Studies in live mice showed that these activated T cell-derived-EVs (AT-_EVs) enhanced antigen processing and presentation (APP) in tumor cells and dendritic cells (DCs) across different immunologically cold tumors. The AT_EVs also synergized with immune checkpoint inhibitors (ICIs) to trigger antitumor immunity and hold back tumor growth.

The discovery extends the scientific understanding of the immune system, identifies a new strategy for boosting immunity against cancers, and potentially offers a new tool for delivering genetic payloads to other cells. “These findings reveal a natural mechanism for treating immunologically silent tumors and other diseases that stem from insufficient immune surveillance,” said David Lyden, MD, PhD, the Stavros S. Niarchos professor in pediatric cardiology and a member of the Gale and Ira Drukier Institute for Children’s Health and the Sandra and Edward Meyer Cancer Center at Weill Cornell Medicine.

Lyden is co-senior author of the researchers’ published paper in Cancer Cell, titled “Activated T cell extracellular vesicle DNA transfer enhances antigen presentation and anti-tumor immunity,” in which they stated, “We uncover a mechanism whereby activated T cell-derived extracellular vesicles (AT_EVs) drive a positive feedback loop that enhances antigen presentation and immune responses in normal physiology and cancer … Notably, AT_EVs hold promise as an acellular immunotherapy, restoring APP and synergizing with checkpoint blockade in immunotherapy-refractory tumors.”

Most animal cells secrete extracellular vesicles which can contain cargo including proteins, snippets of DNA, and other molecules. “Extracellular vesicles (EVs) are nanoparticles naturally released by all living cells, containing proteins, lipids, and genetic material, that facilitate intercellular communication,” the investigators wrote.

The Lyden lab in recent years has made seminal discoveries about extracellular vesicles and their functions, finding for example that vesicles secreted by tumor cells can influence the immune system’s anti-tumor response. Their findings, they noted, “… raised the possibility that EV_DNA from immune cells, such as T cells, may also have immune-related functions.” For their new study the team examined the roles of vesicles secreted by immune cells, and specifically T cells, which are the immune system’s principal tumor-fighters.

In their initial experiments, the scientists found that under physiological conditions, T cell-secreted vesicles tend to home to lymph nodes, spleen and other centers of immune activity. There the vesicles are preferentially taken up by antigen-presenting immune cells, including dendritic cells, which assist in T cell activation, a critical process in the immune response. The researchers found that the overall effect of these vesicles released by activated T cells is to boost the antigen-presenting process, thus promoting T cell priming and broader immune activation. The key payloads in these immune-boosting vesicles turned out to be snippets of T cell DNA.

“These surprisingly abundant DNA fragments are mostly on the surfaces of the vesicles, and are not just random—they are enriched for immune-related genes, including genes that help cells display antigens to the immune system,” said co-senior author Haiying Zhang, PhD, an assistant professor of cell and developmental biology in pediatrics and member of the Lyden lab. “We also found that these vesicles have, attached to their surfaces, a special enzyme that acts as a molecular drill, enabling the transfer of vesicle-carried DNA into the nucleus of the recipient cell where they can be expressed transiently,” added study co-first author Diao Liu, PhD, a postdoctoral research associate in the Lyden Lab.

Infusing DNA-carrying vesicles from activated T cells into mice with tumors, the researchers found that the vesicles were taken up not only by antigen-presenting cells but also by tumor cells themselves. The treated tumors grew more slowly and were better infiltrated by T cells and other immune cells, indicating that the vesicles induced a stronger anti-tumor response. “Our work reveals an EV-mediated mechanism through which activated T cells enhance APP across diverse recipient cells—from DCs in physiological conditions to cancer cells across tumor types,” the authors noted. Although cancers—and viruses—frequently suppress the antigen-presenting process to make malignant or infected cells “invisible” to the immune system, the main effect of the extracellular vesicular DNA was to reverse this process, restoring tumor cells’ visibility.

The team demonstrated the effectiveness of this approach, alone and in combination with existing immunotherapy, in preclinical models of three different immunologically silent cancers: glioblastoma, pancreatic and triple-negative breast cancer. “By boosting APP machinery, AT_EVs enhance tumor immunogenicity and elicit robust anti-tumor responses, particularly when combined with ICIs in otherwise resistant tumors, including pancreatic, breast, and brain cancers,” they stated. “These findings reveal the translational potential of activated T cell-derived extracellular vesicles (AT_EVs) by exploiting a naturally occurring immune-boosting process to overcome immune evasion, particularly in immunologically silent cancers.”

Co-senior author Irina Matei, PhD, an assistant professor of immunology research in pediatrics and member of the Lyden lab, stated, “There seems to be a positive-feedback loop, in which the DNA-carrying vesicles from activated T cells amplify the immune response by acting on both antigen-presenting cells, which increase expression of the machinery processing tumor antigens, and tumor cells, promoting their recognition by the immune system as well as their own production of DNA-laden vesicles.”

The researchers are now working to translate their findings into a new, vesicle-based cancer treatment, which could be used on its own or in conjunction with standard immunotherapies or other cancer treatments. “The surprising ability of these vesicles to transfer DNA from donor T cells into the nuclei of recipient cells suggests their potential as a natural, non-viral platform for transient gene delivery,” said co-first author Mengying Hu, PhD, a postdoctoral research associate in the Lyden Lab who led the research and is now an assistant professor of pharmaceutical sciences at the Ohio State University. “The results point to a broadly applicable gene-transfer strategy that may offer improved safety and efficiency compared with current gene therapy approaches.”

In their paper the authors concluded, “Overall, AT_EVs emerge as an acellular immunotherapy and delivery modality that can prime antitumor immunity, synergize with existing therapies, and serve as a vaccine adjuvant,” they concluded. “Our findings provide a foundation for the therapeutic application of AT_EVs through a deeper understanding of the biological role of AT-EV_DNA.”

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T cells are critical members of the immune system but there are limits to their defensive capabilities. When fighting cancer cells, T cells often burn out and become dysfunctional. A major focus of current cancer immunotherapy efforts is rescuing T cells from this state and getting them back into cancer-fighting shape. The new Cell study led by scientists in the lab of Ananda Goldrath, PhD, a professor of molecular biology at UCSD, and their collaborators elsewhere, suggests that a potential solution to T-cell exhaustion might have to do with protein recycling.

Specifically, their finding has to do with proteostasis, the network of cellular processes that orchestrates the proper construction, movement, and destruction of proteins in cells. A component of this network features a type of recycling function where healthy cells continuously dismantle old and damaged proteins to preserve energy and reuse building blocks to make new proteins. According to the paper, the scientists uncovered an impaired protein recycling function as the surprise culprit in T-cell exhaustion. 

“We found that exhausted T cells’ recycling programs are falling apart, leading to damaged and misfolded proteins that pile up with nowhere to go,” said Nicole Scharping, PhD, a post-doctoral fellow in the Goldrath lab and lead author on the paper. Additionally, the scientists also uncovered a way to reverse the accumulation of misfolded proteins by fixing the broken recycling function and restoring normal proteostasis. As Scharping explained, the issue can be resolved with a “tag and sort” fix. This is accomplished using E3 ligase enzymes which act as labelers at a recycling facility, tagging worn-out proteins so the cell knows to break them down.

“In exhausted T cells, many of these enzymes get switched off, and recycling grinds to a halt,” said Scharping. After examining thousands of proteins, the scientists honed in on NEURL3, RNF149 and WSB1 as the E3 ligases responsible for rescuing T cell recycling functions. “When we restored specific E3 ligases, the buildup cleared, and the T cells regained their function and worked better at clearing tumors.” While the new study was conducted in mice, the researchers indicate that similar strategies could be employed for immunotherapy treatments in human cancer.

Importantly, the findings may have implications in other diseases as impaired protein processing is not unique to exhausted T cells. “We think this loss of proteostasis resembles what occurs in neurons in other protein aggregate diseases such as Parkinson’s and Alzheimer’s,” said Goldrath. “Rescuing these cells from exhaustion could improve the ability of T cells to respond to both chronic infection as well as tumors.”

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“Biopharma teams are under pressure to move more quickly, but their systems are often not built to keep up,” says Weaver. “This collaboration with Cytiva marks a pivotal step in our mission to democratize digital manufacturing, enabling biopharma innovators to deploy SCADA faster, smarter and more affordably.”

Many biopharma teams have long juggled proprietary systems that cannot communicate with one another, creating operational silos, manual workarounds, and data integrity risks. The new system directly addresses this roadblock by having an open architecture, allowing for third-party instrument integration, and real-time oversight of integration capable unit operations from a single interface, notes a Cytiva spokesperson, who explains that the platform features include:

• Native interoperability: The platform is natively integrated with Cytiva bioprocessing equipment and Rockwell Automation’s FactoryTalk software suite, enabling seamless interoperability across systems.
• Scalable growth: A single platform expands from process development to commercial manufacturing without system redesign.
• Cost-effective compliance: A streamlined digital manufacturing system reduces capital and operational costs and enables cGMP compliance.
• Rapid implementation: Pre-engineered templates and modular design shorten deployment and validation timelines.
• Enhanced operational insight: Centralized alarms, real-time monitoring, process intensification and batch reporting tailored to bioprocess workflows.

“This collaboration is designed to empower the next generation of biomanufacturers,” says Nicolas Pivet, manufacturing and digital solutions at Cytiva.

Industry data shows increasing demand for next generation process control systems as organizations transition toward data driven process intensification and continuous manufacturing. Equipment fragmentation remains one of the top pain points cited by biomanufacturers, particularly those advancing programs from R&D to clinical scale. By giving teams a unified digital control layer, the Figurate SCADA reduces the risk of human error, accelerates tech transfer, and supports reliable scaleup as workloads grow in complexity, points out the Cytiva spokesperson.

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Although only 20 or so cell or gene therapies have been FDA-approved, the area holds considerable promise for investment. The global market was valued at nearly $9 billion in 2025, and growth has been projected at over 15% per year from 2026 to 2035. As with any pharmaceutical product, however, the potential of cell and gene therapy relies in large part upon minimizing risks to patient health from adverse effects. Numerous companies, from both prominent names in the field to smaller startups, are developing solutions to mitigate the deleterious consequences of cell and gene therapy.

Reducing cytokine release syndrome

Cytokines are a broad family of small proteins and peptides that cell lineages of the innate and adaptive immune systems employ to communicate with each other and coordinate timely and appropriately scaled responses to foreign antigen-containing cells. Cytokine release syndrome (CRS) occurs when hyperactivation of one or more immune lineages results in the release of excessive quantities of cytokines into the circulation.

“As a scientific community, we’ve been researching CAR T-cell therapy for over 30 years and have grown together in our understanding of the body’s immune response to treatment, from both a safety and efficacy perspective,” says Rosanna Ricafort, MD, vice president and global program lead of hematology and cell therapy at Bristol Myers Squibb. “We have evolved our ability to characterize, stage, and manage potential side effects, allowing for timely and thoughtful interventions of the most commonly associated side effects like CRS.”

Ricafort cited clinical data presented at the 2025 American Society for Clinical Oncology (ASCO) meeting in Chicago demonstrating that over 95% of instances of CRS and other adverse events arising from BMS’s CD19-directed CAR T-cell therapy (Breyanzi^R) occurred in the first two weeks after onset of therapy. “These and other studies have helped establish the largely predictable safety profile of CAR T-cell therapy to date,” Ricafort pointed out.

Minimizing side effects

The NF-κB and prostaglandin E2 pathways are prominent regulators of the activation and differentiation of pro-inflammatory T cell lineages. Excessive signaling through these pathways results in cytokine amplification, which contributes to CRS and immune effector cell-associated neurotoxicity syndrome (ICANS), a complication of some types of CAR T-cell therapy.

CytoAgents, a clinical-stage biotech company, is developing CTO1681, an orally administered prostaglandin signaling inhibitor that has been shown to offset CRS and ICANS toxicities associated with CAR T-cell therapy of lymphoma patients. At the 2025 European Society for Medical Oncology (ESMO) Immuno-Oncology Congress in London, CytoAgents presented non-clinical data showing that CTO1681 treatment reduced secretion of TNF-α, IL6, and other key CRS-associated cytokines with no impairment of CAR T-cell mediated cytotoxicity on lymphoma cells.

“These data suggest CTO1681 could enable safer CAR T-cell therapy administration, support outpatient treatment paradigms, and broaden patient access without compromising anti-tumor efficacy,” said Teresa Whalen, CEO at CytoAgents. CTO1681 is currently in Phase Ib/IIa trials for cancer patients undergoing CAR T-cell therapy, with potential expansion into additional therapeutic spaces including asthma and chronic obstructive pulmonary disease.

Adding immunosuppressants

A potential side effect of adeno-associated virus (AAV)-based gene transfer approaches is acute liver injury resulting in part from CRS in patients receiving AAV therapy. Duchenne muscular dystrophy (DMD) is a progressive, degenerative muscular disorder caused by mutations or changes in the DMD gene, resulting in reduced levels of the protein dystrophin.

Elevidys, developed by Sarepta Therapeutics, is an AAV-based therapy approved for the treatment of DMD that stimulates targeted production of a truncated form of dystrophin in skeletal muscle. “Individuals with non-ambulatory Duchenne face profound unmet need and fewer treatment options,” says Louise Rodino-Klapac, PhD, president of R&D and development and technical operations at Sarepta. Topline data released earlier this year showed that Elevidys treatment resulted in significant improvement in key clinical ambulatory metrics in patients.

As part of its ENDEAVOR clinical trial, Sarepta Therapeutics is evaluating the potential of supplementing Elevidys with sirolimus to reduce potential acute liver injury (ALI) complications. Sirolimus is a mammalian target of rapamycin (mTOR) kinase inhibitor that suppresses responses of T and B cells to interleukin 2, which functions to stimulate proliferation of helper, cytotoxic, and regulatory T cells.

Developing non-integrating therapies

As an alternative approach to supplementing cell and gene therapy modalities with existing immunosuppressants, other companies are modifying CAR T-cell therapy to reduce the risk of CRS and other side effects. Myasthenia gravis, a chronic fatigue-inducing autoimmune disorder in which signals between nerves and muscles are compromised, results in part from the secretion of autoantibodies from B-cell maturation antigen (BCMA)-expressing B plasma cells.

Conventional BCMA-directed CAR T-cell approaches rely on the integration of lentiviral or gamma-retroviral vectors to encode the CAR and typically involve lymphodepletion chemotherapy that can be accompanied by acute and delayed toxicity. In contrast, non-integrating (i.e., mRNA-based) BCMA-directed CAR T-cell therapies may circumvent this toxicity due to the lack of requirement for chemotherapy.

Cartesian Therapeutics is developing an mRNA-based BCMA-targeted CAR T-cell therapy for myasthenia gravis, Descartes-08. At the 2025 American Academy of Neurology (AAN) Annual Meeting in San Diego, results were reported of a Phase IIb clinical trial of Descartes-08 in myasthenia gravis. In the trial, adverse event rates were similar between groups receiving Descartes-08 and the placebo group, and were predominantly mild to moderate in nature, with no cases of CRS or ICANS reported.

“The impressive strength and duration of response shown in the data reinforce our confidence in the potential of Descartes-08 to transform the current treatment landscape in MG, offering patients a safe, flexible, and durable treatment option,” said Carsten Brunn, PhD, president and CEO of Cartesian.

Engineering chimeric receptors

Modifications of CAR T-cell therapy to improve clinical efficacy and reduce side effects can also encompass modification of the molecular structure of the chimeric receptor itself. D domains are highly selective targeting domains incorporated into newer generations of CARs that enhance targeting of pathological cell types and reduce immunogenic responses in patients that give rise to unwanted side effects.

One example of such next-generation CAR T-cell therapies, anito-cell, has been co-developed by Arcellx, Kite Pharma, and Gilead. Anito-cel is an autologous anti-BCMA CAR T-cell therapy for the treatment of relapsed/refractory multiple myeloma patients.

Phase II trial results in multiple myeloma presented at the 2025 American Society of Hematology (ASH) meeting in Orlando showed an overall response rate of 97% and a complete response rate of 68%. Importantly, in the context of side effects, there were no delayed neurological symptoms, and for most patients, only low-grade CRS was observed, which was resolved within a few days.

“The anito-cel D-domain BCMA binder could be important to our work in in vivo cell therapy, further strengthening our potential in oncology and inflammation,” said Daniel O’Day, chairman and CEO of Gilead. “Anito-cel could become a foundational treatment for multiple myeloma over time, including earlier lines of therapy.”

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In February, the FDA unveiled its Plausible Mechanism Pathway draft guidance, a series of initiatives designed to increase regulatory flexibility and spur the development of bespoke gene-editing therapies for rare and ultra-rare disorders, which collectively total about 30 million individuals in the United States.

“The Agency anticipates that substantial evidence of effectiveness for individualized therapies could be established based on a single adequate and well-controlled clinical investigation with confirmatory evidence,” the draft guidance stated.

Last June, at a historic roundtable of cell and gene therapy researchers and clinicians hosted by the FDA, base editing pioneer David Liu, PhD, of Harvard University and the Broad Institute of MIT and Harvard, stated: “With sufficient organization and federal support and partnership with the FDA, I believe it will be possible by 2030 to treat at least 1,000 patients with personalized genetic treatments.”

Meanwhile, conventional gene therapy development continued in 2025. Last year saw four U.S. gene therapy approvals, bringing the number of FDA-approved gene and cell therapies up to 26, according to the American Society of Gene and Cell Therapies (ASGCT)—more than half of the 40 tallied by the organization as being approved worldwide.

Of those 26, 18 were gene therapies, of which 10 had disclosed sales high enough to be included on this A-List, which ranks top-selling gene therapies based on sales and net product revenue figures furnished by the companies in regulatory filings, annual reports, and/or press releases. Each gene therapy is listed with its sponsor(s), type, indication, and initial FDA approval date.

Not included are gene therapies with sales below the top 10, a category that includes two gene therapies approved in 2025: Precigen’s Papzimeos (zopapogene imadenovec-drba), which generated $3.4 million in net product revenue last year after becoming the first-and-only FDA-approved treatment for adults with recurrent respiratory papillomatosis (RRP) in August; and Abeona Therapeutics’ Zevaskyn^® (prademagene zamikeracel), an autologous cell sheet-based gene therapy approved to treat wounds in adults and children with recessive dystrophic epidermolysis bullosa (RDEB).

Three gene therapies did not have disclosed sales in 2025, including:

• Encelto (revakinagene taroretcel-lwey), an allogeneic encapsulated cell-based gene therapy marketed by Neurotech Pharmaceuticals and indicated for the treatment of adults with idiopathic macular telangiectasia type 2 (MacTel).

• Imlygic^® (talimogene laherparepvec), a genetically modified oncolytic viral therapy marketed by BioVex (Amgen) and indicated for local treatment of unresectable cutaneous, subcutaneous, and nodal lesions in patients with melanoma recurrent after initial surgery.
• Waskyra (etuvetidigene autotemcel), a cell-based gene therapy and the first FDA-approved treatment for Wiskott-Aldrich syndrome (WAS). Developer Fondazione Telethon is the first non-profit organization to have successfully led full development of an ex vivo gene therapy from lab research (at Milan’s San Raffaele Telethon Institute for Gene Therapy or SR-Tiget) to regulatory approval.

Also not included this year are sales of three gene therapies that had been marketed by Bluebird Bio: Beta thalassemia treatment Zynteglo (betibeglogene autotemcel), sickle cell disease treatment Lyfgenia^® (lovotibeglogene autotemcel), and cerebral adrenoleukodystrophy (CALD) treatment Skysona^® (elivaldogene autotemcel).

Last year, Bluebird Bio went private after being acquired by funds managed by Carlyle and SK Capital Partners, then rebranded in September as Genetix Biotherapeutics. Genetix does not disclose sales but did announce on March 2 that more than 100 patients received infusions of the three gene therapies during 2025.

Also last year, Pfizer halted development and commercialization of Beqvez (fidanacogene elaparvovec-dzkt), which had been co-marketed with Roche-owned Spark Therapeutics, after it generated no sales in 2024. Last August, Pfizer terminated its license agreement with Spark for Beqvez, an adeno-associated virus (AAV) vector-based gene therapy indicated for forms of moderate to severe hemophilia B in adults.

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Kunwoo Ryan Lee, PhD, knew as early as 2012 that solving the delivery problem would be crucial in fulfilling the promise of the newly discovered CRISPR-Cas9 gene editing technology. He felt strongly that gene editing had potential to transform medicine by curing genetic disorders, but the viral and non-viral vectors available at the time had significant drawbacks. With the support of CRISPR pioneers Jennifer Doudna and Stanley Qi, Lee completed his doctoral thesis on a gold nanoparticle delivery system for Cas9 ribonucleoprotein. He went on to co-found BreezeBio, formerly GenEdit, in 2016 with the aim of creating the next generation of gene editing-based therapeutics. To realize that goal, Lee and his team looked beyond traditional viral gene delivery systems and instead invented a new technology from the ground up.

Most clinical gene therapy trials use viral vectors, including retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. However, viral vectors are limited in the size of the gene they can deliver. They also tend to trigger strong immune reactions and usually can’t be dosed more than once due to acquired immunity.

Non-viral vectors are an alternative technology that offer greater gene loading capacity, more straightforward preparation, and less likelihood of triggering problematic immune reactions. BreezeBio and other biotechnology companies are reimagining gene delivery through non-viral approaches like targeted LNPs, transposons, and novel chemistry.

“Using the platform, we have demonstrated that we can deliver to the spleen, immune system, heart, and lung,” Lee said. The firm also developed a set of nanoparticles targeted to the central nervous system.

Based on those targeted delivery profiles, the Brisbane, California-based BreezeBio has worked with multiple partners to provide delivery solutions for their products, including a multiyear collaboration with Genentech, a member of the Roche Group, signed in 2024. Meanwhile, the company is also advancing its own pipeline of therapeutics built on the NanoGalaxy platform, including a lead candidate for type 1 diabetes, as well as investigational therapies for autoimmune disease and cancer.

Lee said a key advantage of the NanoGalaxy platform for their pipeline, which heavily leans toward autoimmune disease, is that, unlike a viral vector, the company’s studies have shown it does not activate the innate immune system. “That enabled us to use our technology for autoimmune applications and in more targeted oncology applications, as well,” Lee said.

Snug as a bug in a rug

The red flour beetle, a notorious scourge of grain and cereal stores, is the surprising source of Bio-Techne’s transposon-based, non-viral gene delivery system. The system, dubbed TcBuster for the beetle’s scientific name, Tribolium castaneum, was invented by B-MoGen, a spin-out of the University of Minnesota, which was acquired in 2019 by Minneapolis-based Bio-Techne. Researchers at B-MoGen and Bio-Techne developed a hyperactive version of the natural TcBuster transposon by creating a library of three million unique genetic variants and screening each in mammalian cells. In a proof-of-concept study, CAR NK cells engineered using TcBuster demonstrated in vitro functionality and improved survival in a preclinical model of Burkitt lymphoma with a single dose.¹

“The reason you want a hyperactive version is that wild-type transposon systems are fairly low activity,” said Miles Smith, PhD, a product manager for cell and gene therapy at Bio-Techne. “For generating a cell therapy, you want something that’s going to be comparable to the state of the field, and that’s lentivirus.”

Unlike other commercial transposon systems for gene delivery, like Sleeping Beauty and PiggyBac, Smith said TcBuster is not restricted by exclusive licensing. “The turnaround time for GMP material is just a couple of months,” Smith said. “Versus something that might take a lot longer if you have to go through licensing or create a viral batch.”

Gene therapy SORTed

ReCode Therapeutics is developing a pipeline of genetic medicines based on its selective organ targeting (SORT) LNP platform, which adds an additional lipid to the standard LNP formulation, allowing it to zero in on specific organs. Conventional LNPs comprise four lipids—cholesterol, a helper phospholipid, a PEGylated lipid, and an ionizable lipid—that encapsulate a therapeutic gene cassette. These traditional LNPs are primarily taken up by the liver after intravenous administration, limiting their usefulness for other organs and systems. ReCode engineered its SORT LNPs with a biochemically distinct fifth lipid that enables the body to direct the particle to the targeted organ, such as the lung or spleen, bypassing the liver, if necessary.

“Because mRNA in a cell has a relatively short half-life, maybe a day or so, in order to have constant protein production, you need to administer it relatively frequently,” Vladimir Kharitonov, PhD, senior vice president of CMC and pharmaceutical sciences at ReCode, said. “With viral delivery, you can’t really administer it repeatedly.”

In 2024, ReCode presented preclinical data from its cystic fibrosis program showing its mRNA-based therapeutic RCT2100 significantly restored CFTR function in human bronchial epithelial cells derived from patients with cystic fibrosis. In vivo studies using a ferret model demonstrated improvement in mucociliary clearance. ReCode launched the first clinical trial of RCT2100 later that year. The LNP for RCT2100 contains SORT lipids to fine-tune its delivery to the airway epithelial cell types that have a defective or mutated CFTR protein, causing cystic fibrosis. The company is also developing a second mRNA therapy delivered via SORT LNP, RCT1100, for primary ciliary dyskinesia, which targets different cell types in the lung epithelium.

From idea to therapy faster

Gene delivery is just one of many services offered by GenScript to support research from discovery through clinical testing, including gene synthesis, CRISPR reagents, antibodies, and more.

“Gene editing is entering a new era, and the focus has shifted from discovery to translation,” said Jianpeng Wang, PhD, senior director of nucleic acid and peptide R&D at GenScript. “Our goal at GenScript is to help scientists move from idea to therapy faster.”

When it comes to non-viral vectors, the firm offers off-the-shelf and bespoke solutions to fit the customer’s need for delivery of DNA, RNA, siRNA, peptides, and other molecules. Through its targeted LNP service, GenScript offers LNPs designed to enhance precision in directing genetic material to cells. GenScript’s ReadyEdit LNP solutions include Cas9 knock-in and knock-out, Cas12 knock-out, and base or prime editing tailored for the customer’s needs.

“In our ecosystem, we include all of the materials needed,” Wang said. “This integration can help scientists evaluate gene editing efficiency early, both ex vivo and in vivo.”

Wang said the choice of a vector is heavily dependent on the specific therapeutic program. “There isn’t a universally effective or better way to deliver a therapy, either viral or non-viral,” Wang said. He noted, for example, that viral vectors remain a good choice when long-term gene expression is desired. And for viral vectors, the manufacturing process might be more mature, easing transfer to a contract development and manufacturing organization.

However, Wang cautioned that viral vectors still present certain safety concerns. “In recent years, an increasing number of scientists and the FDA have recognized these risks,” he said, “leading to a surge in interest for non-viral delivery methods—particularly for in vivo CAR T therapy and gene editing.”

GenScript has provided LNP services to several customers globally. The most advanced of those is using GenScript’s GMP CRISPR materials (gRNA, HDR templates, and nuclease) alongside a customized LNP encapsulation recipe and is preparing an investigational new drug application.

Foundational LNP science

Vancouver-based Genevant traces its scientific lineage through a string of predecessor companies dating back to the early 2000s and controls foundational intellectual property for the field. Based on its scientists’ work at Protiva Biotherapeutics, the intellectual property comes to Genevant via Arbutus Biopharma, which acquired Protiva in 2015 and partnered with Roivant in 2018 to establish Genevant.

Unlike many companies developing nucleic acid delivery platforms that focus on a single payload modality, Genevant has applied its LNP to many payloads, including mRNA, siRNA, and gene editors in fields spanning antiviral, oncology, and metabolic disorders. The firm’s LNP platform is part of the first RNA-LNP product to achieve regulatory approval, Alnylam Pharmaceuticals’ Onpattro (patisiran), a treatment for polyneuropathy in people with hereditary transthyretin-mediated amyloidosis. Genevant’s LNP technology is also behind Moderna’s COVID-19 vaccines, which were confirmed earlier this month with the resolution of a longstanding patent dispute. An infringement case against Pfizer and BioNTech is pending. Genevant collaborated with Chula Vaccine Research Center and the University of Pennsylvania to develop a COVID vaccine for low- and middle-income countries in Southeast Asia during the pandemic. The program had success, demonstrating non-inferiority to Pfizer and BioNTech’s Comirnaty in clinical trials.

Some key differentiators for Genevant’s LNPs include strategies for optimized delivery in non-human primates instead of mice,² which has resulted in improved gene editing in the liver, and novel chemistries for biodegradable LNPs that prevent accumulation in tissue.³ The company has recently disclosed data showing targeted delivery to T cells for in vivo CAR T therapy, as well as hematopoietic stem and progenitor cells (HSPC) and hepatic stellate cells.

References

1. Skeate JG, Pomeroy EJ, Slipek NJ, et al. Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies. Mol Ther. 2024;32(6):1817-1834. doi:10.1016/j.ymthe.2024.04.024
2. Lam K, Schreiner P, Leung A, et al. Optimizing lipid nanoparticles for delivery in primates. Adv Mater. Published online March 27, 2023. doi: 10.1002/adma.202211420
3. Holland, R, Lam K, Jeng S, et al. 2024. Silicon ether ionizable lipids enable potent MRNA lipid nanoparticles with rapid tissue clearance.” ACS Nano. 2024;18 (15): 10374–87. doi:10.1021/acsnano.3c09028.

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For decades, scientists have struggled to deliver therapeutics to the brain, only to be thwarted by the highly protective blood-brain barrier (BBB). First-generation approaches demonstrated proof of principle but still require advancements to improve the ability to reach specific areas of the brain, or specific cell types, safely, and with sufficient dosage to enable meaningful therapeutic effects.

Although much remains unknown generally about brain biology and its defensive mechanisms, novel therapies for devastating neurological diseases are progressing into clinical trials. There is no magic bullet—no promises, no cures—but a gleaming light can be seen in this particular long and dark tunnel.

Dedicated scientists continue to work on gene therapies for the indications that most benefit from a once-and-done approach, in addition to neurological shuttles to address those disorders that require therapeutic tempering and dosage control.

Expanding platform technologies

In 2021, JCR Pharmaceuticals received regulatory approval for the first biotherapeutic, IZCARGO (pabinafusp alfa), designed to cross the BBB to deliver a therapeutic enzyme for the treatment of a lysosomal storage disorder called mucopolysaccharidosis type II (MPS II) or Hunter syndrome.

The platform technology has been expanded to exploit receptor-mediated transcytosis (RMT) to address other lysosomal storage and neurodegenerative diseases. Still, delivery to specific cells or parts of the brain remains challenging, along with efficient delivery of antisense oligonucleotides or siRNA.

“The issue is not delivery across the BBB, but the endosomal escape to efficiently suppress the target RNA,” said Hiroyuki Sonoda, PhD, representative director, president, and CSO, at JCR Pharmaceuticals. “Small molecule CNS delivery is related to physicochemical properties. The structural design needs to make them lipophilic, yet also able to evade typical transporter clearing mechanisms.”

J‑Brain Cargo^® uses RMT, mainly focusing on the transferrin receptor (TfR). Other promising candidates target different receptors. “We have successfully transported enzymes, antibodies, peptides, decoy receptors, antisense oligos, and siRNA into the CNS,” commented Sonoda. J‑Brain Cargo is particularly suited for enzyme replacement therapies in lysosomal storage disorders and conditions where dose control, reversibility, and titration are important.

For gene therapies, JCR developed the JUST-AAV platform technology. Novel changes in the capsid almost completely eliminate liver tropism. The modified capsids express miniaturized antibodies on the capsid surface against receptors on selected tissues, organs, or the BBB, enhancing targeted delivery. JUST‑AAV is for diseases where continuous transgene expression is desired to achieve the optimal effect.

Several candidates are in global clinical trials, including JR-141 (pabinafusp alfa) for individuals with MPS II (also known as Hunter syndrome), JR-171 to treat MPS I (also known as Hurler, Hurler Scheie, or Scheie syndromes), and JR-441 for individuals with MPS IIIA (also known as Sanfilippo syndrome A).

Programs in collaboration with MEDIPAL HOLDINGS CORPORATION are in different stages of clinical and pre-clinical development for individuals with MPS IIIB (also known as Sanfilippo syndrome B), Fucosidosis, and GM2 gangliosidosis (including Tay-Sachs and Sandhoff disease).

Collaborating with leading pharmaceutical companies is core to JCR’s strategy to bring these platform technologies to broader application. “We enable our partner by turning their biologics into CNS-penetrating versions of their original molecule,” said Sonoda.

JCR manufactures most of its drug products in-house. Last year, they were selected for the Ministry of Economy, Trade and Industry’s “Regenerative CDMO Subsidy” to expand biomanufacturing capacity for regenerative, cell, and gene therapies.

Optimizing BBB transport

“Protein engineering architecture differentiates our delivery technology along with its optimization for efficacy, safety, and tolerability,” said Ryan Watts, PhD, co-founder and CEO of Denali Therapeutics.

The TransportVehicle (TV) technology has the RMT binding site integrated directly into the constant domain (Fc) of an antibody for optimal properties and modularity. This allows the same TV sequences to transport a range of large molecule biotherapeutics such as enzymes, oligonucleotides, and antibodies for systemic administration. The engineered Fc domains bind to specific natural transport receptors expressed at the BBB, such as TfR.

“Our research recently demonstrated that a TV platform-enabled anti-Ab antibody improved distribution in the brain and significantly reduced risk of Amyloid-Related Imaging Abnormalities (ARIA) in a mouse model of Alzheimer’s disease, when compared with a conventional anti-Ab antibody.¹ The study provides the first mechanistic insight for mitigating the risk of ARIA,” detailed Watts.

The Enzyme TransportVehicle (ETV) contains a fusion of a therapeutic enzyme. The Fc portion of the fusion molecule binds the apical surface of the TfR to avoid interference with normal iron transport.

In March 2026, Denali’s lead ETV program, Avlayah (tividenofusp alfa-eknm), received FDA accelerated approval for the pediatric treatment of the lysosomal storage disorder MPS II. Avlayah is the foundation for their broader ETV franchise, addressing other lysosomal storage disorders such as MPS IIIA. Results from the open-label Phase I/II clinical trial are available.²

Their Oligonucleotide TransportVehicle (OTV) platform is an engineered TV conjugated to an oligonucleotide for the systemic delivery of genetic medicines to the brain. Extensive characterization and research demonstrate the ability of OTV to elicit broad biodistribution of oligonucleotide therapies throughout the CNS following systemic exposure.

“For example, our investigational therapy DNL628 for the treatment of Alzheimer’s disease is designed to cross the BBB and reduce the tau protein by targeting the MAPT gene that encodes for tau,” explained Watts.

Lastly, the Antibody TransportVehicle (ATV) platform is designed to enable brain delivery of antibodies capable of selective immune activation and a targeted therapeutic approach after intravenous administration. The investigational anti-Ab antibody therapy DNL921, for example, is designed to reduce amyloid plaques and avoid ARIA.

The TV-enabled clinical development portfolio also includes candidates for frontotemporal dementia-granulin and Pompe disease.

Advancing clinical options

“It is exciting to begin to see that delivery through the BBB is possible using gene therapy or shuttle approaches,” said Todd Carter, PhD, CSO at Voyager Therapeutics. Although first-generation therapeutics are demonstrating meaningful levels of delivery, optimization, and improvement of the functionality, exposure duration, and therapeutic effects are still needed.

“For some diseases, gene therapy is the preferred treatment modality, as both the capsid and the payload can be modified to perform a specific job,” said Carter. But viral vector delivery for gene therapy has had problems with liver-based toxicity.

For the best human translation opportunities, Voyager developed a model in non-human primates (NHPs) requiring cross-species activity across multiple NHP species. This strategy resulted in the company’s TRACER (Tropism Redirection of AAV by Cell-type-specific Expression of RNA) technology, used to screen tens of millions of vector variants using barcoded libraries in which capsids were modified with slight insertions of seven to nine amino acids.

Successful expression in neurons demonstrated that the capsids crossing the BBB worked. Directed evolution improved them. “Next, we needed to determine the mechanism—the receptors they were targeting,” said Carter. This led to the identification of the receptor, alkaline phosphatase (ALPL), tissue nonspecific.

Now, Voyager has multiple families of capsids that mediate delivery into the brain, are detargeted from the liver, and, for the most advanced, have improved the capsid’s ability to target the brain using ALPL. “Using the ALPL receptor elevates delivery to the brain and allows us to substantially reduce dosage,” said Carter.

“I would not have picked ALPL just on face value,” added Mihalis Kariolis, PhD, vice president of non-viral therapeutics at Voyager Therapeutics. “It highlights the power of the unbiased TRACER approach. Expanding the number of brain delivery receptors provides highly differentiated options to reduce side effects and expand the diversity of treatment modalities.”

Both gene therapy and shuttle approaches have opportunities in different indications. Once-and-done gene therapy is not tweakable, whereas shuttle-based dosing is. “In our APOE gene therapy program, we want to reduce existing APOE4 and replace it with APOE2 permanently,” said Carter. “The shuttle has advantages in situations where permanent ongoing delivery is not required.”

Voyager’s most advanced program (VY7523) is a tau monoclonal antibody that is exquisitely specific for pathological tau. Data will be available in the second half of the year. A gene therapy (VY1706) moving into the clinic this year is designed to knock down tau mRNA and protein intracellularly. A collaboration with Neurocrine Biosciences focuses on Friedreich’s ataxia (FA) and is also expected to enter the clinic this year.

Combining transport receptors

The protective BBB is crucial for maintaining homeostasis and ensuring proper neurological function. Comprised of both cellular and acellular components, this sophisticated structure tightly regulates information flow between the periphery and the brain. According to Tanya Wallace, PhD, vice president of neuroscience discovery research at AbbVie, despite the BBB’s importance, many seemingly basic biological questions remain unanswered, fueling additional global research.

The complexity of the BBB also represents a significant bottleneck for advancing therapeutics targeting brain-related disorders. Historically, achieving therapeutically relevant levels of drugs in the brain has been a major challenge in treating serious diseases such as Alzheimer’s and Parkinson’s diseases. “A notable success story is the development of L-DOPA, a prodrug that leverages existing transport mechanisms to cross the BBB,” said Wallace. Once in the brain, L-DOPA is metabolized into dopamine, offering a key symptomatic treatment for Parkinson’s disease.

Breakthroughs in delivery now allow scientists to leverage more technologies that can bring not only small molecules but also complex biologics into the brain. The Modular Delivery (MODEL^TM) platform exemplifies this progress. The platform enables engineering of bispecific antibodies, capable of targeting naturally expressed BBB receptors such as TfR and CD98. TfR and CD98 are well-characterized at the BBB, and, together, they offer distinct advantages for increasing brain exposure to therapeutics.

“By engaging these transport pathways, the platform can enhance the uptake of a variety of therapeutics, including antibodies and oligonucleotides,” highlighted Wallace. “This multi-receptor strategy provides flexibility to optimize the balance of uptake, release, and distribution in the brain, paving the way for potentially more effective treatments across neurological disease areas.”

This platform technology facilitated the development of ABBV-1758, which is progressing in clinical development. ABBV-1758 utilizes TfR to transport a 3pE-Ab antibody across the BBB to enable the removal of amyloid beta plaques, a pathological hallmark of Alzheimer’s disease.

As scientists aspire to further refine delivery strategies, ongoing research is exploring additional receptors and innovative approaches, including insulin-like growth factor 1 receptor (IGF-1R) and brain cell-type-specific targeting. The field is rapidly evolving to advance more precise, personalized interventions for challenging neuroscience conditions.

“Successful brain delivery requires more than just advances in transport technology; it demands interdisciplinary collaboration, novel preclinical models, and thoughtful clinical translation,” Wallace pointed out. Continued biological research and investment into innovative discovery platforms will be crucial for bringing transformative therapies to patients with the greatest unmet needs.

References

1. Pizzo ME, Plowey ED, Khoury N et al. Transferrin receptor-targeted anti-amyloid antibody enhances brain delivery and mitigates ARIA. Science. 2025 Aug 7;389(6760):eads3204. doi: 10.1126/science.ads3204.
2. Muenzer J, Burton BK, Harmatz P et al. An intravenous brain-penetrant enzyme therapy for mucopolysaccharidosis II. N Engl J Med. 2026 Jan 1;394(1):39-50. doi: 10.1056/NEJMoa2508681.

The post Breaking Through the Barrier appeared first on GEN - Genetic Engineering and Biotechnology News.

IDT played a pivotal role in manufacturing the personalized gene editing therapy given to baby KJ Muldoon to treat his rare metabolic disorder. Today, KJ is free from the toxic ammonia buildup that drives a 50% mortality rate for his condition in infancy. While his story highlights the life-changing potential of gene editing, the field now wrestles with the next challenge: expanding these therapies to benefit broader patient populations.

In contrast to KJ’s urea cycle disorder, which stemmed from a single disease-causing mutation that could be precisely targeted, many genetic disorders arise from numerous mutations scattered across a gene where individualized corrections are too resource-intensive to scale.

Gannerkote says turning powerful gene editing tools into broadly accessible clinical therapies requires progress across multiple fronts. Many CRISPR therapies are still bespoke, with manufacturing processes that are not yet standardized or easily repeatable, leading to long timelines and high costs. In regulation, therapy developers and government regulators face a learning curve when evaluating new modalities, particularly when speed is critical for patients with life-threatening conditions.

Today’s gene editing companies reflect on what’s required to scale personalized CRISPR therapies for maximized impact in the clinic.

End-to-end

Sadik Kassim, PhD, CTO of Genomic Medicines at Danaher, explains that personalized therapies do not naturally lend themselves to traditional drug-development models. Gene editing companies are now seeking “platformization,” where common manufacturing processes are standardized, and limited elements, such as guide RNAs, are customized for each patient to reduce costs and speed timelines.

“Baby KJ’s treatment succeeded because multiple elements aligned simultaneously,” explained Kassim. The foundational science, which achieved successful gene corrections in animal models of phenylketonuria (PKU), an inherited metabolic disorder caused by mutations in the PAH gene that impair the enzyme responsible for breaking down phenylalanine, had already been developed in the academic labs led by Children’s Hospital of Philadelphia (CHOP) physician scientists, Rebecca Ahrens-Nicklas, MD, PhD, and Kiran Musunuru, MD, PhD. Teams were then able to move quickly when the clinical need became clear.

Regulatory engagement was also critical. Danaher teams worked directly with the FDA to streamline the treatment approval process without compromising patient safety. That collaboration compressed a timeline that would normally take 18–24 months down to roughly six months.

“Replicating this for future patients will require moving away from one‑off efforts and toward repeatable platforms with established processes, validated assays, and clearer regulatory precedents, so that speed becomes the norm rather than the exception,” Kassim said.

Amy Pooler, PhD, CSO of ElevateBio, agrees that the transition steps between therapy design and manufacturing are often where the greatest delays occur. ElevateBio seeks to address this bottleneck by building an end-to-end genetic medicine platform.

“A critical driver for the company is making sure we have a clear line of sight into manufacturing from the very beginning,” Pooler said. “One reason Baby KJ’s case was successful is that Danaher managed the handoffs smoothly.”

Pooler also describes developing genetic medicines as “building the plane while you’re flying it.” The field still lacks enough data to reliably predict patient outcomes. Every clinical trial readout provides a valuable lesson for the field.

“I’m excited about the clinical evidence that’s starting to accumulate, showing gene editing can be transformative for patients, which we didn’t have five to ten years ago,” she said.

Large gene, generalizable therapy

ElevateBio’s expanding CRISPR toolbox includes base, prime, and epigenetic editing. Notably, the Durham-based company’s AI platform generates novel recombinases for targeted gene insertion, an approach that holds promise as a generalizable medicine that could treat patients regardless of their underlying disease-causing mutation.

Using AI-guided design, ElevateBio explores entirely new regions of protein space to discover potent and highly specific recombinases that expand the range of diseases amenable to gene editing. These engineered enzymes, which possess 50% or less homology to known proteins, can access novel genomic regions that remain difficult to target with existing CRISPR technologies.

Ben Kleinstiver, PhD, associate investigator at Massachusetts General Hospital (MGH) and co-author of the NEJM study describing KJ’s case, says the FDA’s Plausible Mechanisms Pathway has helped address some of the regulatory challenges to streamline the path to the clinic. Yet, there remains a major motivation for pan-mutation approaches that are more widely applied across patients.

Kleinstiver’s research group, in collaboration with Full Circles Therapeutics, recently developed a circular single-stranded DNA donor (ssDNA) that enables safer kilobase-scale integration for human cells.¹ The technology provides an alternative to double-stranded DNA (dsDNA) donors that evoke harmful immune responses yet are required for recognition by the diverse suite of genome editing enzymes. Notably, the new circular donor maintains recombinase compatibility by attaching a short region of dsDNA that can go undetected by the cytosolic DNA sensor and immune system activator, cGAS.

Patients now

While the gene editing field often concentrates on large indications driven by a single common mutation, Edward Kaye, MD, CEO and director of Aurora Therapeutics, aims to extend these technologies beyond the “lucky few” who share the same mutation.

Aurora is pursuing an “umbrella IND” strategy that allows multiple guide RNAs to be evaluated within a single clinical trial. The company’s initial focus is on PKU.

PKU offers several advantages for early clinical development. Patients are routinely identified through newborn screening programs shortly after birth, which facilitates trial participant identification and enrollment. The condition also benefits from a clear regulatory precedent: reductions in phenylalanine levels are an established clinical endpoint used to move therapies toward approval.

“What we learn from PKU will be used for many other diseases because we have the systems in place,” said Kaye. “It expands gene editing into many more patients, by going after one disease first.”

Kaye also stresses the importance of engaging patient communities, whose input can ensure studies and regulatory processes are not overly burdensome for patients and families.

Maher Masoud, CEO of MaxCyte, emphasizes putting patients at the forefront. He adds that most gene-editing therapies in the clinic require significant patient conditioning, which can lead to lengthy treatment cycles and clinical trial timelines. Yet he sees these barriers to scale being eroded over the near term. As an example, modalities, such as allogeneic cell therapies, require far less patient conditioning and easier dosing regimens to support cheaper therapies.

In 2013, MaxCyte partnered with CRISPR Therapeutics on early work that led to the first FDA-approved therapy based on CRISPR-Cas9, Casgevy, with MaxCyte’s ExPERT electroporation platform enabling the efficient delivery of gene editing machinery into cells.

More than a decade later, the company has developed more than 1,000 applications and protocols. The broad engineering platform can repeatedly engineer batches of at least 20 billion cells using CRISPR-Cas9 in addition to base and prime editing.

Masoud says low-significant gene editing commercial success has been a bottleneck to scaling personalized therapies. Yet, he reiterates that CRISPR and other gene editing technologies were discovered a short 12 years ago.

“With CRISPR, we are finally seeing cures, Casgevy, LYFGENIA, and baby KJ are proof of that,” he says. “This is just the beginning.”

References

1. Tou, C.J., Xie, K., Ferreira da Silva, J., et al. Invasive DNA donors and recombinases license kilobase-scale writing. Nature. 2026. DOI: 10.1038/s41586-026-10241-z.

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Despite these growing pains, gene therapy remains one of the most promising strategies for treating genetic diseases. Beneath the headlines, researchers and technology developers are quietly transforming the infrastructure that makes these therapies possible.

At the center of that transformation is the adeno-associated virus (AAV), a tiny viral vector that has become the dominant platform for directly delivering therapeutic genes into human tissues. AAV-based therapies have already shown encouraging clinical results in diseases ranging from inherited retinal disorders to neurological syndromes.

But the rapid expansion of AAV-based therapies has also exposed important limitations. Manufacturing these complex biological products at scale is difficult, ensuring consistent product quality requires advanced analytical tools, and naturally occurring AAV variants often lack the tissue-specificity needed to efficiently deliver genes without high doses.

Across the cell and gene therapy ecosystem, scientists and companies are now addressing these challenges through innovations in bioprocessing, vector engineering, and computational design. These advances could determine whether gene therapy evolves into a sustainable therapeutic platform capable of reaching larger numbers of patients.

Building scalable workflows

Manufacturing viral vectors remains one of the most technically demanding aspects of gene therapy development. Producing AAVs requires complex biological systems, typically involving cultured mammalian cells that generate the virus after being supplied with the necessary genetic components.

As gene therapy programs move from laboratory research into clinical trials, production requirements expand rapidly. Processes that work well at a small scale often struggle to maintain efficiency and consistency when scaled to large bioreactors.

One crucial challenge involves balancing upstream production—where the virus is generated—with downstream processing—where the vector is purified and formulated for clinical use.

Pouria Motevalian, PhD, director, CMC development, pharma services, at Thermo Fisher Scientific, says that successful scale-up depends on developing a detailed scientific understanding of the process early in development.

“Effective downstream scale-up begins with the establishment of reliable scale-down models,” Motevalian explains. “These models are then used to define the design space—the scientifically established operating region in which critical process parameters and material attributes can vary without compromising product quality.”

This concept of design space comes from quality by design, a development philosophy that emphasizes understanding how manufacturing variables influence product attributes. By mapping these relationships early, developers can design processes that remain robust as they scale.

“Parameters are defined not only by target setpoints, but also by proven operating ranges,” Motevalian says. “As a result, facility, equipment, and material constraints can be accommodated during scale-up without compromising process performance or product quality.”

Such flexibility becomes increasingly important as upstream technologies improve. New cell-culture strategies, optimized media formulations, and refined production platforms are allowing developers to generate higher quantities of AAV particles. But these gains can introduce downstream bottlenecks if purification systems are not prepared to handle the additional load.

“As upstream yields increase, downstream bottlenecks related to loading capacity, filtration throughput, buffer demand, and facility fit can be proactively addressed while maintaining recovery and product quality,” Motevalian notes.

Automation and high-throughput development platforms are also helping accelerate process optimization. These technologies enable rapid testing of multiple process conditions, allowing researchers to evaluate purification strategies and more efficiently optimize workflows.

Transforming viral-vector analytics

Manufacturing improvements alone cannot support large-scale gene therapy production. Developers must also demonstrate that each batch of viral vectors meets strict quality standards, which places heavy demands on analytical technologies. “The latest innovation in viral-vector analytics is centered on faster turnaround, lower sample consumption, and more multiplexed methods,” Motevalian says.

Traditional techniques, such as analytical ultracentrifugation (AUC), remain essential for evaluating vector composition, but emerging complementary technologies are providing additional insights. “Increasing emphasis is being placed on rapid orthogonal tools, such as mass photometry, as complements to gold-standard methods, such as AUC,” Motevalian explains. “At-line approaches, such as size-exclusion chromatography with multi-angle light scattering, also enable quicker assessment of vector-genome and capsid titers.” These methods allow developers to monitor vector quality more quickly during development, reducing delays and enabling faster decision-making.

Meanwhile, molecular techniques are becoming more efficient. Multiplex droplet digital PCR (ddPCR) assays, for example, allow researchers to simultaneously analyze several genomic regions. “Multiplex ddPCR strategies are expanding analytical efficiency by enabling simultaneous interrogation of multiple genome regions and selected impurities,” Motevalian says.

Another emerging trend is the adoption of multi-attribute analytical methods based on liquid chromatography–mass spectrometry (LC-MS). These approaches allow several critical quality attributes to be monitored within a single analytical workflow.

The ability to track multiple parameters simultaneously strengthens process understanding and supports regulatory comparability assessments.

Engineering better vectors

Although manufacturing and analytics are improving rapidly, gene-therapy developers must still contend with the biological limitations of naturally occurring AAV vectors. Many widely used AAV serotypes were discovered decades ago and did not originally evolve for precision gene delivery in humans. As a result, they often lack the targeting specificity needed for efficient therapy.

“There are three main roadblocks, and they are all interdependent: delivery, safety, and the cost of scaling up manufacturing,” says Amos Gutnick, PhD, associate director of product development at PackGene. “Natural AAVs, like AAV9 or AAV2, still struggle with precision, so patients currently require massive doses in order to benefit from a therapeutic effect.”

Those high doses can “introduce serious safety risks, like liver toxicity and adverse immune reactions,” Gutnick notes. “They also drive production costs through the roof, often making the program commercially untenable.”

To overcome these obstacles, developers are working simultaneously on improving vector design and optimizing manufacturing efficiency. “At PackGene, our company motto is literally: ‘Make Gene Therapy Affordable,’” Gutnick says. Part of that effort involves improving bioprocessing platforms to generate higher yields and better quality vectors.

“On the manufacturing end, we’re constantly innovating our bioprocessing platforms to boost overall yields and purity,” Gutnick explains. “These innovations empower us to manufacture GMP-grade plasmids at record-breaking low costs and maximize the percentage of full, functional AAV capsids at every scale.”

AI accelerates vector design

Artificial intelligence (AI) is increasingly shaping how new viral vectors are developed. Machine-learning algorithms can analyze large datasets describing how capsid sequences behave in biological systems and use those insights to design improved variants.

As Gutnick says, “AI is a cornerstone of our π-Icosa Capsid Engineering Platform.” These models are trained to optimize both biological performance and manufacturability. “Biologically, the AI helps us design novel AAV capsids with incredible tissue targeting while actively detargeting the liver,” Gutnick explains.

Avoiding liver accumulation is particularly important because the liver often receives a large share of systemically delivered vectors and can become a source of toxicity. Equally important is ensuring that engineered capsids can be produced efficiently. “But just as importantly, we use AI to design for high manufacturability from day one,” Gutnick says.

Historically, some promising vectors discovered in the laboratory proved difficult to manufacture at scale. Designing with production in mind helps reduce that risk.

Faster effective-vector discovery

Identifying the right AAV capsid for a particular therapeutic application can take years. To accelerate this process, researchers are developing tools that allow large numbers of vector variants to be tested simultaneously.

As Gutnick says, “One of the biggest bottlenecks for translational researchers is simply the time and money it takes to find the right vector.”

To help address that challenge, PackGene collaborated with the Children’s Medical Research Institute to develop capsid-discovery kits that enable multiplex screening experiments. “This partner used this off-the-shelf kit to run high-throughput, multiplexed in vivo screens on dozens of capsids all at once,” Gutnick explains.

The screening system uses next-generation sequencing (NGS) to track both where vectors travel in the body and whether they successfully express their genetic payload. “By using dual NGS readouts to track both where the AAV went and if it actually worked, they were able to quickly pinpoint a lead capsid that hit their target tissue perfectly while avoiding off-target areas,” Gutnick says. “By giving researchers access to these kits, we’re helping them cut months—even years—off their discovery timelines.”

Platforms streamline production

Although improvements in vector design and analytics are essential, the infrastructure needed to manufacture gene therapies at scale is also evolving.

For example, Catalent developed an AAV production platform designed to accelerate the path from gene discovery to clinical manufacturing. The approach relies on standardized suspension HEK293 cell culture systems and integrated supply chains that combine plasmid production, process development, and manufacturing services.

Charles River Laboratories introduced its nAAVigation vector platform to streamline viral-vector development. Built around a high-productivity HEK293 suspension cell line and optimized upstream and downstream processes, the platform is intended to reduce development timelines while enabling scalable production.

Synthetic biology company Asimov is exploring another strategy through its AAV Edge Stable Producer System. Rather than relying on transient transfection for each production run, the system uses engineered producer cell lines in which viral genes are integrated directly into the genome. These stable cell lines can produce AAV vectors more consistently and might reduce manufacturing costs by eliminating the need for multiple plasmids.

Meanwhile, Andelyn Biosciences is applying its AAV Curator manufacturing platform to support gene-therapy programs targeting rare diseases. The company is collaborating with the Drake Rayden Foundation and researchers at the University of Texas Southwestern to manufacture clinical-grade vectors for a potential therapy aimed at nonketotic hyperglycinemia, a severe inherited metabolic disorder.

Toward a sustainable ecosystem

The rapid evolution of AAV technology reflects a broader shift in gene therapy from experimental science toward industrial-scale medicine.

Manufacturing innovations are enabling more efficient production of viral vectors. Advanced analytics are providing deeper insight into vector quality and performance. And computational approaches are unlocking new possibilities for designing safer, more precise delivery systems.

Together, these advances suggest that the next chapter of gene therapy will be defined not only by scientific discovery but also by the ability to manufacture and deliver treatments reliably and at scale. If those efforts succeed, AAV vectors could help transform the treatment of genetic disease—turning once-experimental therapies into widely accessible medicines capable of changing patients’ lives.

The post Smarter AAVs Drive Gene Therapy’s Next Chapter appeared first on GEN - Genetic Engineering and Biotechnology News.

Life sciences solutions providers are well aware of the challenge and are responding with innovations of their own. New solutions include customized reagents that scale from bench to clinical trials, industrialized lentiviral vector manufacturing techniques, and innovations to boost lentiviral transduction.

Scalable reagents

Scalability is one hurdle. “The infrastructure for reagents is set up for blockbuster drugs that are made in 10,000 L batches. But, when you produce CAR T therapeutics and other personalized therapies for rare diseases, you may need only one liter of (a given) reagent…that may have a minimum order of 350 L,” Stephen Gunstream, CEO of Teknova, says. “Generally, these aren’t stock reagents that last a few years. They are made to order [for specific products].”

Manufacturers’ dilemma, therefore, is that the custom, research-grade buffers they used for development lack the Good Manufacturing Practice (GMP)-level safeguards needed for human trials and may not be animal-free, but the animal-free, GMP products developers need aren’t available in small batches.

Continuing to use research-use only (RUO) reagents through scale-up creates other challenges. Aside from the obvious sterility control and consistency concerns, “Changes in mixing and manufacturing during scale-up change the product. Making one liter of product is different from making 1,000 L.” The ability to use the same raw materials and manufacturing processes, but at the appropriate grade, would help manufacturers tremendously.

Flexible small-batch facility

To that point, three years ago, Teknova completed a new facility specifically for small batch, modular manufacturing. It produces custom reagents quickly and at scale and—importantly—at various grades. Using this new facility, “We’ve done custom batches as small as one liter,” Gunstream says.

Step one involved designing a flexible, modular, regulatory-compliant manufacturing facility for RUO, RUO+, and GMP-grade reagents. This enables manufacturers to use the same reagents start to finish, selecting the appropriate grade for each step. That ability minimizes reagent costs, reduces risks, increases speed to market, and enhances product quality and consistency.

The facility, Gunstream points out, has been validated under multiple production scenarios, and features a “robust microbial contamination strategy that scales across manufacturing grades.”

Things you didn’t know you’d make

Teknova employs a bracketed validation strategy to create “a quality system designed to work with things you didn’t know you’d be making,” Gunstream says. That entailed analyzing raw materials used for custom reagents, knowing which have been used successfully in approved products, and validating these materials themselves at certain ranges.

To smooth development and scale-up, Gunstream advises selecting a supplier that:

• Can scale from beginning to clinical trials
• Is flexible and fast
• Has experience making small, custom batches
• Shares their insights

Manufacturing LVVs at scale

With roughly one dozen CAR T therapies in or nearing clinical trials, no single delivery technology dominates.

“From a manufacturing standpoint, this [plethora of options] places strong demands on vector production, purification, analytics, and scalability. As the field evolves, robust, flexible manufacturing platforms are essential,” Brian Tomkowicz, PhD, vice president and head of R&D and virology fellow, SK pharmteco, said in a recent webinar.

SK pharmteco is optimizing production of lentiviral vectors (LVVs), one of the go-to delivery CGT options, by industrializing ex vivo LVV production even as CAR T therapeutic delivery shifts toward in vivo modalities. With that shift, LVVs are defined as a drug product rather than an intermediate. Consequently, “The demands placed on antiviral vector manufacturing are shifting dramatically,” Tomkowicz said.

“An under-appreciated reality in LVV manufacturing is that downstream yield is overwhelmingly constrained by ion exchange chromatography (IEX) recovery,” he pointed out. “Although upstream harvest and clarification stems preserve the majority of vector material, a substantial loss occurs during the ion exchange step…even in processes that are optimized.” Consequently, “Total [functional particle] recovery often plateaus around 15% to 40%, with 20% being typical.”

In contrast, SK pharmateco’s high-throughput convective IEX delivers a functional particle recovery rate that ranges from 60% to 90%. Charts show a 98% host cell protein clearance and a transducing unit recovery rate exceeding 85% across multiple runs. To obtain such results, SK pharmateco replaced IEX’s traditional packed-bed resin with a membrane adsorber.

IEX, using a membrane absorber, cut total processing time to one hour or less (down from two to four hours using resin). That’s thanks in part to load capacities up to 250 mL/MV and residence times of less than six seconds, which help make throughput more than five times faster than packed-bed resin-based IEX. “This enables same-day processing,” Tomkowicz said.

Importantly, this high-throughput, membrane absorber-based IEX performance is maintained as process stress increases. Multiple tests involving a variety of input concentrations and higher viral particles measured physical recovery and functional titers. The highest levels of recovery peaked at 60–85%.

“Membrane [IEX] enables smaller footprints, disposable formats, and substantially lower material and buffer costs across both the 50L and 500 L production scales. This results in a six- to 20-fold cost reduction,” Tomkowicz explained.

The total cost of producing a 50 L batch using membrane adsorber IEX is about $3,000, he says, down from the $60,000 to $70,000 cost of a resin-based approach.

The most important optimization lesson, he said, is that “Recovery is governed by mass transfer physics rather than brand-specific attributes.”

As the industry moves from ex vivo to in vivo modalities, “The key difference is not just how we manufacture an LVV, but what we package inside it. Therefore, architecture and rational payload design become essential to allow manufacturers to exploit variants,” Tomkowicz emphasized. “While our focus is on scalable manufacturing, payload design and process requests must develop together.”

Tomkowicz called this a simple, scalable, fit-for-purpose platform for clinical and commercial manufacturing, predicting it will be “a manufacturing inflection point.” Before this method is widely adopted, however, Tomkowicz said he would like to see the work expanded to more vectors and more patients.

LVVs for adoptive cell therapy

“Perhaps the greatest challenge in the field of CGT today is the high cost of developing and manufacturing lentiviral vectors,” Chris Lowe, PhD, LentiBOOST business leader at Revvity, says. “One of the key drivers of this cost is the difficulty in achieving reproducible lentiviral transduction efficiency across a range of clinically relevant cell types (including T cell, hematopoietic stem cells, and progenitor cells) without compromising viability, phenotype, or function,” he tells GEN.

The issue manufacturers face is that even optimized vectors may require a high copy number if transduction is inefficient. “Transduction efficiency, therefore, can be the difference between a program that reaches the clinic and one that gets stuck in the lab because vector supply, manufacturing costs, or product quality are not sustainable at scale,” Lowe points out.

To address this challenge, industry scientists are exploring approaches that include optimizing vector design and production, improving physical and process-based activities, and developing chemical and biological enhancers.

For example, transduction can be improved by refining envelope pseudotypes, promoter selection, and genome architecture to improve tropism, especially when those technologies are accompanied by advances in vector manufacturing that increase functional titer and consistency. “While beneficial,” Lowe says, “these strategies enhance transduction indirectly and can increase manufacturing complexity.”

Likewise, spinoculation, controlled temperature shifts, and other physical and process-based methods to increase virus-cell contact (often combined with media and serum optimization) may improve cell fitness during transduction but are not without their own challenges. “Specifically,” Lowe cautions, “care must be taken to avoid issues with cell health, and these techniques can be challenging to scale up. These methods are often best paired with complementary chemical and biological enhancers.”

Such enhancers typically increase lentiviral transduction by promoting virus-cell interaction of membrane fusion, Lowe elaborates. “Traditional approaches such as polycations and extracellular matrix components are well established but can be associated with cytotoxicity, activation of cellular stress pathways, variable performance across primary cell types, and limited availability in consistent GMP-grade formats.

“Polymer-based enhancers offer an alternative approach that addresses some of these limitations,” Lowe continues. “Non-ionic poloxamer formulations, including technologies such as [Revvity’s] LentiBOOST enhancer, have been shown to improve transduction efficiency and increase vector copy number while maintaining cell viability.”

By providing a receptor-independent way to modulate the interface between viral and cellular membranes, the poloxamer in Revvity’s LentiBOOST technology promotes fusion. This increases the odds that each particle will deliver its cargo successfully across lentiviral constructs and multiple cell types—including primary T cells and human stem cells.

Multiple options

Today the industry is exploring “a variety of different approaches to enhance lentiviral transduction, including nanoparticle-assisted delivery, transient pathway modulation, and next-generation surface or matrix technologies that improve virus—cell contact or intracellular trafficking,” Lowe says.

That said, there are strong reasons to optimize the cellular environment. “Lentiviral transduction is most efficient when cells are metabolically active, not stressed, and can support membrane fusion and reverse transcription,” he says. Revvity, through BioLegend, which it acquired in 2021, also offers cell culture reagents in its Cell Vive family.

As CGT developers look to increasingly challenging cell types and ever-more-complex engineering, “there is a corresponding need to improve transduction efficiency…to increase therapeutic response rates, and to lower manufacturing costs,” Lowe says.

In the future, he adds, “Lentiviral transduction will increasingly coexist with non-viral and hybrid delivery systems. In that context, effective, GMP-grade enhancers will help ensure that lentiviral vectors remain a cornerstone technology where stable, durable gene delivery is required.”

With the plethora of advances being developed today, CGT manufacturers are on the cusp of improvements that will reduce process times, cut costs, enhance flexibility, and support scalability.

Fouad Atouf, PhD, is the chief science officer at United States Pharmacopeia.

References

1. Murimi-Worstell B, Ballreich M, Seamans G, Alexander, C. Association between US Pharmacopeia (USP) monograph standards, generic entry and prescription drug costs. Published: November 12, 2019.

2. Gupta, RK. The vital role of biological standardization in ensuring efficacy and safety of biological products–Historical perspectives. Journal of Pharmaceutical Sciences, 2025; 114(2): 690–700.

3. Atouf F and Venema J. Do Standards Matter? What is Their Value? Journal of Pharmaceutical Sciences, 2020; 109(8): 2387-2392.

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