Molecular Therapy - Methods & Clinical Development

Recombinant adeno-associated virus (rAAV) vectors are pivotal for gene therapy; however, the encapsidation of residual DNA, particularly plasmid backbone sequences, pose significant safety risks. Recent studies have identified the p5 promoter, which contains a Rep-binding element and a terminal resolution site (TRS), as a cryptic origin of replication that facilitates packaging of upstream sequences. In this study, we investigated the effect of p5 TRS modifications on impurity DNA levels in a single-plasmid All-in-One (AiO) AAV production system. Wild-type p5 (p5wt) promoted significant packaging of upstream plasmid backbone DNA, especially when the backbone was positioned between p5wt and the inverted terminal repeat. Introducing mutations or deletions in the p5 TRS significantly reduced encapsidation of plasmid-derived sequences, including kanamycin resistance genes, and improved the ratio of full to partial particles, as seen with the p5{Delta}loop variant. Furthermore, the p5{Delta}loop-AiO system showed higher rAAV yields than both conventional triple-transfection methods and previously reported p5-spacer variants. Thus, our findings suggest a robust vector design strategy for minimizing DNA impurities, thereby enhancing the safety and efficacy of AAV-based gene therapy.

While adeno-associated virus (AAV) vectors have shown therapeutic benefit in clinical applications, noted challenges include low transduction efficiencies, poor cellular targeting, and vector related adverse events. Recently, it was demonstrated that a rationally designed synthetic inverted terminal repeat (SynITR) altered the AAV vector-induced DNA damage response and abrogated apoptosis in human embryonic stem cells. To explore the utility of AAV-SynITR for diverse gene therapy applications, vector production, transduction, and the cellular response were evaluated in various contexts. Regarding production, SynITR preparations exhibited comparable titers to wtITR in a serotype/transgene-independent manner. Despite slightly decreased transduction efficiency in various cell lines, intravenous administration of AAV8 vectors showed SynITR enhanced transduction in a tissue-specific manner in liver (>7-fold) and kidney and pancreas (>2-fold) at equivalent vector copy numbers; however, no differences were observed in muscle/heart/spleen tissues. Interestingly, persistent {gamma}H2AX, a marker of aging/chronic inflammation, was abundant in the liver and spleen following wtITR (but not SynITR) transduction. In human corneas, SynITR enhanced transduction up to 16-fold over wtITRs. These data demonstrate that SynITRs elicit tissue-specific transduction enhancement and alter the cellular stress response. Importantly, the SynITRs offer an alternative context to elucidate wtITR biology for targeted, enhanced, and potentially safer human gene therapy.

Efficient systemic delivery to the lung remains a major barrier for adeno-associated virus (AAV)-mediated pulmonary gene therapy, particularly when pre-existing immunity limits the use of conventional capsids. Here, we evaluated Bovine AAV, a phylogenetically divergent capsid, as candidate vector for lung-directed gene transfer. In adult C57BL/6J mice, intravenous delivery of Bovine AAV resulted in robust and preferential lung transduction comparable to AAV4, with predominant targeting of alveolar type I pneumocytes and pulmonary endothelial cells. In primary human lung-resident cells, Bovine AAV was particularly effective in microvascular endothelial cells, a target poorly transduced by AAV4 in vitro. Bovine AAV demonstrated scalable production with yield, purification performance, capsid quality, and genome integrity comparable to AAV9. In sera from healthy adults from the United States and Switzerland, Bovine AAV showed intermediate neutralization frequencies, lower than AAV2 and AAV4 but higher than AAV5 and AAV9. Of relevance, Bovine AAV maintained in vivo transduction efficiency in mice previously immunized with a pool of human and non-human primate-derived AAV capsids, including AAV4. Together, these results position Bovine AAV as a promising lung-tropic and immune-distinct vector for pulmonary gene therapy, with particular relevance for applications requiring systemic delivery in the presence of pre-existing immunity to conventional serotypes.

Lentiviral vectors are commonly used to introduce chimeric antigen receptor transgenes into T cells, but routine assays quantify vector copy number or integration sites without sequencing full-length integrated vectors. HIV-1 proviruses often acquire large deletions and cytidine deaminase-driven hypermutation; whether similar variation occurs in therapeutic lentiviral vectors is unclear. We adapted a novel long-read capture approach to enrich long fragments spanning vector DNA and adjacent human sequence, enabling simultaneous integration-site mapping and proviral integrity analysis with single-molecule resolution. In research-grade CAR T cells produced with an experimental, transient-transfection lentiviral vector workflow, 40% of integrated vectors carried recurrent deletions that removed the internal promoter or parts of the chimeric antigen receptor cassette. The dominant promoter deletion was present in the viral stock. In clinical chimeric antigen receptor T cell products, promoter deletions were less frequent, but detectable pre-infusion and post-infusion. Across datasets we observed widespread G-to-A substitutions consistent with restriction factor editing, including changes predicted to introduce premature stop codons within the transgene open reading frame. Our method reveals proviral variants invisible to standard quality-control assays and provides a framework to improve vector production and monitor transgene integrity in clinical products.

BackgroundMicroglia have become a cell type of interest in the neurodegenerative field given both genetic and pathological evidence for their role in disease development and progression. There has been a rapid growth of studies using iPSC-derived microglial models to understand the molecular mechanisms driving these neurological diseases. However, it remains difficult to transduce myeloid cells effectively which is critical when aiming to study the role of disease associated genes and pathways. Current methods require exposure to multiple viruses which is not suitable for all experimental paradigms. We have therefore sought and characterised a high efficiency promoter and plasmid design to allow high transduction efficacy with a single lentivirus. ResultsUsing the spleen focus-forming virus (SFFV) promoter in combination with central polypurine tract (cPPT) and Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) plasmid elements gave significantly higher transduction efficiency and transgene expression than was achieved with commonly used promoters CMV and EF1. This could then be further improved if required to over 90% transduction efficiency with the removal of lentivirus restriction factor SAM and HD domain-containing protein 1 (SAMHD1) by adding VPX. ConclusionsOur findings allow for a simpler, more efficient and streamlined approach to transgene expression in iPSC-derived microglia and macrophages using only a single lentivirus. This minimises potential unintended side effects such as additional cellular activation and increased cell death.

One barrier to developing an HIV-1 cure is viral reservoirs persisting within B cell follicles of lymphatic tissues, partly due to failure of HIV-specific cytotoxic cells to express the follicular-homing receptor CXCR5. Our group explores CAR cell therapies which also express CXCR5 as a potential cure strategy for HIV. Although previous studies have mostly explored CAR T cell therapies, CAR NK cells may be an attractive alternative as they can be used in allogeneic settings and are naturally cytotoxic towards HIV-infected cells. Here, we developed a novel and innovative multiplex engineering method for rhesus macaque NK cells to create virus-specific CAR NK cells multiplexed (MP) with CAR/CXCR5/IL-15/PD-1 KO/transient-CCR7. We first evaluated MP NK cells in vitro for functionality. MP NK cells were then infused into one chronically SIV-infected rhesus macaque to observe tolerance and localization of therapeutic cells. Finally, we performed a larger primate study in which SIV-infected rhesus macaques were infused with two doses of MP NK cells to study long-term localization, safety, and efficacy. In vitro, MP NK cells were expanded to clinically relevant numbers, migrated to chemokine signaling, and secreted cytotoxic cytokines in response to SIV-Env-expressing cells. In the preliminary rhesus macaque study, the therapy caused no adverse reactions, and CAR+ NK cells localized to sites of SIV replication within the spleen and lymph nodes. In the larger primate study, two doses of MP NK cells at 1.2 x 108 cells/kg were safe and increased the levels of NK cells and CAR+ NK cells found within lymphatic tissues. Importantly, the CAR+ NK cells detected in lymph nodes were predominantly CCR7+, demonstrating the importance of CCR7 and CXCR5 in combination for migration to SIV viral reservoirs in follicles of lymphatic tissues. This study is the first to demonstrate this type of complexity and combination of engineering techniques in NK cells. With further optimization, these techniques could lead to the development of novel NK cell therapies to treat HIV and other diseases.

Virus-free genome engineering provides a flexible alternative to viral vectors for generating genetically modified cell models. Here, we establish an integrated biosafety level 1-compatible CRISPR/Cas9 homology-directed repair (HDR) workflow for stable transgene knock-in in neuroblastoma cell lines using non-viral delivery approaches. We systematically evaluated donor cassette architecture and delivery conditions across electroporation-based Cas9 ribonucleoprotein (RNP) delivery and lipid nanoparticle (LNP)-mediated co-delivery of Cas9 mRNA, sgRNA, and donor DNA. Modular AAVS1-targeting donor constructs identified a compact EF1(s)-Donor-Q8-Tag-sPA cassette that consistently yielded the strongest HDR-associated knock-in readouts, achieving up to 60% stable reporter-positive cells following electroporation without HDR enhancers. While LNP-mediated delivery enabled efficient CRISPR cargo co-delivery and generation of genetically modified tumor cell populations, knock-in efficiencies remained lower than those observed with electroporation. Subsequent enrichment approaches enabled generation of highly pure edited cell populations following both delivery strategies. Functional validation demonstrated stable transgene expression in vitro, including in three-dimensional bioprinted tumor models, and in vivo in xenograft mice without impairing tumor growth or viability. Together, these findings establish a practical non-viral HDR platform for stable engineering of solid tumor models and provide a framework for further optimization of genome editing workflows across distinct delivery modalities. Key findings- We establish a complete, virus-free CRISPR/Cas9 HDR workflow that reliably enables stable knock-in in solid tumor cell lines, demonstrated here in two neuroblastoma models under biosafety level 1 conditions. - We establish and evaluate LNP-mediated co-delivery of Cas9 mRNA, gRNA, and donor DNA for non-viral HDR knock-in in solid tumor models, revealing delivery modality-specific differences in editing efficiency, toxicity, and expression dynamics. - By systematically varying donor architectures, we identify a compact HDR template - combining a shortened custom EF1 promoter, the minimal Q8 surface reporter, and a synthetic polyadenylation signal (sPA) - that markedly improves knock-in efficiency in solid tumor cell lines, outperforming conventional cassettes. - Virus-free edited tumor cells generated using this workflow retain stable transgene expression and functional fitness in 3D bioprinted tumor constructs and xenograft mouse models, directly linking in vitro knock-in optimization to in vivo relevance. - The resulting biosafety level 1 compatible, end-to-end pipeline - integrating donor design, digital PCR-based quantification of precise integration, and enrichment strategies-offers a practical and transferable platform for engineering transgenic solid tumor models without viral vectors.

Successful AAV-expressed antibody therapy for HIV-1 requires broadly neutralizing antibody (bNAbs) concentrations and reduced immune responses to sustain viral suppression without ART. We have previously demonstrated that co-delivery of AAV-expressed PD-L1 reduces immune responses against HIV-1 bNAbs in rhesus macaques. Here we systematically evaluated six AAV9 transgene cassettes encoding 10-1074 with different promoter/intron combinations (CMV, CMV/R, CBA, CASI, CB7, EF1) across in vitro systems, immune-deficient mice, and in rhesus macaques. We show that both promoter and species selection, leads to differences in 10-1074 concentrations with the CB7 promoter leading to greatest expression in mice and CMV/R promoter in macaques. In addition to differences observed, loss of 10-1074 serum concentrations in macaques resulted in higher anti-drug antibody responses and antigen specific IFN-y T cell responses were focused on the 10-1074 heavy-chain variable region. Furthermore, inclusion of the WPRE greatly impacted 10-1074 expression leading to higher concentrations in both mice and nonhuman primates. Lastly, circulating 10-1074 in macaques retained neutralizing activity against diverse HIV-1 pseudovirus isolates. Together these results demonstrate how expression elements influence AAV-expressed antibodies in the context of co-delivery and highlight the need for further improvements to AAV transgene cassettes when co-delivered with AAV expressed PD-L1.

Capsid engineering has greatly improved the performance of recombinant AAV vectors used for gene therapy. One commonly used strategy is the insertion of a short, 7-mer, peptide into surface-exposed loops to modify receptor interactions and enhance cell entry. While effective in receptor retargeting and improved transduction, these insertions might destabilize the capsid protein, hinder assembly, and thus limit production. While previous attempts have used deep mutational scanning and AI to predict which insertions are viable, there is lack in understanding the structural consequences of these peptide insertions at the amino-acid level. Here we combined experiments, deep sequencing and large protein language models to gain insight on the impact of 7-mer insertions on the VR-VIII region. We first characterize the biochemical properties of viable insertions, thus identifying which residues are well tolerated, and which should instead be avoided. We then focus on the nearby context of those insertions, by studying the effect of the linkers, either for highly diverse libraries or for individual variants known for their efficiency. Next, we study the broader context, by extending our analysis to the whole capsid sequence, and identifying regions that can tolerate insertions without long-ranged structural deformations that could affect capsid functionality. We conclude with a cross-serotype comparison and a viability analysis of tens of previously engineered variants. Our work showcases how AI can uncover structure-function rules governing the success of engineered AAV capsids.

Recombinant adeno-associated viruses (rAAVs) have gained increasing importance in gene therapy due to their safe and precise gene delivery. However, certain indications require substantially higher vector doses, pushing manufacturing capacity and cost of goods (COG) to its limits. In this study, we present for the first time a continuous twin-column capture process (CaptureSMB) enabling direct purification of rAAV5 from unprocessed perfusion harvest without prior concentration or processing. This approach differs fundamentally from conventional batch workflows which typically mandate clarification and concentration before affinity capture and offers a novel process integration in viral vector manufacturing. A single-column batch capture process was developed first and subsequently compared to continuous CaptureSMB configurations. Optimized CaptureSMB operation achieved consistent yields over four cycles, with recoveries exceeding batch operation (+ 14.3%) with concomitant higher productivity (+ 11.4%) and reduced buffer consumption (- 79.2%). Critical quality attribute analysis showed lower host cell protein levels and lower residual DNA in early CaptureSMB cycles, while full capsid ratios, thermal stability and transduction efficiency of rAAV5 particles remained unaltered across cycles and process modes. These findings highlight that continuous twin-column CaptureSMB directly from perfusion harvest can not only improve yield and manufacturing efficiency but also maintain and in some respects enhance product quality. This novel strategy provides a promising route to address manufacturing capacity and cost challenges in rAAV gene therapy production.

Chimeric antigen receptor (CAR)-T cell therapies have transformed the treatment of various tumor types by redirecting and activating T cells against tumor cells. However, CAR-T cell manufacturing approaches remain challenging and limit their widespread use in clinical settings. In vivo CAR-T therapy bypasses ex vivo cell manufacturing and patient preconditioning limitations; however, it faces a significant safety concern as CAR proteins on viral packaging cells are incorporated into budding virions, leading to off-target transduction of tumor cells. Here, we address this risk by developing the CAR-Less ER-Anchor Vector (CLEAN-V) system. By exploiting endoplasmic reticulum (ER) retention, CLEAN-V prevents the CAR protein from trafficking to the cell surface during viral packaging, thereby blocking its incorporation into the viral envelope. CLEAN-V particles exhibit near-complete loss of CAR-mediated tumor cell transduction. Furthermore, CLEAN-V integrates seamlessly into existing third-generation LVV workflows in four- or five-plasmid formats and generates CAR-T cells with preserved phenotypic and functional integrity. These results establish CLEAN-V as a robust platform for developing safe, targeted lentiviral vectors for in vivo CAR-T therapy.

The clinical translation of CRISPR gene editing is challenged by the lack of delivery systems that are both safe and efficient in therapeutically relevant cell types such as mesenchymal stem cells (MSCs). Non-viral delivery avoids the immunogenicity and genomic integration risks of viral vectors but faces fundamental trade-offs between editing efficiency and cytotoxicity. Here, we present a comprehensive multiomic analysis of four non-viral CRISPR delivery modalities including cell-penetrating peptide- (CPP), lipid-, and polymer-based nanoparticles and electroporation; across mRNA and ribonucleoprotein (RNP) molecular formats. We systematically evaluate each modality, demonstrating that lipid-based delivery achieved the highest editing rates at the cost of genomic instability risks, interferon pathway activation, and a pro-inflammatory shift in MSC paracrine activity. Alternatively, CPPs yield moderate editing rates while reducing these unintended side-effects, whereas polymers and electroporation consistently yielded the lowest efficiencies. CRISPR molecular format and delivery method interacted in a stress-dependent manner, with RNP delivery reducing editing rates under high-stress systems while improving them in lower-stress modalities such as CPP and electroporation. These findings establish that editing efficiency alone is an insufficient metric for delivery system selection, and that genomic stability, transcriptomic dysregulation, and inflammatory response must be treated as primary design criteria for CRISPR therapies.

Protein truncating variants caused by UGA stop codons are the most prevalent class of rare variant mutations in neurodevelopmental diseases. Suppressor transfer RNA (sup-tRNA) have therapeutic potential for premature termination codon (PTC) repair, but have thus far underperformed by traditional AAV delivery platforms and progress has been hampered by the lack of methods to non-invasively assess in vivo activity in mammalian brain. To fill this material gap, we utilize transcranial in vivo bioluminescence imaging data from a luciferase-UGA mouse model to enable payload optimization. These data demonstrate that U6 promotor and AAV2/9 capsids have the lowest in vivo activity, whereas self-complementary AAV2/9 with the tRNA in a minimal 100bp genomic context provide broad and efficacious PTC rescue. Further, payload tRNA multiplexing and use of tRNA introns enable efficacy of low viral titers and sustained rescue. tRNA sequencing of scAAV delivered ArgUGA sup-tRNA in brain demonstrate no effects on endogenous tRNA levels, their acylation or processing, and these features are also maintained in scAAV delivered ArgUGA sup-tRNA. Collectively, this work defines a scalable strategy for precision UGA stop codon suppression, supporting development of durable genetic rescue therapies for neurodevelopmental disorders in the mammalian brain. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/724978v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1a48274org.highwire.dtl.DTLVardef@170b999org.highwire.dtl.DTLVardef@1a8fdfcorg.highwire.dtl.DTLVardef@1bacb04_HPS_FORMAT_FIGEXP M_FIG C_FIG

Research on targeted genetic therapies for myocardial diseases, such as cardiomyopathies, currently focuses on (r)AAVs as the delivery method. Despite substantial efforts and advances in animal trials, predicting biodistribution and transduction efficacy in human tissue remains challenging due to interspecies differences in tissue tropism and the difficulty of accurately assessing alternative delivery routes and vector differences. The application in humans has also proven challenging, in part due to severe adverse events associated with systemic administration of (r)AAVs. This necessitates implementing alternative trial designs and stringent evaluation methods that minimize harm or risk to patients. Applying a predesigned vector carrying a gene-editing tool to a normothermic machine-perfused living beating heart in an ex vivo setting could overcome conventional obstacles and limitations. This can serve as a basis for safe and effective gene-therapy testing and assist in evaluating effects at the molecular level. Boxed-Breathing-Heart is a translational trial assessing the feasibility of ex vivo gene editing and gene translation in normothermic machine-perfused human hearts. Human hearts explanted from cardiomyopathy patients undergoing heart transplantation are donated for research and immediately placed in an Organ Care System, where they are surgically connected. The viability of the heart is maintained through normothermic perfusion of system solutions and donor blood. A predesigned AAV containing a CRISPR-Cas system is infused into the circulation and dispersed throughout the tissue via coronary perfusion. The changes at the cellular and molecular levels are assessed continuously via frequent sequential myocardial biopsies. Furthermore, after the pre-planned 72-hour perfusion, the heart is sectioned and analyzed using spatial and single-cell omics. The aim is to provide a proof-of-concept for genetic therapeutic options delivered to the human heart via AAV in an ex vivo perfusion setup. In summary, Boxed-Breathing-Heart provides an ex vivo translational platform for evaluating targeted cardiac gene therapies, enabling molecular analysis directly in human hearts and accelerating clinical translation without posing risks to patients.

BackgroundBiallelic loss-of-function mutations in PRKN gene (encoding Parkin protein) cause early-onset Parkinsons disease (EOPD). Parkin is a crucial component of PINK1-Parkin pathway, which marks damaged mitochondria for degradation via mitophagy. Without functional Parkin, damaged mitochondria accumulate, causing oxidative stress and neurodegeneration. ObjectiveInvestigate Parkin gene replacement via AAV gene therapy as a potential treatment for Parkin-dependent EOPD. MethodsWe initially validated phosphorylated ubiquitin Ser65 (pUbSer65) as an indicator of Parkin-mediated mitophagy initiation. We evaluated AAV-mediated PRKN replacement (hereafter, AAV-Parkin) in a Parkin knockout neuroblastoma cell line (SH-SY5Y cells) and feasibility of delivery in mouse and rat models. ResultsOur research showed pUbSer65 signal was reduced in Parkin-KO SH-SY5Y cells when compared to wild-type cells after mitochondrial stress, indicating deficiency in initiation of mitophagy. AAV-mediated human PRKN gene replacement successfully restored these pUbSer65 levels in knockout cells. We saw restoration in patient-derived fibroblasts following AAV-Parkin overexpression. We developed a translatable gene therapy approach using rodents. We demonstrated the feasibility of delivering AAV-Parkin directly into the substantia nigra (SN) of wild-type rats. Using an AAV1 capsid with Ef1a promoter, we achieved dose-dependent Parkin expression and identified a well-tolerated dose. We also evaluated multiple promoters in a proprietary Spark100 capsid, finding Ef1a and Synapsin1 (Syn1) were most effective for transducing dopaminergic neurons in the SN of mice without causing adverse effects. These findings established a well-tolerated vector dose and an optimal capsid-promoter combination. ConclusionsOur results support the potential of AAV-Parkin gene therapy as a disease-modifying approach for Parkin-deficient EOPD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/737487v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@16dd13corg.highwire.dtl.DTLVardef@c3dfcdorg.highwire.dtl.DTLVardef@19a310dorg.highwire.dtl.DTLVardef@a66f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Adeno-Associated Viruses (AAVs) are powerful platforms for delivering therapeutic transgenes via recombinant AAV (rAAV) vectors. However, a limited understanding of the regulation of AAV gene expression has narrowed the ability to efficiently express therapeutic transgenes from rAAV vectors. Since rAAVs retain only the wtAAV inverted terminal repeats (ITR), we hypothesized that regulatory elements outside the ITR that govern wild-type AAV (wtAAV) gene expression can be used to modify rAAV genomes to enhance vector performance. Through in silico analysis, biochemical pulldowns, and high-throughput sequencing, we have identified that the host architectural protein CCCTC-binding Factor (CTCF) associates with the wtAAV type 2 (wtAAV2) genome but is absent from rAAV vectors. Global knockdown and site-specific deletion revealed that the CTCF binding element (CBE) on the wtAAV2 genome, located upstream of the viral P5 promoter, regulates expression of the viral Rep68/78 genes. We have re-engineered new rAAV vectors expressing a GFP reporter transgene to contain the wtAAV2-CBE upstream of the vector promoter. Our results show that CTCF binding dramatically increased rAAV transduction efficiency and GFP expression by up to four-fold across multiple cell types. This enhancement was independent of the AAV capsid serotype used for packaging rAAV vectors. CUT&RUN analysis revealed that this CBE was necessary and sufficient to regulate the chromatin landscape of wtAAV2 and rAAV2. Finally, we observed that CTCF-mediated chromatin remodeling of rAAV2 led to increased production of nascent RNA transcripts from the vector genome. Based on our findings, we propose that CTCF supports wtAAV2/rAAV gene expression by shaping the local chromatin landscape. SIMPLE ABSTRACTRecombinant Adeno-Associated Viruses (rAAV) gene therapy vectors have been engineered from wild-type AAV (wtAAV) by inserting the viral telomeres (that serve as replication and packaging signals) on either side of therapeutic transgenes. However, efficient expression of transgenes using current rAAV technologies require high doses, which can lead to sporadic toxic side effects. We hypothesized that uncharacterized regulatory elements in the wtAAV2 genome drive efficient viral gene expression and are absent from the current generation of rAAV vectors. Using in-silico analysis combined with biochemical pulldowns, high-throughput sequencing, and mutant viral systems, we have identified a novel cis-acting element bound by the cellular architectural protein CCCTC-binding Factor (CTCF). This CTCF binding element is necessary for wtAAV2 gene expression and is sufficient to enhance the rAAV vectors ability to express reporter transgenes. This CTCF-binding element regulates the chromatin landscape of the virus and its vectors. Our discovery that adding the 19-bp AAV2 CTCF-binding element enhances transgene expression without affecting vector production efficiency presents a promising new rAAV gene therapy platform that is likely to reduce clinical doses and minimize toxicity in therapeutic applications.

Key PointsO_ST_ABSQuestionC_ST_ABSDoes the circadian timing of stem cell infusion influence the risk of aGVHD after allo-PBSCT? FindingsIn this randomized prospective clinical trial that included 198 patients, infusion stem cell at 12:00 pm at noon was associated with a significantly lower incidence and less severity of aGVHD compared with infusion at 6:00 pm, without influencing engraftment or relapse. MeaningScheduling stem cell infusion at an earlier time-of-day may reduce aGVHD risk after allo-PBSCT. IMPORTANCEAcute graft-versus-host disease (aGVHD) remains a major complication following allogeneic peripheral blood stem cell transplantation (allo-PBSCT), compromising patient survival and quality of life. OBJECTIVETo evaluate the effect of stem cell infusion time-of-day on aGVHD after allo-PBSCT. DESIGNA multicenter, randomized, open-label, phase 3 clinical trial was conducted from March 18, 2024, through June 11, 2025, with follow-up through December 31, 2025 (median, 462 days among survivors). SETTINGSix transplantation centers in China. PARTICIPANTSPatients aged 12 to 60 years with malignant hematologic diseases undergoing first allo-PBSCT were screened; 198 eligible patients were randomized. INTERVENTIONSPatients were randomly assigned in a 1:1 ratio to receive stem cell infusion at either 12:00 pm at noon ({+/-} 0.5 hour) or 6:00 pm ({+/-} 0.5 hour). MAIN OUTCOMES AND MEASURESThe primary end point was the cumulative incidence of grade II-IV aGVHD within 100 days after transplantation. Secondary end points included grade III-IV aGVHD, hematopoietic recovery, transplant-related mortality (TRM), relapse, and survival outcomes. RESULTSAmong 198 randomized patients (median age, 38 years; 119 [60.1%] male), grade II-IV aGVHD within 100 days occurred in 11 of 99 patients (11.1%) in the 12:00 pm group and 22 of 99 patients (23.2%) in the 6:00 pm group. The cumulative incidences of grade II-IV and III-IV aGVHD were significantly lower in the 12:00 pm group (II-IV: 11.1% [95% CI, 5.9%-18.2%] vs 23.2% [95% CI, 15.4%-32.0%], P = 0.029, hazard ratio, 2.18 [95% CI, 1.06-4.48]; III-IV: 2.0% [95% CI, 0.4%-6.5%] vs 12.2% [95% CI, 6.7%-19.5%], P = 0.006, hazard ratio, 6.25 [95% CI, 1.39-28.15]). There were no significant differences in hematopoietic recovery, TRM, or relapse between groups. The estimated probability of GVHD-free, relapse-free survival (GRFS) at 360 days favored the 12:00 pm group (66.7% [95% CI, 56.2%-75.2%] vs 56.5% [95% CI, 46.1%-65.5%]). CONCLUSIONS AND RELEVANCEStem cell infusion at 12:00 pm was associated with a lower incidence and severity of aGVHD compared with infusion at 6:00 pm, without influencing engraftment or relapse. Optimization of infusion timing may represent a simple strategy to reduce aGVHD risk. TRIAL REGISTRATIONClinicalTrials.gov Identifier: NCT06294678.

PurposeOculocutaneous albinism type 1 (OCA1) is an inherited disorder caused by tyrosinase (TYR) gene mutations. Affected individuals experience visual impairment and severe photosensitivity from ocular hypomelanosis, with no current treatments. We evaluated the safety and efficacy of a TYR-encoding adeno-associated virus (AAV) vector in healthy rhesus macaques as a potential OCA1 treatment. MethodsA novel AAV2-based capsid (ATX002) was packaged with the human VMD2 promoter and TYR (hTYR) fused with mGreenLantern (mGL). Two adult rhesus macaques were injected with ATX002-hVMD2-hTYR-mGL subretinally (OD) and intravitreally (OS). Safety and efficacy were assessed via comprehensive ophthalmic examination, fundus photography, spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinography at baseline and defined timepoints up to 12 weeks post-injection, followed by post-mortem immunohistochemistry (IHC). ResultsBoth subretinal doses induced localized hypermelanosis by 3 weeks post-injection, which persisted through the study endpoint and was accompanied by measurable thickening of the retinal pigment epithelium (RPE) on SD-OCT. Histological IHC confirmed successful RPE transduction via robust mGL fluorescence, corroborating in vivo findings by revealing localized RPE hyperplasia and transgene-expressing cells adjacent to regions of de novo hypermelanosis. Intravitreal delivery did not induce any changes to the RPE. Transient uveitis was observed but successfully managed with anti-inflammatory treatment. ConclusionsSubretinal AAV-TYR delivery is a safe and effective approach with the potential to induce RPE pigmentation. These findings support the use of AAV-TYR gene therapy for OCA1, demonstrating efficacy and a manageable safety profile in a large-animal model, and provide a critical bridge toward human clinical translation.

The success of Chimeric Antigen Receptor (CAR) T cell therapy is heavily dependent on the quality of the final cellular product. Current expansion protocols often rely on reagents that require removal from cell culture media, posing logistical challenges in manufacturing, and can also lead to terminal differentiation. Here, we evaluate the use of a soluble, bead-free T cell activator, T cell expansion protein (T-CEP), as a streamlined alternative for generating potent CAR-T cells. Human T cells were activated with T-CEP or known T cell activators (Dynabeads and TransAct) and transduced with either CD19 or interleukin-13 (IL-13) mutein (tetravariant-13; TV-13)-based CAR lentiviral vectors. Our results demonstrate that T-CEP supports robust CAR-T cell expansion and achieves transduction efficiencies comparable to commercial reagents for both types of CAR-T cells. Notably, T-CEP significantly favored the expansion of CD8+ T cells, yielding an enhanced CD27+ phenotype and a lower CD4:CD8 ratio compared to TransAct. Cytotoxicity assays confirmed that T-CEP-expanded CAR-T cells possess cytolytic function equivalent to commercial reagents for both CARs, while exhibiting lower levels of inflammatory cytokine secretion. In summary, T-CEP represents a competitive alternative to existing expansion agents, as it does not require its removal during CAR-T manufacturing and generates a CD8+ dominant, less-differentiated phenotype without compromising efficacy.

To facilitate the translation of NF1 exon 17 skipping as a mutation-specific therapy for Neurofibromatosis type 1 into in vivo testing, we have continued to develop more efficient antisense oligonucleotides (ASOs), humanized mouse models, and explored multiple delivery platforms including an adeno-associated virus (AAV)-U7-SnRNA vector approach. We evaluated both biodistribution and exon skipping efficacy of a U7-SnRNA targeting NF1 exon 17 with an SFFV-driven cassette containing T2A-linked Luciferase (Luc) and eGFP packaged in AAV-9, AAV-F and AAV-B1 capsids. We show that AAV-F is superior to AAV-9 and AAV-B1 for mouse brain delivery based on DNA transduction, GFP expression, and luciferase activity, but AAV-B1 delivers 2-4 fold more to sciatic nerve (SCN). In terms of exon skipping, AAV-F appears to induce the most skipping in liver and optic nerve (ON), while AAV-B1 mediates highest skipping in the liver, SCN, and ON. The identification of AAV serotypes that allow efficient transduction and delivery of transgenes to the mouse CNS and PNS is impactful for preclinical research in murine models of other diseases. Furthermore, this is both the first report of NF1 exon skipping efficacy in vivo and the first successful application of an U7-SnRNA for the restoration of functional neurofibromin for NF1.