The logic of therapeutic vaccination differs fundamentally from preventive immunization. Rather than blocking infection, these vaccines are designed to treat existing cancers by activating tumor-specific immunity and strengthening immune surveillance. Yet the review is candid about the field’s central frustration: even though endogenous T-cell responses against tumors exist in most patients, they typically fail to control disease, and immune checkpoint blockade (ICB) therapy—drugs that release the molecular brakes on T cells—often cannot rescue those responses on its own. Vaccines are meant to supply what the tumor microenvironment suppresses: a large, specific, and activated pool of anti-tumor T cells. Combining vaccines with checkpoint inhibitors has produced encouraging outcomes, including improved survival and tumor reduction across multiple cancer types, but low immunogenicity and tumor heterogeneity remain stubborn obstacles.

Nucleic acid vaccines are the fastest-moving segment of the field. DNA vaccines deliver bacterial plasmids encoding tumor-associated antigens (TAAs) or neoantigens into the cell nucleus, where they are transcribed into mRNA and translated into target proteins. They are stable, cheap to manufacture, easy to store, and can encode multiple antigens to hedge against tumor heterogeneity. The trade-offs are significant: DNA must cross the nuclear envelope, an extra step that lowers antigen production efficiency, and theoretical genomic integration remains a safety consideration unique to DNA. Because their intrinsic immunogenicity is low, DNA vaccines generally require adjuvants, electroporation, or nanoparticle delivery, and few clinical successes have been reported. One notable exception is GNOS-PV02, a DNA plasmid encoding up to 40 personalized neoantigens, which is being tested with an IL-12-encoding plasmid and the PD-1 antibody pembrolizumab in a phase 1/2 trial in hepatocellular carcinoma with promising early results.

mRNA vaccines have captured most of the attention, and the review argues the hype is at least partly earned. mRNA only needs to reach the cytoplasm to begin producing antigen, avoiding the nuclear barrier, and it cannot integrate into the genome. Advances in lipid nanoparticle (LNP) delivery have dramatically improved mRNA stability and cellular uptake, while self-amplifying mRNA (samRNA) promises prolonged antigen expression at lower doses, though cancer applications remain at an early preclinical and clinical stage. A double-edged sword is innate immune sensing: mRNA triggers pattern recognition receptors such as RIG-I and Toll-like receptors, which can act as a built-in adjuvant but can also induce type-I interferon responses that inhibit translation of the vaccine itself. The flagship candidate is mRNA-4157, which encodes up to 35 neoantigen epitopes and is delivered in LNPs. Combined with pembrolizumab, it improved recurrence-free survival versus pembrolizumab alone in a phase 2 trial of patients with completely resected, high-risk cutaneous melanoma, and phase 3 trials are now underway in melanoma, non-small cell lung cancer, and cutaneous squamous cell carcinoma.

Peptide vaccines take a more direct approach, administering synthetic polypeptides that mimic TAAs or neoantigens, which are captured by antigen-presenting cells and used to prime CD8-positive and CD4-positive T cells. Their Achilles’ heel is size: short peptides can load directly onto MHC molecules on any cell, including non-professional antigen-presenting cells that induce tolerance rather than immunity. Synthetic long peptides containing both MHC class I and class II epitopes avoid this trap and reliably induce both T-cell compartments. To overcome weak immunogenicity, researchers pair peptides with Toll-like receptor agonists, checkpoint inhibitors, or nanoparticle carriers such as virus-like particles and liposomes. Clinical results are accumulating: EVX-01, a personalized vaccine of up to 15 neoantigen epitopes with the CAF09b adjuvant, is in phase 1/2 testing alongside checkpoint inhibitors, and SurVaxM, targeting survivin in newly diagnosed glioblastoma, significantly prolonged patient survival in a phase 2 trial combined with pembrolizumab. A new phase 1 trial is evaluating a nine-peptide vaccine with the TLR1/2 ligand XS15 in acute myeloid leukemia patients in first remission.

Cell-based vaccines exploit whole cells rather than defined antigens. Dendritic cell (DC) vaccines harvest a patient’s own antigen-presenting cells, load them ex vivo with tumor lysates, peptides, or antigen-encoding RNA, and reinfuse them to activate cytotoxic T cells. Whole tumor cell vaccines, using irradiated or genetically modified tumor cells, present a broad antigen repertoire that hedges against heterogeneity, and are often supercharged with adjuvants such as GM-CSF. The category’s proof of principle is Provenge (sipuleucel-T), the FDA-approved autologous DC vaccine for prostate cancer. GVAX, an irradiated GM-CSF-secreting tumor cell vaccine, improved disease-free and overall survival in pancreatic cancer patients when combined with the antibodies urelumab and nivolumab in a phase 2 trial. Ongoing phase 2 studies are testing the neoantigen-loaded DC vaccine NeoDC-Vac after chemoradiotherapy in locally advanced esophageal squamous cell carcinoma, and an autologous tumor lysate-loaded cDC1 vaccine in ovarian cancer. The review notes that high production costs, labor-intensive manufacturing, and variable patient responses still constrain this platform.

Virus-based strategies come in two flavors. Oncolytic viruses preferentially infect and replicate inside tumor cells, exploiting abnormal surface receptor expression and the weakened antiviral signaling—blunted interferon and PKR pathways—characteristic of cancer. As infected cells lyse, they release reactive oxygen species, cytokines, and damage-associated molecular patterns that ignite both innate and adaptive immunity, and epitope spreading helps the immune system recognize tumor antigens beyond those the virus carries. The landmark agent is T-VEC, the first FDA-approved oncolytic therapy: an attenuated herpes simplex virus deleted for ICP34.5 to enhance tumor-selective replication and reduce neurovirulence, and engineered to carry the GM-CSF gene to recruit dendritic cells and macrophages into the tumor. Viral vector vaccines, by contrast, use engineered adenoviruses, lentiviruses, or vaccinia viruses to deliver antigen genes into host cells, mimicking natural infection so effectively that adjuvants are often unnecessary. Their vulnerability is pre-existing vector immunity, which can neutralize the vaccine—a problem managed by heterologous prime-boost strategies that switch vectors between doses.

Two technological currents are reshaping the entire field. The first is artificial intelligence: machine learning tools such as NetMHC and DeepHLA mine genomic and transcriptomic data to predict which tumor mutations will yield immunogenic neoantigens that bind a patient’s HLA molecules, bypassing central immune tolerance and reducing off-target risk. GNOS-PV02 already uses such algorithms to select neoantigens by predicted binding affinity. But the review cautions that limited training datasets and ethnic bias in HLA haplotype data can undermine the generalizability of predictions. The second is delivery engineering: LNP surface charge can be tuned to redirect biodistribution to different organs, cholesterol analogues such as C-24 alkyl phytosterols enhance in vivo mRNA delivery, hydrogels act as depots for sustained antigen release—with one study finding that D- versus L-glutamate chirality in polypeptide hydrogels profoundly altered immune cell infiltration and T-cell exhaustion—and lentiviral particles pseudotyped with Sindbis virus glycoprotein can target mRNA directly to dendritic cells, boosting both humoral and T-cell responses.

The review’s most sobering conclusion is that no vaccine platform, however elegant, is likely to deliver durable clinical benefit alone. Combination is the operative word: pairing vaccines with anti-PD-1 and anti-CTLA-4 antibodies, cytokines such as IL-2 and IL-15 superagonist ALT-803, adoptive cell transfers like tumor-infiltrating lymphocyte therapy, or even radiotherapy and chemotherapy that can convert immunologically cold tumors into inflamed ones by breaking down physical barriers of tumor-associated fibroblasts and poor vascularization. Underlying biology still constrains outcomes: HLA diversity between patients complicates neoantigen prediction and blocks some patients from accessing promising TAA epitopes, while individual T-cell receptor repertoires and pMHC densities produce highly variable T-cell avidity—and recent work suggests low-avidity T cells actually drive endogenous tumor immunity. Toxicity, patient variability, and treatment resistance complicate combination regimens. Still, with 15,000 accesses and growing citations since its May 2025 publication, the review captures a field at an inflection point: after decades of disappointment, therapeutic cancer vaccines—personalized, AI-designed, and rationally combined—are finally delivering on their immunological promise.

Subject of Research: Therapeutic cancer vaccine platforms and their combination with immunotherapy

Article Title: Therapeutic vaccine strategies in cancer: advances, challenges, and future directions

Article References: Therapeutic vaccine strategies in cancer: advances, challenges, and future directions. (n.d.). https://doi.org/10.1007/s44272-025-00039-x

Image Credits: AI Generated

DOI: 10.1007/s44272-025-00039-x

Keywords: therapeutic cancer vaccines, cancer immunotherapy, mRNA vaccines, DNA vaccines, neoantigens, peptide vaccines, dendritic cell vaccines, oncolytic viruses, viral vectors, immune checkpoint inhibitors, lipid nanoparticles, artificial intelligence

The study, published in the Journal of Translational Medicine, describes how the researchers retooled the AAV5 capsid using rapid directed evolution. Their strategy centered on variable region VIII of the viral capsid protein, a surface-exposed loop that governs how the virus engages cellular receptors. At residue Q574 within this region, they inserted libraries of random heptapeptides, seven-amino-acid sequences that collectively presented an enormous diversity of receptor-binding profiles. Each randomized capsid was packaged with its own encoding genome, so that the viral particles effectively carried the blueprint of their own success. The library was then subjected to iterative selection on 661W cells, a well-established mouse photoreceptor-derived cell line, allowing only those variants that entered these cells efficiently to amplify through successive rounds.

This selection scheme is a classic example of artificial Darwinism applied to a gene therapy vector. Rather than rationally designing a single capsid modification and hoping it improves tropism, the team let the biology choose. Millions of capsid variants competed for entry into photoreceptor-like cells, and the fittest clones were recovered, re-amplified, and challenged again. After several rounds, one variant emerged as the clear winner and was designated AAV5-AlyT. The name reflects its tropism for the outer retinal layers, and the variant carries a heptapeptide insertion at the Q574 position that presumably alters the capsid’s interaction with receptors on the inner limiting membrane and on photoreceptor cells themselves, the two principal barriers that natural AAV5 must overcome after intravitreal delivery.

In vitro comparisons confirmed that AAV5-AlyT transduced 661W cells far more efficiently than its parental AAV5 scaffold. The critical question, however, was whether that cellular advantage would translate into living eyes, where the vitreous, the inner limiting membrane, and the layered architecture of the retina impose constraints that cell culture cannot reproduce. In mice, a single intravitreal injection of AAV5-AlyT carrying a reporter transgene produced robust gene expression concentrated in the outer retina, the compartment containing photoreceptor inner and outer segments. The expression was not a transient burst; it persisted for more than six months after the single injection, an essential property for any therapy aimed at chronic degenerative diseases in which the treated cells must be supported for years.

The vector’s performance extended beyond mice. In rabbits, whose eyes are larger and anatomically closer to the human eye in several respects, AAV5-AlyT achieved efficient transduction across retinal layers after intravitreal delivery. Cross-layer transduction is a notable feature, because it suggests the capsid can penetrate the inner limiting membrane and then distribute genetic cargo to cells in multiple retinal strata rather than being confined to a narrow band. For translational purposes, this matters enormously: rabbit and non-human primate eyes are the standard large-animal models used to de-risk retinal gene therapy before human trials, and a vector that fails to scale from mouse to larger eyes rarely survives the development pipeline.

Having established delivery efficiency, the researchers tested whether AAV5-AlyT could actually treat disease. They chose two complementary models of retinal degeneration. The first was the ABCA4 knockout mouse, a model of Stargardt disease, an inherited macular dystrophy caused by mutations in the ABCA4 transporter gene that lead to toxic lipofuscin accumulation in photoreceptors and the retinal pigment epithelium. The second was light-induced retinal degeneration, a widely used model that recapitulates aspects of dry age-related macular degeneration, in which excessive light exposure triggers photoreceptor death and inflammatory degeneration. In both models, the therapeutic cargo was not a replacement gene but the complement regulator Crry, a mouse analogue of human complement regulatory proteins that dampens the cascade of complement activation driving tissue damage.

The results were striking. AAV5-AlyT-mediated delivery of Crry markedly rescued visual function in both disease models, as measured by electroretinography, the standard electrophysiological test of retinal responsiveness to light. Immunofluorescence and histological analyses showed that the treatment suppressed pathological complement activation and reduced damage to the retinal pigment epithelium, the support layer whose dysfunction is central to both Stargardt disease and dry AMD. These findings position the vector not merely as a delivery tool but as a validated platform for anti-inflammatory and neuroprotective gene therapy in the outer retina, where complement dysregulation is increasingly recognized as a key driver of degeneration.

One of the most clinically consequential findings concerns pre-existing immunity. A significant fraction of the human population carries neutralizing antibodies against common AAV serotypes, generated by prior natural infection, and these antibodies can neutralize therapeutic vectors before they reach their target cells, rendering treatment ineffective or forcing exclusion of patients from trials. The team screened 300 human serum samples for neutralizing activity against AAV5 and against the engineered AAV5-AlyT. The engineered variant showed a 34.9 percent lower rate of pre-existing neutralizing antibody response than parental AAV5. The heptapeptide insertion, while enhancing retinal tropism, apparently also perturbed an antibody epitope on the capsid surface, a serendipitous but valuable outcome that could broaden the eligible patient population for future therapies built on this capsid.

The technical achievement rests on a few principles worth underscoring. Directed evolution of AAV capsids has produced several notable vectors in recent years, but the field has struggled to find variants that combine outer retinal tropism with the practicality of intravitreal delivery. The Q574 locus in variable region VIII proved to be a productive engineering site, likely because it sits within a capsid region that influences both receptor binding and antibody recognition. The iterative selection on a photoreceptor cell line provided a clean, high-throughput fitness landscape, and the two-animal-species validation, followed by therapeutic proof of concept in two distinct degeneration models, gives the work an unusually complete translational arc for a single study.

Caveats remain, as they always do at this stage. The efficacy data come from mouse and rabbit models, and the human eye presents additional barriers, including a thicker inner limiting membrane and a much larger vitreous volume, that will require testing in larger animals and eventually in clinical trials. The Crry protein used as therapeutic cargo is mouse-specific, so human applications would require an equivalent human complement regulator. The authors also note a patent application related to the capsid, reflecting the commercial interest that engineered AAV vectors typically attract. Even so, AAV5-AlyT addresses the central logistical obstacle that has limited retinal gene therapy to specialized surgical centers: the need for subretinal injection. A vector that reaches photoreceptors through an intravitreal shot, sustains expression for over six months, evades a meaningful fraction of pre-existing neutralizing antibodies, and has already demonstrated functional rescue in models of both inherited and age-related retinal degeneration represents a substantial step toward making outer retinal gene therapy as routine as the injections ophthalmologists deliver every day.

Subject of Research: Directed evolution of an adeno-associated virus capsid for outer retinal gene delivery

Article Title: An engineered AAV variant with high-efficiency outer retinal tropism for gene delivery

Article References: Li, Y., Zhao, Z., Chen, Y., Zai, X., Chen, Z., Zhang, J., Zhang, Z., Wang, B., Song, X., Long, J., Wu, S., & Hou, L. (2026). An engineered AAV variant with high-efficiency outer retinal tropism for gene delivery. Journal of Translational Medicine. https://doi.org/10.1186/s12967-026-09022-z

Image Credits: AI Generated

DOI: 10.1186/s12967-026-09022-z

Keywords: adeno-associated virus, directed evolution, gene therapy, retinal degeneration, intravitreal injection, photoreceptors, Stargardt disease, age-related macular degeneration, viral vectors, complement cascade, neutralizing antibodies, AAV5-AlyT

The intellectual foundations of the field reach back to classic experiments showing that the differentiated state of a cell is not permanently fixed. Nuclear transfer and cell fusion studies demonstrated that somatic cell identity could be reset, and the discovery that the transcription factor MyoD could convert mouse fibroblasts directly into myoblasts established the concept of direct reprogramming. The landmark identification by Takahashi and Yamanaka of four transcription factors capable of generating induced pluripotent stem cells, or iPSCs, then opened a path to generating nearly any cell type in the laboratory. Several iPSC-derived therapies have since received regulatory approval or entered clinical trials. Yet the review emphasizes that iPSC-based approaches face persistent challenges, including tumorigenesis risk, inefficient differentiation, and poor survival and integration of transplanted cells within host tissues.

Direct reprogramming, also called transdifferentiation, offers an alternative by converting one somatic cell type into another without passing through a pluripotent intermediate. Even this strategy, however, has struggled when performed in vitro. Cells reprogrammed in culture often remain incompletely or immaturely converted, show poor tissue homing after transplantation, and suffer low long-term survival. In vivo direct reprogramming flips the logic entirely: rather than making cells in a dish and delivering them, researchers deliver reprogramming factors directly to the injury site, converting tissue-resident cells in their native environment. The review attributes the superior maturity of in vivo reprogrammed cells to pro-developmental and regenerative cues within the tissue microenvironment, which help drive cells toward fully functional states and promote integration with surrounding tissue.

At the molecular level, direct reprogramming works by rewriting gene regulatory networks, the hierarchical webs of transcription factors, signaling pathways, non-coding RNAs, and chromatin remodelers that define cellular identity. Central to this process are pioneer factors, a class of transcription factors capable of binding compacted heterochromatin. During neuronal reprogramming of fibroblasts, for example, the pioneer factor Ascl1 binds neural-lineage targets regardless of chromatin accessibility, locally opens the chromatin, and recruits the master regulators Brn2 and Myt1l to establish a neuron-specific regulatory circuit. Pioneer factors begin by scanning chromatin through non-specific electrostatic interactions, then distort and unwrap DNA at specific binding sites, recruiting co-regulators that remodel histones and create a transcriptionally permissive state. Successful reprogramming culminates in activation of peripheral network components that govern terminal fate and maturity, such as the factors Mafa and Errγ, which drive glucose-responsive insulin secretion in reprogrammed pancreatic beta cells.

The reprogramming toolkit extends well beyond transcription factors. Small molecules can activate developmental signaling pathways and modify the epigenetic landscape; a cocktail of CHIR99021, SB431542, DAPT, and LDN193189, for instance, converted human fetal astrocytes into cortical neurons by suppressing glial programs and activating neural master regulators. MicroRNAs offer another route: a combination of miRNAs 1, 133, 208, and 499 reprogrammed cardiac fibroblasts into cardiomyocytes by reinforcing cardiac lineage programs while silencing fibroblast signatures. Short hairpin RNAs and antisense oligonucleotides against the RNA-binding protein PTB have converted astrocytes into neurons, with stereotactically injected PTB antisense generating dopaminergic neurons that alleviated Parkinsonian symptoms in mouse models. Even exosomes, tiny membrane vesicles released by cells, can carry reprogramming cargo; insulinoma-derived exosomes enriched in miR-127 and miR-709 converted exocrine cells into pancreatic beta-like cells in vitro.

Delivering these factors into living tissue remains one of the field’s central engineering problems, and the review systematically compares the options. Viral vectors dominate current practice. Adenoviruses carry large payloads and achieve high transduction of dividing and non-dividing cells, as demonstrated when delivery of the transcription factors Ngn3, Pdx1, and Mafa converted more than 20 percent of infected pancreatic exocrine cells into insulin-positive cells within ten days. Adeno-associated viruses persist episomally for months to years and have reprogrammed retinal Müller glia into rod photoreceptors and astrocytes into neurons at efficiencies near 20 percent. Lentiviruses integrate into the host genome, enabling sustained expression but raising insertional mutagenesis concerns. Non-viral alternatives include plasmids, which can carry very large cargoes with minimal immunogenicity but express transgenes only transiently, and nanoparticles, particularly lipid nanoparticles, which can be surface-engineered for tissue-specific targeting. In one striking example, neutrophil-membrane-coated mesoporous silicon nanoparticles carrying cardiac reprogramming miRNAs homed to injured heart tissue via fibrinogen-derived targeting peptides, improving cardiac function and reducing fibrosis after myocardial infarction in mice.

The therapeutic results assembled in the review span nearly every major organ system. In the brain, delivery of the transcription factors Ascl1, Lmx1a, and Nurr1 converted striatal glial cells into functional neurons that integrated into host circuits within three months. AAV-mediated suppression of PTB converted astrocytes into neurons with efficiencies rising from roughly 20 percent at three weeks to 80 percent at twelve weeks, replenishing lost dopaminergic neurons and restoring striatal dopamine in Parkinson’s disease models. In the heart, retroviral delivery of Gata4, Mef2c, and Tbx5 generated induced cardiomyocytes that developed mature sarcomeres, fired action potentials, and electrically coupled with endogenous tissue, improving cardiac function after infarction. A seven-molecule chemical cocktail administered orally and intraperitoneally achieved lower but measurable conversion with functional recovery. In the pancreas, reprogrammed beta cells secreted insulin and improved glucose tolerance in diabetic mice, while in adipose tissue, exosome-driven conversion of inflammatory macrophages and browning of white fat improved insulin sensitivity and energy expenditure in obese mice.

Despite these preclinical successes, the review is unflinching about why in vivo direct reprogramming has yet to enter a single clinical trial. Reprogramming kinetics and efficiency in living tissue lag far behind culture conditions: a small-molecule cocktail that converted 94 percent of neonatal astrocytes into neurons in vitro achieved only about 11 percent in adult mouse brain, and in vitro-optimized doses proved insufficient in vivo, sometimes requiring tripled concentrations. The complex three-dimensional tissue microenvironment, with its biophysical cues such as matrix stiffness and shear stress, and its biochemical milieu of injury-induced inflammatory signals, reactive oxygen species, and cellular senescence, can either promote or antagonize reprogramming in ways that two-dimensional cultures cannot predict. Safety concerns compound the problem: reprogramming factors such as c-Myc carry oncogenic potential, integrating vectors risk insertional mutagenesis, transient progenitor-like states may mimic cancer phenotypes, and non-target cells sharing lineage history with targets may be inadvertently converted. Partially reprogrammed cells that revert or drift into undefined states represent a further unresolved risk.

The path forward, the authors argue, will require a convergence of emerging technologies. CRISPR-based transcriptional activators offer programmable activation of lineage-specific gene networks, as shown when AAV-delivered CRISPR repression of Nrl converted rod photoreceptors into cone-like cells in a retinitis pigmentosa model. Artificial intelligence and machine learning models trained on single-cell multi-omics data can predict gene perturbation effects, identify bifurcation points where reprogramming trajectories succeed or fail, and screen for novel factor combinations. Organ-on-chip platforms that replicate tissue-specific stiffness, fluid flow, and electrical properties may finally allow preclinical optimization of dose and exposure time under realistic three-dimensional conditions. Stimuli-responsive delivery systems, including microneedle patches, hydrogels, and electromagnetic-field-activated gene switches, promise controlled and targeted release. Before first-in-human trials, the review concludes, the field must validate outcomes in large animal models, establish GMP-compliant manufacturing, define patient stratification criteria, and develop regulatory frameworks that evaluate the integrated platform of factors and delivery vehicles as a single therapeutic entity. If those challenges can be met, in vivo direct reprogramming could move regenerative medicine beyond repair toward genuine rejuvenation of organs currently considered untreatable.

Subject of Research: In vivo direct reprogramming of tissue-resident cells for regenerative therapy

Article Title: In Vivo Direct Reprogramming: Current Progress and Future Prospects from Mechanisms to Therapeutic Application

Article References: Singh, R. D., Calvoli, M., & Kim, K. K. (2026). In Vivo Direct Reprogramming: Current Progress and Future Prospects from Mechanisms to Therapeutic Application. Advanced Science, Article e77370. https://doi.org/10.1002/advs.77370

Image Credits: AI Generated

DOI: 10.1002/advs.77370

Keywords: direct reprogramming, in vivo reprogramming, regenerative medicine, transcription factors, pioneer factors, gene regulatory networks, iPSCs, cardiac regeneration, neuronal reprogramming, viral vectors, lipid nanoparticles, CRISPR

The review begins where the technology itself began: in prokaryotes. CRISPR-Cas systems evolved as adaptive immune defenses in bacteria and archaea, storing fragments of viral DNA in genomic memory banks and using them to recognize and destroy invading genetic material. When researchers realized that this bacterial immune machinery could be reprogrammed with a single guide RNA to target nearly any sequence of interest, the implications were enormous. Early CRISPR-Cas9 experiments demonstrated remarkable efficiency and ease of use compared with earlier genome-editing platforms such as zinc-finger nucleases and transcription activator-like effector nucleases, which required laborious protein engineering for each new target.

Yet the honeymoon was short-lived. As clinical ambitions grew, so did concern over off-target effects, the unwanted edits that occur when the Cas9 nuclease acts at sites that only partially match the guide RNA. Variable editing outcomes, unpredictable insertions and deletions at the target site, and the risk of large genomic rearrangements all limited the technology’s broader application to human therapy. The review emphasizes that these technical shortcomings, alongside delivery inefficiencies that make it difficult to get editing machinery into the right cells in the right tissues, remain among the field’s most persistent challenges even as the toolkit has grown far more sophisticated.

The evolution from scissors to editors is best embodied by the rise of base editing and prime editing. Rather than relying on double-strand breaks and the cell’s error-prone repair pathways, these newer approaches chemically convert individual DNA letters or write short new sequences with minimal collateral damage. The review highlights work demonstrating that prime editing can correct oncogenic KRAS variants without the constraints imposed by traditional CRISPR-Cas9 workflows, and that CRISPR-based strategies have achieved efficient correction of both KRAS and TP53 mutations, two of the most frequently mutated genes in human cancer. Such results illustrate the conceptual shift: the goal is no longer simply to disrupt a gene but to rewrite it precisely, restoring normal function rather than merely silencing a faulty instruction.

Cancer therapy has emerged as one of the most active arenas for these refined tools. The review surveys how CRISPR-Cas9 library screening has accelerated anti-cancer drug discovery by systematically identifying genes that tumors depend on, and how ex vivo editing of immune cells is reshaping immunotherapy. Chimeric antigen receptor T cells engineered with CRISPR have shown that targeting the CAR construct to a defined genomic locus, such as the TRAC locus, can enhance tumor rejection compared with randomly integrated vectors. Editing of natural killer cells and T cells from human donors is being pursued for cancer immunotherapy, and strategies to improve CAR-T cell persistence through CRISPR-mediated knockout of exhaustion-related pathways are advancing through preclinical and clinical pipelines.

Beyond cancer, the review catalogs applications across viral disease, autoimmune conditions and inherited disorders. CRISPR-based approaches are being developed to engineer resistance to viruses, with HIV-1/AIDS therapy representing a prominent example in which editing aims to remove or disable viral genomes integrated into host DNA. Diagnostic platforms derived from CRISPR nucleases, including SHERLOCK and DETECTR, exploit the sequence-specific recognition of Cas enzymes to detect viral nucleic acids with high sensitivity, offering rapid tools for emerging infectious diseases. In autoimmune disease research, genome editing is being used to dissect and potentially correct the immune dysregulation that underlies conditions such as lupus and rheumatoid arthritis, pointing toward a future in which the immune system itself can be retuned at the genetic level.

None of these therapeutic visions can be realized without solving the delivery problem, and the review devotes substantial attention to the technologies that ferry editing machinery into cells. Viral vectors, particularly adeno-associated virus and lentiviral vectors, offer efficient gene transfer but raise questions about payload capacity, insertional risks and immune responses. Non-viral alternatives include physical methods such as electroporation and microinjection, lipid nanoparticles and solid lipid nanoparticles, cell-penetrating peptides, DNA nanocarriers and extracellular vesicles. Nanotechnology-based delivery of CRISPR components for cancer treatment is a particularly active frontier, with gold nanoparticles and other platforms being evaluated for biocompatibility and cytotoxicity under evolving regulatory frameworks. The choice of delivery vehicle, the review makes clear, often determines whether an elegant editing strategy can ever reach the clinic.

Even when editing succeeds, the human body may object. Immunogenicity of the bacterial Cas9 protein is a recognized concern, since many people carry pre-existing antibodies and T cells reactive to common Cas variants from environmental exposure to the microbes that harbor them. The review also confronts the ethical dimensions that shadow every advance in the field. The prospect of human germline and heritable genome editing has prompted a global policy debate about where the lines should be drawn, and ethicists have argued that the moral considerations raised by such a powerful tool demand attention to accessibility, consent and the long-term consequences of permanent genomic change. Who will be able to afford these therapies, and how societies will govern their use, remain unresolved questions that the technical community cannot answer alone.

What emerges from the review is a portrait of a technology on a clear trajectory. Engineered Cas variants, such as PAM-flexible chimeric Cas9 proteins that relax the sequence constraints on where editing can occur, are expanding the addressable space of the genome. High-fidelity nucleases reduce off-target activity, base and prime editors increase the precision of the intended change, and improved delivery systems broaden the range of treatable tissues. Together these refinements position CRISPR as an increasingly precise platform for both fundamental research and therapeutic use, supporting targeted therapies for genetic disorders in ways that the original scissor paradigm could never achieve. The authors, based at the University of Science and Culture and the Royan Institute in Tehran, frame this progression not as a completed project but as an ongoing evolution toward greater specificity, safety and predictability.

The clinical momentum is already visible. Approved CRISPR-based therapies for hemoglobin disorders have demonstrated that genome editing can deliver durable benefit in patients, and dozens of trials are testing edited cell therapies, in vivo editing and CRISPR-enabled diagnostics across a widening spectrum of disease. The remaining obstacles, off-target editing, delivery inefficiency, immune reactions and equitable access, are formidable but no longer conceptual; they are engineering and policy problems with active research programs attached to each. As Rahbari, Lohrasbi and Amiri-Yekta conclude, the story of therapeutic genome editing is one of steady refinement, in which the crude power of the original molecular scissors is being progressively replaced by the controlled, predictable hand of a true genome editor. The precision revolution they describe is redefining what medicine can attempt at the level of DNA itself, and the pace of that redefinition shows no sign of slowing.

Subject of Research: The evolution of CRISPR-based genome editing from early nuclease cutting tools toward precise therapeutic platforms for genetic disease

Article Title: From scissors to editors: how the evolution of precision is redefining therapeutic genome editing

Article References: Rahbari, M., Lohrasbi, R., & Amiri-Yekta, A. (2026). From scissors to editors: how the evolution of precision is redefining therapeutic genome editing. Molecular Genetics and Genomics, 301(1), Article 193. https://doi.org/10.1007/s00438-026-02467-3

Image Credits: AI Generated

DOI: 10.1007/s00438-026-02467-3

Keywords: CRISPR-Cas9, genome editing, gene therapy, base editing, prime editing, off-target effects, cancer immunotherapy, CAR-T cells, viral vectors, nanoparticle delivery, precision medicine, genetic disorders

The research, led by Franziska Köhler and Sonja Kallendrusch of Leipzig University and the HMU Health and Medical University, together with colleagues in Erfurt and Potsdam, compared two complementary patient-derived culture systems side by side. The first, patient-derived neurospheres (PDNS), are compact, free-floating spheroids grown from dissociated tumor cells under serum-free conditions. They offer a controlled, tumor-cell-intrinsic system in which variables can be manipulated with relative ease. The second, patient-derived tissue slice cultures (PDTC), preserve the tumor’s native architecture: thin, 350-micrometer slices of fresh surgical tissue are cultured at an air–liquid interface, retaining the extracellular matrix, vascular structures, immune cells, and stromal compartments that neurospheres inevitably discard. The team systematically exposed both models to two AAV serotypes, AAV2 and AAV6, each carrying a green fluorescent protein (GFP) reporter gene, and then tracked transduction with live confocal microscopy, quantitative PCR, flow cytometry, and immunofluorescence.

The technical rigor of the workflow is central to the study’s credibility. Thirteen surgical specimens from 25 collected samples ultimately entered the analysis, twelve of them IDH-wildtype glioblastomas under the WHO 2021 classification. Tissue not required for diagnosis was obtained with informed consent and ethical approval. For neurospheres, tumor tissue was mechanically and enzymatically dissociated with Accutase, filtered, and seeded at 100,000 cells per well; after 10 to 15 days, spheroids of 100 to 150 micrometers in diameter were ready for experiments. Slice cultures were punched into 2 to 3 millimeter discs and maintained on membrane inserts. After testing several media formulations, the researchers selected NeuroCult-XF, a serum-free proliferation medium, because it preserved tissue architecture and histopathological features better than serum-containing alternatives or other defined formulations. Blinded pathological review confirmed that fibrillary architecture and even characteristic gemistocytic features—enlarged cell bodies with eccentric nuclei—were retained in both models.

The cellular composition analysis revealed stark differences between the two systems, exactly as the team had hypothesized. Slice cultures retained expression of immune markers such as CD68, the vascular marker CD31, and the hypoxia marker HIF1α in organotypic patterns closely mirroring the original tumors. Neurospheres, by contrast, showed virtually no CD31, scarce and disorganized CD68-positive cells, and only weak HIF1α restricted to larger spheroids. More subtly, sphere-forming conditions selectively enriched stem-like populations: the mean SOX2-positive cell fraction rose from 0.114 in native tissue to 0.328 in PDNS, and Nestin positivity climbed from 0.132 to 0.393, while the mesenchymal marker CD44 fell. This finding matters because it confirms that neurosphere culture is not a neutral act of preservation but an active selection process that reshapes the cellular landscape before any vector is ever introduced.

When the vectors were applied, the serotype differences were unambiguous. In neurospheres, AAV6 drove significantly greater GFP expression than AAV2 across a dose range of 10^6 to 10^9 particles, with significant signal appearing as early as day 2 at higher doses, while AAV2 reached significance only at the highest dose after 5 days. Quantitative PCR corroborated the fluorescence data: five days after exposure to 10^8 particles, PLAT-normalized vector genome abundance averaged 146.7 copies per cell for AAV2 but 1,074.0 for AAV6 in EGF-containing medium. Notably, epidermal growth factor supplementation itself proved to be a critical experimental variable. Because AAV6 is known to use EGFR as a co-receptor for cellular entry, the team deliberately compared EGF-containing and EGF-free conditions. In neurospheres, EGF presence modified both the magnitude and temporal stability of AAV6-mediated GFP expression, and marker-defined analysis showed that EGF deprivation broadened GFP positivity across GFAP-, CD44-, Nestin-, and SOX2-positive populations, whereas under EGF-containing conditions only the Nestin-positive fraction showed significant GFP signal.

Exploratory flow cytometry from two independent experiments added an intriguing layer. GFP positivity was markedly enriched among SOX2-positive cells (40.8 percent versus 7.2 percent in SOX2-negative cells) and among cells triple-positive for CD133, Nestin, and SOX2 (41.0 percent versus 7.5 percent). The authors are careful to stress that these figures, based on limited sample numbers, are descriptive and cannot distinguish preferential vector uptake from differences in population abundance, survival, or promoter activity. Still, the pattern suggests that the most stem-like, aggressive cellular states within a glioblastoma may be among those most efficiently engaged by AAV6—a hypothesis with obvious therapeutic implications if it can be confirmed with larger datasets and mechanistic studies.

The most sobering results came from the tissue slice cultures. Overall GFP readouts were comparable to, or in some cases higher than, those in neurospheres, but interpatient variability was dramatically greater. Some patient specimens transduced robustly; others showed little or no detectable GFP despite identical conditions. Mean fluorescence intensities of 4,989 for AAV2, 4,232 for AAV6, and 6,448 for AAV6 without EGF in slice cultures contrasted with far lower values in matched neurospheres, but the spread between patients was wide enough that no single-patient result could be generalized. The authors argue that this variability should not be dismissed as experimental noise. Instead, it may represent a biologically faithful reflection of the heterogeneity that any real-world gene therapy for glioblastoma would confront. A vector that performs uniformly in a homogeneous spheroid panel may still fail in a substantial fraction of patients, and only multi-patient tissue-based validation can expose that vulnerability before clinical translation.

Spatial analysis added another dimension. In neurospheres, GFP-positive cells clustered predominantly at the spheroid periphery, consistent with diffusion-limited vector transport through densely packed three-dimensional tissue, although the authors caution that GFP fluorescence reflects the downstream consequence of delivery and expression rather than physical particle penetration itself. Slice cultures showed a more homogeneous distribution of fluorescence across the tissue, an effect not associated with preferential vascular localization and therefore likely driven by preserved microarchitecture rather than vascular entry routes. Equally notable was the observation of occasional GFP-positive cells in two samples of adjacent non-neoplastic brain tissue, a preliminary but cautionary signal that off-target transduction of tumor-adjacent cells can occur under experimental conditions, consistent with the broad central nervous system tropism of naturally occurring AAV serotypes.

The study’s self-imposed limitations are candidly acknowledged. Vector dose escalation could not be performed in slice cultures because of limited tissue availability, preventing fully matched comparisons between models. Flow cytometric findings rested on only two experiments. Vector genome quantification cannot distinguish entry, trafficking, persistence, or processing, and GFP expression additionally depends on transcription and translation. Slice cultures themselves, while architecturally faithful, remain ex vivo systems that cannot reproduce systemic immunity, vascular dynamics, or long-term tumor evolution. The significant increase in SOX2 expression observed in neurospheres after AAV6 treatment without EGF likewise supports an association rather than a demonstrated causal effect of vector exposure on tumor-cell state.

What the study ultimately delivers is a framework rather than a verdict. The authors explicitly do not claim that both models are mandatory for every preclinical AAV program, nor that either predicts in vivo performance. Instead, they advocate a question-driven approach: neurospheres, with their lower variability and experimental tractability, are well suited to initial serotype screening and dissection of tumor-cell-intrinsic effects; candidate vectors that pass that filter can then be challenged against panels of slice cultures from multiple patients, where preserved architecture, stromal and vascular compartments, and honest interpatient heterogeneity reveal how performance holds up in tissue that more closely resembles what a surgeon would leave behind in a resection cavity. In a disease where nearly every promising preclinical result has eventually collided with clinical reality, the insistence that model selection itself is a determinant of apparent vector efficacy may prove to be one of the more consequential methodological messages glioblastoma gene therapy has received in years.

Subject of Research: Preclinical evaluation of AAV-mediated gene delivery in patient-derived glioblastoma cultures

Article Title: Patient-derived tissue cultures complement neurospheres for preclinical evaluation of AAV-mediated gene delivery in glioblastoma

Article References: Köhler, F., Hess, K., Koloske, C., Gaunitz, F., Rosahl, S. K., Gerlach, R., & Kallendrusch, S. (2026). Patient-derived tissue cultures complement neurospheres for preclinical evaluation of AAV-mediated gene delivery in glioblastoma. Journal of Neuro-Oncology, 179(3), Article 93. https://doi.org/10.1007/s11060-026-05807-w

Image Credits: AI Generated

DOI: 10.1007/s11060-026-05807-w

Keywords: glioblastoma, AAV vectors, gene therapy, patient-derived models, neurosphere cultures, tissue slice cultures, tumor microenvironment, viral vectors, EGF, transduction, preclinical testing, gene delivery