of ssRNA via a positively charged cleft on the outside of the NP helix. The NP forms the core of a repeating asymmetric unit consisting of two NPs associated with two oppositely-oriented VP24 proteins, one of which in turn associates with a VP35 protein.

Image adapted from reference 8 (Sugita et al.)

In the cryo-EM structure8 above, the nucleocapsid helix of NP and ssRNA is displayed. VP24 and VP35 are not shown, though they also associate with the nucleocapsid.

VP24

VP24’s interactions and association with NP are required for nucleocapsid formation as well as for helping package the nucleocapsid into virions.4 It is involved in the initiation of viral budding. VP24 additionally inhibits the host cell’s immune responses. It inhibits IFN responses by blocking p38 phosphorylation, which inhibits the p38 MAPK pathway. It also can block NF-κB activation, precluding multiple downstream IFN gene expression pathways. VP24 can inhibit nuclear translocation of the phosphorylated transcription factor STAT1 by interacting with importins of the NPI-1 subfamily of importin-α.

VP35

VP35 is a tetrameric protein which plays a structural role in ebolavirus by associating with the surface of the nucleocapsid. It furthermore acts as a polymerase cofactor which bridges the NP-RNA complex with L (the polymerase) during replication.7 It has helicase and NTPase activities, which indicate that it may unwind RNA helices and hydrolyze NTPs to facilitate transcription and replication.4 VP35 also helps facilitate genome packaging and nucleocapsid assembly.

In addition, VP35 inhibits host cell immune responses.4 It interferes with the dimerization, phosphorylation, and nuclear localization of interferon regulatory factor 3 (IRF-3). It accomplishes this by preventing proteins TBK-1 and IKKε from interacting with IRF-3. Under normal circumstances, phosphorylation and dimerization of IRF-3 causes it to translocate to the nucleus and induce transcription of IFNα, IFNβ, and other genes. VP35 furthermore suppresses interferon transcription by enhancing SUMOylation of IRF-7 via interaction with PIAS1 (a type of SUMO ligase). VP35 also blocks PACT (which prevents activation of PACT-induced RIG-I ATPase) as well as inactivating PKR.

VP30

VP30 forms a hexamer composed of three dimers.4 It is required for RNA transcription initiation. It should be noted that VP30 has a disordered arginine-rich region in the middle of its sequence which interacts with the viral RNA. VP30 also interacts with NP, an interaction which must occur at a certain threshold level for optimal transcriptional activity. VP30 binds zinc, an essential capability for viral transcription initiation.

For transcription to occur, VP30 must either exhibit no phosphorylation (on serines and threonines) or have only partial phosphorylation along with constant phosphorylation-dephosphorylation activity. Partial phosphorylation is only acceptable at some stages of viral replication. By contrast, when it is phosphorylated, VP30 binds NP more robustly. This allows it to tightly associate with the nucleocapsid as new ebolavirus particles are synthesized.

VP40 (matrix protein)

Ebolavirus VP40 is abundantly expressed and associates with the plasma membrane of the host cell, where it facilitates viral assembly and budding.4 It contains two late budding domains (L-domains) of four amino acids each: PTAP and PPEY. The PTAP domain interacts with tumor susceptibility gene 101 protein (tsg101), which recruits VP40 to lipid raft domains on the plasma membrane. PPEY interacts with ubiquitin ligase Nedd4 and ubiquitin ligase ITCH E3, causing ubiquitination of the matrix proteins in certain ways, a requirement for budding.

VP40 can form dimers, hexamers, filaments, and octamers. Dimerization of VP40 is essential for binding to Sec24c and trafficking to the plasma membrane. Sec24c is a component of the coat protein complex II (COPII) which facilitates formation of transport vesicles traveling from the endoplasmic reticulum to the Golgi apparatus, enabling eventual transport to the plasma membrane.9 Dimers can assemble into filaments via VP40’s C-terminal domain residues, which is crucial for matrix assembly and budding.4

VP40 contains a C-terminal domain with a hydrophobic interface which penetrates the plasma membrane to anchor the matrix protein and facilitate assembly and budding.10 Interestingly, VP40 has been shown to selectively anchor onto the plasma membrane via interactions with the enriched anionic phospholipids like phosphatidylserine found in the plasma membrane. At the membrane, the dimers assemble into linear hexamers which are also important for assembly and budding. VP40 can additionally form octameric rings which are essential for VP40-ssRNA binding. Oligomers of VP40 have also been implicated as inhibitory regulators of viral transcription.11

L protein

The L protein is the RNA-dependent RNA polymerase (RdRP) of the ebolavirus.4 It is a fairly large (2212 amino acids) protein consisting of five domains: (i) the RdRP domain which facilitates transcription and replication and polyadenylation, (ii) the capping domain which has polyribonucleotidyl transferase activity, (iii) a connector domain, (iv) a methyltransferase domain, and (v) a small C-terminal domain. The capping domain transfers a GDP to the 5’ phosphate of the viral mRNA. The methyltransferase then methylates the first nucleotide at the 2’-O position and the guanosine cap at the N-7 position. The small C-terminal domain plays a role in recruiting RNAs before methylation.

Additionally, the first 450 amino acids contain a homo-oligomerization domain which overlaps with the RdRP domain. The first 380 amino acids furthermore contain a domain for interaction with the VP35 protein, allowing localization of the L protein into viral inclusion bodies during assembly.

GP

GP (glycoprotein) is a fusogenic transmembrane protein.4 It has a cathepsin binding site which is cleaved inside the endosome as a step in viral infection. It is also post-translationally cleaved by furin from its precursor GP0 to make GP1 and GP2 subunits, which remain linked by disulfide bonds. Three GP1,2 complexes associate to form the trimeric GP that is displayed on the ebolavirus envelope surface.5

GP1 mediates attachment to host cell receptors via its receptor binding domain (RBD).4 There is a heavily glycosylated mucin-like domain (MLD) in GP1, which can stimulate host dendritic cells by activating their MAPK and NF-κB pathways. GP1 furthermore contains another important heavily glycosylated domain called the glycan cap (though it is in the middle of the GP1 sequence).12

GP2 facilitates fusion of viral envelopes with host cell membranes. It does this by inserting a hydrophobic loop domain into the endosomal membrane, bringing the envelope into close contact with said endosomal membrane.4 GP2 also contains a transmembrane anchor domain to help tether the GP to the envelope. GP2 furthermore can inhibit cellular antiviral responses. Firstly, it interferes with tetherin activity (tetherins are host cell proteins that aim to prevent viral budding by “tethering” the virus). It also interferes with NF-κB signaling pathways. GP2 can additionally trigger lymphocyte apoptosis and cytokine dysregulation via an immunosuppressive C-terminal motif.

GP is subject to heavy post-translational glycosylation, protecting the protein against host antibodies.4 GP1 contains 95 glycosylation sites and GP2 contains an additional 2 known glycosylation sites. In particular, MLD is highly glycosylated, allowing it to mask cell-surface proteins like MHC-I (thus inhibiting CD8+ T cell responses).

GP1,2 can be cleaved away from the viral envelope by the host enzyme TACE (TNF-α converting enzyme), leading to shed GP.4,13 The shed GP can sequester antibodies, acting as an immunological decoy. Shed GP furthermore contributes to triggering various inflammatory cytokines.

GP1,2 expression makes up only 20% of the total expressed protein from the GP gene.4,5 The other 80% consists of soluble secreted glycoprotein (sGP), Δ peptide, and small soluble secreted glycoprotein (ssGP). Both the GP1,2 and the ssGP are transcribed only when different ribosomal stuttering events occur during transcription as described earlier (in the genome section).

The sGP is a secreted protein which may serve as an immunological decoy which (as with shed GP) binds antibodies and thereby reduces the available antibodies that can bind to the virus itself.4,5 It has 7 glycosylation sites. In addition, sGP might inhibit inflammatory cytokines and chemokines, further helping the virus evade immunological responses.4

A small C-terminal region of sGP can be cleaved off to form Δ peptide.4,5 The Δ peptide also acts as an immunological decoy. Δ peptide can inhibit entry of ebolavirus into certain cells, preventing superinfection. In addition, Δ peptide may act as a viroporin, forming pores in mammalian cells.

The ssGP consists of an N-terminal region of 295 amino acids which are identical to GP0 and sGP and a C-terminal region of 3 amino acids which are distinct. It is secreted as a disulfide bonded homodimer which undergoes glycosylation. Its function remains unknown.4

Life Cycle

Attachment

Ebolavirus begins its life cycle by leveraging GP1,2 to attach to host cell receptors.2 There are three known mechanisms for attachment including (i) binding of C-type lectins, (ii) interaction with phosphatidylserine-binding receptors, and (iii) antibody-dependent enhancement.

C-type lectins bind the GP’s glycans found on the MLD as well as the glycan cap.2 Such lectins are mainly expressed on antigen presenting cells (dendritic cells, monocytes, macrophages, etc.) which are a primary target cell of ebolaviruses. However, they are not required or sufficient for entry, so they act as accessory receptors.

During budding, ebolavirus incorporates the host scramblase XKR8 into its envelope (via interactions with GP1,2),2 which randomly swaps phospholipids between inner and outer membrane leaflets. It should be noted that other scramblases like TMEM16F might also be used by the virus in the same way. The scramblases expose phosphatidylserine on the envelope’s surface (phosphatidylserine is normally found on the inner leaflet rather than the outer leaflet). As a result, phosphatidylserine receptors (TIM-1, TIM-4, Axl, and Mer) on the host cell membrane can bind the phosphatidylserine on the viral envelope. Since exposure of phosphatidylserine is normally used by the host to induce phagocytosis of apoptotic cell debris, the presence of phosphatidylserine on the ebolavirus envelope targets it for uptake into phagocytes. This is called “apoptotic mimicry”.

Antibody-dependent enhancement is when anti-ebolavirus antibodies bind to the virus and immune cell Fc receptors bind the antibodies.2  Complement factor C1q can also bind the ebolavirus-antibody complexes and attach virions to immune cell surfaces. Though these pathways normally facilitate clearing of viruses by endocytic uptake and degradation, ebolavirus may leverage the process for infection instead.

Endosomal trafficking and fusion

After binding the cell surface, the ebolavirus is endocytosed via macropinocytosis, preferentially near host cell membrane lipid rafts.2 Virions are trafficked from the early endosome to the late endosome. In the late endosome, the GP’s MLD and glycan cap are cleaved off by cathepsin B, cathepsin L, and/or other host cell proteases. This allows the GP to bind the intracellular receptor NPC1 (Niemann–Pick C1), which is found on the inner surface of the endosome. Low pH in the endosome causes acidification in the virus, which triggers disassociation of the VP40 matrix protein from the envelope, granting the virus more flexibility. It is thought that this flexibility may represent an additional prerequisite for fusion. Finally, the GP experiences a conformal change that causes insertion of GP2’s hydrophobic loop domain into the endosomal membrane, facilitating fusion with the envelope (a process dependent on certain pH and Ca2+ levels). After fusion, the nucleocapsid is released into the cytosol.

Transcription

Condensed nucleocapsids in the cytosol next begin RNA synthesis.2 To do this, they use a ribonucleoprotein complex consisting of L, NP, VP35, and VP30. Primary transcription relies on proteins from the incoming virion while secondary transcription can also utilize proteins newly produced inside the host cell. L (along with its VP35 cofactor) catalyzes RNA polymerization as well as methylation (capping) of viral mRNAs as discussed earlier.

Cytosolic transcription initiated by the polymerase complex is assisted by VP30.2 L starts at a site at the 3’ end of the genome and scans for the start signal of the first gene, which is the NP gene. The mRNA’s polyadenylation is triggered via polymerase slippage at the poly-uridine end signals which were discussed previously. L continues scanning the genome for the next start signal. It should be noted that scanning can occur in both directions along the genome. If the polymerase disassociates from the genome during scanning, it must return to the 3’ end to reinitiate. Because of this, genes close to the 3’ end of the genome are transcribed at a higher level than genes towards the 5’ end, a transcriptional gradient which might have functional significance.

Primary transcription occurs within 1-2 hours after infection.2 After ~10 hours post-infection, ebolavirus causes the formation of cytosolic inclusion bodies that serve to facilitate secondary transcription and genome replication. These inclusion bodies are rich in L, NP, VP35, and VP30 as well as VP24, VP40, and certain host proteins such as CAD, STAU1, SMYD3, RBBP6, PEG10, hnRNP L, and RUVBL1. Ebolavirus inclusion bodies occur as membraneless phase-separated condensates driven by NP oligomerization interactions.  

Viral mRNAs are exported from inclusion bodies by recruiting host NFX1 (nuclear RNA export factor 1).2 NFX1 binds mRNAs within inclusion bodies and transports them out into the cytosol, where translation can occur. It has been shown that hypusinated eIF5A (eukaryotic initiation 5A) is required for viral mRNA translation. Note that hypusination is a post-translational modification where a lysine in eIF5A is converted to a non-canonical amino acid called hypusine.14 In addition, ADAR1 (adenosine deaminase acting on RNA 1) edits 3’ untranslated regions within viral mRNAs and thus alters some of their negative regulatory elements to no longer downregulate translation.2

The transition from transcription to replication is thought to involve VP30 phosphorylation.2 Non-phosphorylated VP30 associates more strongly with the L-VP35 complex than its phosphorylated form. Phosphorylation of VP30 (and its lower affinity for L-VP35 in this form) may shift the focus of L-VP35 towards replication. When VP30 is phosphorylated, it also interacts more strongly with NP, which helps VP30 incorporate itself into new virions. That said, this process is not fully understood. Cellular kinases (SRPK1 and SRPK2) and phosphatases (PP2A-B56 and PP1) are sequestered into viral inclusion bodies to facilitate VP30 phosphorylation and dephosphorylation.

Replication

Only L, VP35, and NP are needed for ebolavirus replication (unlike transcription, which also needs VP30).2 For replication, the genome is copied into an antigenome. Replication is initiated at the first C in the genome, which is actually position 2 in the sequence. As a result, the copies initially lack the 3’-terminal nucleotide. To fix this, it is believed that the 3’ region of the RNA folds into a hairpin structure which back-primes addition of the missing nucleotide. Both the genome and the antigenome are encapsidated by NP.2 During the replication process, VP35 acts as a chaperone for monomeric NP that has not yet bound RNA. VP24 may cause nucleocapsids to transition from a relaxed state to a more condensed state.

Assembly and budding

After release from inclusion bodies, the nucleocapsids are transported along actin filaments to the plasma membrane where budding takes place.2 GP1,2 reaches the plasma membrane through the secretory pathway since it is a transmembrane protein. VP40 mediates budding by taking over parts of the host’s ESCRT (endosomal sorting complex required for transport) pathway. VP40 has a motif which recruits Tsg101 (an ESCRT-I component) to lipid rafts in the membrane. VP40 also has a motif which interacts with ubiquitin protein ligases (NEDD4, ITCH, WWP1, and SMURF2). These ubiquitin ligases ubiquitinate VP40, which facilitates its activity in budding. VP40 may also induce curvature across membrane phospholipids via its oligomerization. VP40 has a basic patch in its C-terminal domain which interacts with phosphatidylserine, which causes phosphatidylserine to cluster. Indeed, it has been shown that proper matrix layer formation requires phosphatidylserine clustering, so the interaction likely has functional importance.

References

1.      Ghosh, S., Saha, A., Samanta, S. & Saha, R. P. Genome structure and genetic diversity in the Ebola virus. Curr. Opin. Pharmacol. 60, 83–90 (2021).

2.      Bodmer, B. S., Hoenen, T. & Wendt, L. Molecular insights into the Ebola virus life cycle. Nat. Microbiol. 9, 1417–1426 (2024).

3.      Martin, B., Hoenen, T., Canard, B. & Decroly, E. Filovirus proteins for antiviral drug discovery: A structure/function analysis of surface glycoproteins and virus entry. Antiviral Res. 135, 1–14 (2016).

4.      Jain, S., Martynova, E., Rizvanov, A., Khaiboullina, S. & Baranwal, M. Structural and Functional Aspects of Ebola Virus Proteins. Pathogens vol. 10 at https://doi.org/10.3390/pathogens10101330 (2021).

5.      Lee, J. E. & Saphire, E. O. Ebolavirus Glycoprotein Structure and Mechanism of Entry. Future Virol. 4, 621–635 (2009).

6.      Mehedi, M. et al. Ebola Virus RNA Editing Depends on the Primary Editing Site Sequence and an Upstream Secondary Structure. PLOS Pathog. 9, e1003677 (2013).

7.      Fujita-Fujiharu, Y. et al. Structural basis for Ebola virus nucleocapsid assembly and function regulated by VP24. Nat. Commun. 16, 2171 (2025).

8.      Sugita, Y., Matsunami, H., Kawaoka, Y., Noda, T. & Wolf, M. Cryo-EM structure of the Ebola virus nucleoprotein–RNA complex at 3.6 Å resolution. Nature 563, 137–140 (2018).

9.      Mancias, J. D. & Goldberg, J. Structural basis of cargo membrane protein discrimination by the human COPII coat machinery. EMBO J. 27, 2918–2928 (2008).

10.    Adu-Gyamfi, E. et al. The Ebola Virus Matrix Protein Penetrates into the Plasma Membrane. J. Biol. Chem. 288, 5779–5789 (2013).

11.    T., H. et al. Oligomerization of Ebola Virus VP40 Is Essential for Particle Morphogenesis and Regulation of Viral Transcription. J. Virol. 84, 7053–7063 (2010).

12.    Peng, W. et al. Glycan shield of the ebolavirus envelope glycoprotein GP. Commun. Biol. 5, 785 (2022).

13.    Ning, Y.-J., Deng, F., Hu, Z. & Wang, H. The roles of ebolavirus glycoproteins in viral pathogenesis. Virol. Sin. 32, 3–15 (2017).

14.    McKenna, S. The first step of hypusination. Nat. Chem. Biol. 19, 664 (2023).

Selection of Intriguing Nontraditional Funding Opportunities

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Selection of Intriguing Nontraditional Funding Opportunities

During my pursuits, I’ve come across an increasing number of exciting nontraditional routes for funding scientific research. The efforts of Adam Marblestone and Benjamin Reinhardt have been particularly instrumental in stimulating this ecosystem, but many other great people have contributed as well. These new funding routes are a welcome relief since many of the most innovative and far-reaching projects are not especially suited for receiving governmental NIH, NSF, etc. funding. If you would like to find a more comprehensive list of such alternative funding sources, you should check out https://arbesman.net/overedge/. My own list (below) consists of funding sources that stand out to me as particularly promising. I hope you find this useful and feel free to reach out if you have any questions!

PDF version

Amaranthe Foundation https://amaranth.foundation/bottlenecks-of-aging “We outline initiatives which, if executed, could meaningfully accelerate the advancement of aging science and other life-extending technologies. The resulting document is a philanthropic menu, for which Amaranth is seeking both talent to execute on and co-funders. If you are a founder, researcher, or philanthropist interested in executing or co-sponsoring one or several of the projects or proposals below, please reach out to us”.

Arc Institute https://arcinstitute.org/ “Headquartered in Palo Alto, California, Arc is a nonprofit research organization founded on the belief that many important scientific programs can be enabled by new organizational models. Arc operates in partnership with Stanford University, UCSF, and UC Berkeley. Arc gives scientists no-strings-attached, multi-year funding, so that they don’t have to apply for external grants and invests in the rapid development of experimental and computational technological tools. As individuals, Arc researchers collaborate across diverse disciplines to study complex diseases, including cancer, neurodegeneration, and immune dysfunction. As an organization, Arc strives to enable ambitious, long-term research agendas. Arc’s mission is to accelerate scientific progress, understand the root causes of disease, and narrow the gap between discoveries and impact on patients.”

ARIA (Advanced Research + Invention Agency) https://www.aria.org.uk/ “ARIA empowers scientists to pursue breakthroughs at the edge of the possible. We have a big mission, and getting there won’t be easy. That’s why we’re building a new kind of research agency that does things differently. Created by an Act of Parliament, and sponsored by the Department for Science, Innovation, and Technology, ARIA will fund projects across the full spectrum of R&D disciplines, approaches, and institutions. ARIA’s programmes and projects are directed by our Programme Directors, scientific and technical leaders with deep expertise and a focused, creative vision for how technology can enable a better future. While Programme Directors are tasked with deeply exploring a topic and designing funding opportunities, they won’t get to breakthroughs alone. To maximise their chance at success, they’ll develop their thinking through direct calls for feedback, source projects through open solicitations, and have their programmes reviewed by experts against a clear set of evaluation criteria.”

Astera Institute https://astera.org/ “We empower visionary, high-leverage science and technology projects with the capacity to create transformative progress for human civilization. We target programs in Artificial General Intelligence, Science, and Climate that currently lack a natural home in the existing innovation ecosystem.”

Brains https://spec.tech/brains “Brains is a training program to provide the skills and opportunities to translate ambitious research visions that aren’t a good fit for a company but are too big for a single academic lab into impact. These visions could be anything from upending the way we make carbon-based products to how we understand the brain or build air- breathing fusion engines. Think YCombinator for coordinated research programs.”

Convergent Research https://www.convergentresearch.org/ “New types of organization are needed to accelerate scientific progress. Academic research groups and startup companies are essential to science and technology development. But there are some projects they just aren’t suited for. A university astronomy lab couldn’t have launched the Hubble Space Telescope on its own, nor would a venture-backed startup have built the Large Hadron Collider at CERN. Hubble and CERN illustrate a common pattern in science: a need for projects that are bigger than an academic lab can undertake, more coordinated than a loose consortium or themed department, and not directly profitable enough to be a venture-backed startup or industrial R&D project. Focused Research Organizations (FROs) are a new type of scientific institution designed to fill this gap.”

Emergent Ventures Grant/Fellowship https://www.mercatus.org/emergent-ventures “We want to jumpstart high-reward ideas—moonshots in many cases—that advance prosperity, opportunity, liberty, and well-being. We welcome the unusual and the unorthodox. Our goal is positive social change, but we do not mind if you make a profit from your project. (Indeed, a quick path to revenue self-sufficiency is a feature not a bug!) Projects will either be fellowships or grants: fellowships involve time in residence at the Mercatus Center in Northern Virginia; grants are one-time or slightly staggered payments to support a project. We encourage you to think big, but we also will consider very small grants or short fellowships if they might change the trajectory of your life. We encourage applications from all ages and all parts of the world.”

Flux Capacitor https://1517.substack.com/p/the-flux-capacitor-time-funds-and “Flux Capacitor is a 3-month break away from academia to pursue out-there ideas to build into a startup OR moonshot science… You want to hit pause on the academic rat race and spend 3 months on first-principles exploration of either applied, practical problems that can be commercialized within a VC-funded startup in the near-medium term (5 years) or moonshot fundamental science. To help you do this, we’ll give you up to $100k in funding.”

Foresight AI Safety Grant Program https://foresight.org/ai-safety/ “This grant program seeks to support projects working to make progress on three areas we consider underexplored when it comes to AI Safety… 1. Neurotechnology, Whole Brain Emulation and lo-fi Uploading for AI safety; 2. Security, Cryptography, and Auxiliary Approaches for Infosec and AI Security; and 3. Safe and Beneficial Multipolar AI Scenarios… Projects will be evaluated by a mix of Foresight staff and external advisors. We aim to focus on projects that have a chance of being successful within short AI timelines. Rather than funding many projects with the potential of making a small difference in the long-run, we may be more inclined to fund projects that are high-risk high-reward, in the sense that they are more speculative but would make a big difference if successful. Generally, we are interested in proposals for scoping/mapping opportunities in this area, especially from a differential technology development perspective.”

Hypothesis Fund https://www.hypothesisfund.org/ “The Hypothesis Fund advances scientific knowledge by supporting early stage, innovative research that increases our adaptability against systemic risks to the health of people and the planet. We make seed grants to fund research projects at their earliest stages, typically before there is any preliminary data. Our funding is intended to be catalytic — a fast path to enable a scientist to ‘turn over the card’ and see what’s there.  And we focus on bold new ideas in basic research, not continuations of existing research. The Hypothesis Fund approach is different.  We empower a world-class and diverse network of scientist Scouts to identify the high-risk, high-reward ideas at the edge of the network that would otherwise be un-pursued or underfunded.”

Long-Term Future Fund https://funds.effectivealtruism.org/funds/far-future “The Long-Term Future Fund aims to positively influence the long-term trajectory of civilization by making grants that address global catastrophic risks, especially potential risks from advanced artificial intelligence and pandemics. In addition, we seek to promote, implement, and advocate for longtermist ideas, and to otherwise increase the likelihood that future generations will flourish.”

OpenResearch https://www.openresearchlab.org/ “OpenResearch is a nonprofit research lab. We fund work that requires a very long time horizon, seeks to answer open-ended questions, or develops technology that shouldn’t be owned by any one company.”

Renaissance Philanthropy https://renaissancephilanthropy.org/ “Renaissance Philanthropy’s mission is to fuel a 21st century renaissance by increasing the ambition of philanthropists, scientists, and innovators. We do this by advising philanthropists, surfacing breakthrough ideas, and incubating ambitious initiatives. Our aim is to activate a virtuous loop of increasing ambition and impact between philanthropists and innovators: by identifying frontier experts both in science and in new ways of solving problems; by tapping into the growing number of emerging philanthropists; and by building multi-sector initiatives that can harness the power of philanthropy, markets, and governments.”

Survival and Flourishing Fund https://survivalandflourishing.fund/speculation-grants.html “SFF Speculation Grants are expedited grants organized by SFF outside of our biannual grant-recommendation process (the S-process). “Speculation Grantors” are volunteers with budgets to make these grants. Each Speculation Grantor’s budget grows or increases with the settlement of budget adjustments that we call “impact futures” (explained further below). Currently, we have a total of ~20 Speculation Grantors, with a combined budget of approximately $10MM (up from $4MM initially). Our process and software infrastructure for funding these grants were co-designed by Andrew Critch and Oliver Habryka.”

1517 Fund https://www.1517fund.com/ “1517 is a venture capital fund and community supporting college dropouts, renegade students, and deep tech scientists with investment at the earliest stages of their companies. Founded by the cofounders of the Thiel Fellowship, it supports founders across software, hardware, and deep tech verticals and also provides a community to hackers, makers, and scientists from across the world.”