The exponential expansion of metagenomic data obtained through high-throughput sequencing technologies has surpassed the petabyte-scale threshold, resulting in an unprecedented abundance of data now enabling the discove...The exponential expansion of metagenomic data obtained through high-throughput sequencing technologies has surpassed the petabyte-scale threshold, resulting in an unprecedented abundance of data now enabling the discovery of previously unknown viral and bacterial species. Here, we demonstrate the power and promise of mining sequencing data to uncover natural adeno-associated virus (AAV) cap(sid) genes, with the synergistic aims to expand our repertoire of templates for vector development and to enhance our understanding of the AAV space and of virus evolution across species. Specifically, we harnessed the Serratus Explorer to identify 29 AAV variant genomes from publicly accessible raw metagenomic data generated from birds, nonhuman primates, or human samples, of which 16 were classified as high-quality based on the high coverage of their region. To this end, we devised a comprehensive computational pipeline comprising (i) reference candidate selection, (ii) prealigned data acquisition, (iii) variant calling and frequency estimation, (iv) consensus calling, (v) variant resolution, (vi) phylogenetic analysis, and (vii) protein structure analysis steps. Eight representative genes from four different host organism species were synthesized and used to produce so-called metAAV vectors, which exhibited intriguing and biomedically relevant properties including partial escape from neutralizing anti-AAV antibodies and muscle tropism combined with robust liver detargeting in systemically injected mice. We concurrently pursued a conventional, reference-independent metagenome-based genome assembly, which also successfully reconstructed AAV genes but solely for abundant variants. Together with the fact that this traditional reference-independent method necessitates substantial computational resources and misses to accurately resolve multiple closely related variants, this highlights the assets and superiority of our original consensus-based reconstruction pipeline for fundamental virus research and for future gene therapy vector bioengineering.
Immune checkpoint inhibitors (ICI), such as antibodies against Programmed Death 1 (PD-1), are widely used to treat different types of cancer. Unfortunately, only a small subset of patients benefits from such treatments,...Immune checkpoint inhibitors (ICI), such as antibodies against Programmed Death 1 (PD-1), are widely used to treat different types of cancer. Unfortunately, only a small subset of patients benefits from such treatments, and the systemic administration of ICIs can cause severe immune-related adverse events. To overcome these hurdles, we performed an proof-of-concept investigation of recombinant adenoviral vectors encoding different variants of secreted PD-1 (sPD-1), differing in size, cysteine mutations, oligomerization, and effector functions. First, eight sPD-1 variants were screened, followed by the generation of recombinant adenoviruses with four of the highest-performing sPD-1 constructs based on qualitative analysis of sPD-1 secretion and ligand binding. The secretion of sPD-1 was analyzed using immunoblotting, while binding to PD-L1 and PD-L2 ligands was assessed using a pull-down assay and ELISA. In addition, the inhibition of PD-1:PD-L1 interaction was studied using cell-based signaling bioassays. It was demonstrated that the Fc-fusion sPD-1 variants resulted in the highest sPD-1 yields and were the most efficient in ligand binding. In particular, the single-chain Fc-fusion sPD-1 constructs were the most potent variants, as they effectively blocked PD-1-mediated signaling in T cells in two different coculture assays. The results of this proof-of-concept study indicate that stable and well-secreted sPD-1 constructs have significant potential for site-specific immune gene therapy.
Systemic delivery of adeno-associated viral (AAV) vectors is a promising approach for brain gene therapy, particularly in combination with emerging techniques such as focused ultrasound (FUS), which can transiently and n...Systemic delivery of adeno-associated viral (AAV) vectors is a promising approach for brain gene therapy, particularly in combination with emerging techniques such as focused ultrasound (FUS), which can transiently and noninvasively open the blood-brain barrier to facilitate delivery of AAVs to the brain. However, off-target vector accumulation, particularly in the liver, remains a significant safety concern. To address this, we introduced a mutation into variable region 1 of the AAV9 capsid (G266A) and assessed its effect on liver and brain transduction, testing both direct intraparenchymal injection and intravenous injection with FUS. Interestingly, we found that G266A mutation strongly decreased liver transduction while having only a modest effect on transduction of the brain and other tissues. We also found that the G266A mutation had minimal impact on cell attachment and uptake but strongly decreased binding of the capsid to both human and mouse AAV receptor (AAVR), which likely explains the decrease in liver transduction. Overall, our findings suggest that the G266A mutation and modification of AAVR binding could be a useful strategy to mitigate liver toxicity associated with systemic brain gene therapy.
Recombinant adeno-associated viruses (rAAVs) are useful vectors for clinical gene therapy. It is crucial to examine DNA impurities, such as plasmid DNA, for quality control of rAAV products. In this study, we examined hi...Recombinant adeno-associated viruses (rAAVs) are useful vectors for clinical gene therapy. It is crucial to examine DNA impurities, such as plasmid DNA, for quality control of rAAV products. In this study, we examined highly purified rAAV1, rAAV2, rAAV5, and rAAV6 samples produced on a three-plasmid platform, using a high-throughput sequencer. These samples contained 0.49-3.80% detectable DNA impurities derived from the three plasmids, as estimated by the ampicillin resistance gene (). The plasmid impurities consisted of 90.62-95.84% pAAV, 3.21-6.83% pRC, and 0.95-2.55% pHelper DNA. These trends were consistent with those of DNA impurities determined by droplet digital PCR (ddPCR), indicating that cleaved pAAV backbone DNA was the primary source of DNA impurities encapsulated into the capsids. To examine the preferential encapsulation of pAAV backbone DNA into capsids, short sequencing reads were mapped to the entire pAAV backbone sequence, and we found that the reads were relatively evenly distributed across the backbone sequence, with occasional sharp drops. Furthermore, the read length distribution containing the pAAV backbone sequence showed a main peak at 3.2 kb in Oxford Nanopore Technologies sequencing. This length was consistent with that of backbone DNA nicked at two terminal resolution sites by Rep78/68. Analysis of the terminal sequences of the long reads containing backbone sequences revealed that 92.7-98.4% of them contained Rep-binding elements. These results indicate that the pAAV backbone in linear DNA form was cleaved from pAAV-ZsGreen1 by Rep78/68 nicking at in the nucleus, and that the single-stranded DNA was efficiently translocated into the capsids through the encapsulating machinery, similar to the rAAV genome.
Polyethylenimine (PEI) is widely employed as a transfection reagent in recombinant adeno-associated virus (rAAV) manufacturing, but it must be removed from the final product due to its potential toxicity. Accurate quanti...Polyethylenimine (PEI) is widely employed as a transfection reagent in recombinant adeno-associated virus (rAAV) manufacturing, but it must be removed from the final product due to its potential toxicity. Accurate quantification of PEI in complex biological matrices such as rAAVs is challenging, largely because the strong electrostatic attraction between PEI and nucleic acids can hinder the accuracy of its quantification. Here, we report a robust high-performance liquid chromatography method with charged aerosol detection for the quantification of residual linear PEI in purified AAV samples. Sample preparation includes treatment with trifluoroacetic acid and hydrochloric acid at 60°C to denature capsid protein, disrupt PEI-DNA polyplexes, and hydrolyze nucleic acids. The method achieves a limit of detection of 5 µg/mL and a limit of quantitation of 10 µg/mL in spike-and-recovery studies, with quantification confirmed via visual peak identification. This approach enables sensitive, specific, and reproducible PEI measurement and provides a valuable tool for process monitoring and quality control in gene therapy manufacturing.
Piotrowski-Daspit AS, Lynn AY, Eaton DA
… +10 more, Bracaglia LG, Markey AI, Mortlock RD, Stitelman DH, Martinez ML, Lee CC, Kliman HJ, Glazer PM, Tarantal AF, Saltzman WM
Hum Gene Ther
· 2026 Mar · PMID 41645533
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Preliminary investigations focused on biodistribution of polymeric nanoparticles (NPs) shortly after ultrasound-guided delivery via the portal vein in early second-trimester fetal rhesus monkeys. Results demonstrated tha...Preliminary investigations focused on biodistribution of polymeric nanoparticles (NPs) shortly after ultrasound-guided delivery via the portal vein in early second-trimester fetal rhesus monkeys. Results demonstrated that poly(lactic-co-glycolic acid) (PLGA) NPs ( = 3; 3 mg administered at 75-80 days gestational age) and poly(amine-co-ester)-polyethylene glycol (PACE-PEG) NPs ( = 3; 3 mg at 75-80 days gestational age) distributed to fetal tissues when assessed 24 h post-administration. No adverse findings were observed. PLGA NPs were found primarily in the fetal liver and spleen, whereas PACE-PEG NPs showed more widespread biodistribution to a range of anatomical sites. In another fetal subset with PACE-PEG NPs ( = 2; 90 days gestational age) assessed within 48-h post-administration, results demonstrated enhanced green fluorescent protein reporter mRNA expression in select tissues. These early-stage short-term studies suggest that polymeric NPs, particularly those composed of PACE-PEG, can be safely administered and are potential candidates for fetal delivery of therapeutic nucleic acids. While preliminary, these studies provide evidence to support further investigations in this species to address long-term safety and efficiency.
Recently, our research group generated induced tissue-specific stem/progenitor (iTS/iTP) cells. Compared with induced pluripotent stem (iPS) cells, iTS/iTP cells offer several advantages, that is, easy generation, higher...Recently, our research group generated induced tissue-specific stem/progenitor (iTS/iTP) cells. Compared with induced pluripotent stem (iPS) cells, iTS/iTP cells offer several advantages, that is, easy generation, higher differentiation efficiency, and no teratoma formation. In this study, iTS cells were generated from mouse pancreatic tissues (iTS-P cells) using two different methods. Plasmid vectors were used for expressing , , , (), or () to evaluate the efficiency and differentiation potential of the resulting cells. No significant difference in reprogramming efficiency between the - and -based methods was observed. Among the established clones, iTS-P OSKM2 and iTS-P YAP9 cells, which demonstrated high efficiency in differentiating to insulin-producing cells (IPCs), were selected for further comparison. Both iTS-P OSKM2 and iTS-P YAP9 cells expressed genetic markers of endoderm and pancreatic progenitors and differentiated into IPCs more efficiently than the embryonic stem (ES) cells. Genomic bisulfite sequencing revealed that the pluripotency factors and were partially methylated in both iTS-P OSKM2 and iTS-P YAP9 cells. Unsupervised hierarchical clustering of gene expression profiles showed that iTS-P YAP9 cells clustered more closely with the ES cells than with the iTS-P OSKM2 cells. However, the expression levels of and were significantly lower in both iTS-P OSKM2 and YAP9 cells than in the ES cells. These results conclude that no substantial difference is present in the characteristics between iTS-P cells induced by or , and that their higher differentiation efficiency than the ES cells indicates promising potential for clinical applications.
Hearing impairment, one of the most prevalent sensory disorders, remains a major risk factor for dementia in the aging population. Although interventions such as hearing aids and cochlear implants provide partial benefit...Hearing impairment, one of the most prevalent sensory disorders, remains a major risk factor for dementia in the aging population. Although interventions such as hearing aids and cochlear implants provide partial benefit, they do not address the underlying pathology of sensorineural hearing loss. Inner ear gene therapy has attracted significant attention as a promising approach; however, its clinical translation requires minimally invasive and controllable methods for gene activation. We previously developed a photoactivatable Cre recombinase (PA-Cre) system for spatiotemporal regulation of gene expression. In this study, we evaluated the feasibility of irradiating the external auditory canal (EAC) and tympanic membrane (TM) as minimally invasive approaches for activating cochlear gene expression. Tyrosine-mutant AAV9/3 vectors (AAV.GTX) encoding PA-Cre and a Cre-dependent reporter (sfGFP-to-tdTomato) were injected via the round window membrane in 9-week-old C57BL/6J mice. Seven days later, light irradiation was applied using three approaches: (1) Direct cochlear irradiation via postauricular access, (2) TM irradiation with a fiber-optic probe, and (3) noninvasive EAC irradiation through the intact TM. Recombination efficiency in inner hair cells (IHCs) was quantified using whole-mount immunohistochemistry. AAV.GTX efficiently transduced IHCs and drove robust sfGFP expression. In the absence of light, tdTomato expression remained minimal (<5%), indicating low basal Cre leak activity. Direct cochlear irradiation produced strong recombination (conversion rate: 88.4 ± 1.5%), confirming the functionality of PA-Cre in the mouse inner ear. TM and EAC irradiation yielded high conversion efficiencies (95.8 ± 1.7% and 97.6 ± 1.2%, respectively), comparable to direct irradiation, while preserving cochlear integrity. These findings indicate that PA-Cre functions effectively in the mouse cochlea with minimal leak activity and that TM and EAC irradiation enable robust, minimally invasive gene activation. This strategy highlights the light-mediated, noninvasive modulation of cochlear gene expression, informing future translational development.
The adenovirus region's immune-modulating genes (, , ) are frequently modified in oncolytic adenoviruses (OAds) through deletion and transgene insertion like granulocyte-macrophage colony-stimulating factor (). However,...The adenovirus region's immune-modulating genes (, , ) are frequently modified in oncolytic adenoviruses (OAds) through deletion and transgene insertion like granulocyte-macrophage colony-stimulating factor (). However, the synergistic effects of dual-gene deletions on antitumor efficacy and transgene capacity remain unexplored. To address this, we constructed three E3-modified OAds including OAd5-delgp19K (del), M20 (del and ), M22-0 (del and ), and their -armed derivatives, systematically evaluating the impact of and deletions on viral replication, tumor cell lysis, immune modulation, and antitumor activity. Key findings revealed that deletion OAd prolonged intracellular viral replication, creating a "viral bomb" effect that delayed cell lysis, evading anti-adenovirus antibodies, sustained GM-CSF expression, and culminating in superior tumor suppression. deletion OAd accelerated viral dissemination but triggered rapid antibody-mediated clearance in immunocompetent hosts, resulting in transient GM-CSF expression and diminished therapeutic persistence. In immunocompetent Syrian hamster Hap-T1 subcutaneous tumor models, deletion OAd demonstrated potent tumor inhibition, durable immune microenvironment remodeling, robust viral replication, and evading anti-adenovirus antibodies. These results underscore the critical role of coordinated deletion in optimizing viral replication, transgene expression, and immune evasion, providing a strategic framework for engineering next-generation OAds.
Although the safety of retroviral vector (RV) gene therapy has been improved over the last years, insertional mutagenesis is still a risk factor, as seen in some of the clinical trials targeting hematopoietic stem cells....Although the safety of retroviral vector (RV) gene therapy has been improved over the last years, insertional mutagenesis is still a risk factor, as seen in some of the clinical trials targeting hematopoietic stem cells. This highlights the necessity of appropriate preclinical genotoxicity assays. Our group previously developed the Immortalization Assay (IVIM) and Surrogate Assay for Genotoxicity Assessment (SAGA) to evaluate the risk of side effects by integrating vectors. In this study, murine hematopoietic stem and progenitor cells are transduced with RVs, and genotoxicity can be detected by a proliferation advantage under limiting dilution conditions (IVIM) or the activation of genes associated with oncogenesis and stem cell-like properties (SAGA). A limitation of SAGA is the costly microarray technology. In this study, we present the digital droplet-based SAGA-Quantification (SAGA-Q) as a cost-efficient and faster alternative. Murine samples transduced with known mutagenic vector designs consistently showed upregulation of genotoxicity predictor genes. Based on a training set of 140 IVIM samples (including untransduced controls and samples transduced with long terminal repeat-driven γRV, SIN-LV.SF, SIN-LV.EFS, SIN-LV.PGK.RAG2, SIN-LV.MND.RAG1, and SIN-LV.MND.RAG2), we used random forest prediction for reliable and fast identification of genotoxic vector designs. The relevance of the predictor genes for the immortalization process was further highlighted by an elevated expression in immortalized clones. By simplifying SAGA to SAGA-Q, we aim to increase the accessibility of genotoxicity assessment and, thus, support the safer translation of gene therapy products to clinical trials.
Genome and RNA editing modalities have revolutionized precision gene therapy, offering a safer alternative to traditional gene replacement approaches. Alpha-1 antitrypsin deficiency (AATD) is a compelling model for preci...Genome and RNA editing modalities have revolutionized precision gene therapy, offering a safer alternative to traditional gene replacement approaches. Alpha-1 antitrypsin deficiency (AATD) is a compelling model for precision medicine because the disease mechanism is well defined-mutations in a single gene are responsible for both liver and lung pathology. In this review, we summarize the current preclinical and clinical efforts for AATD, with an emphasis on genome and RNA editing strategies.
As impact of adeno-associated virus (AAV) empty capsids on drug product safety and quality remains inconclusive, downstream purification strategy has been focusing on empty capsid removal. Anion-exchange chromatography (...As impact of adeno-associated virus (AAV) empty capsids on drug product safety and quality remains inconclusive, downstream purification strategy has been focusing on empty capsid removal. Anion-exchange chromatography (AEX) has made significant progress in separating empty from full capsids in recent years. Still, achieving baseline resolution between different AAV subpopulations remains challenging due to subtle charge differences. With a certain AAV construct design, this difficulty is compounded when upstream packaging efficiency is low or when empty and full capsids of a particular serotype have similar electrostatic charge profiles. To improve separation and product purity, secondary interaction mechanisms using multimodal (mix-mode) chromatography are often introduced.In this study, we present a case study on developing a polishing chromatography step to remove empty capsids from AAV7 and AAV8 preparations. To create a challenging feed material for the polishing step, we used small gene-of-interest (GOI) and poorly packaged starting materials. We investigated multiple critical process parameters, including buffer matrix, salt concentration, pH, peak fractionation strategies, and column chemistry (strong AEX vs. mix-mode). Mass photometry (MP) and charge detection mass spectrometry (CDMS) were used to characterize capsid populations. Optimized AEX conditions for AAV8 achieved 80% full capsids by MP and 90% GOI-containing capsids by CDMS. For AAV7, the mix-mode column demonstrated improved resolution compared with the standard AEX gradient method. These results demonstrate that mix-mode chromatography provides an alternative polishing option for serotypes where traditional AEX fails to achieve the desired separation.
Ruangsiriluk W, Jacobo SMP, Iskenderian A
… +33 more, Modi M, Chen N, Felice B, Yuan S, Mukherji ST, Rivera Hernandez M, Sunkara S, MacDonald A, Rajarshi G, Palmieri K, Crooker R, Wu R, Dalrymple M, Chheda M, Megahid D, Arce C, Shen M, Dasari B, Zhao X, Yalavarthi M, Rompikuntal P, Ayyadurai S, Fang J, Hozanovic A, Pescatore B, Bassous D, Romashko A, Galbreath E, Maccani A, Steinkellner J, Ehmann DE, Choi VW, Islam R
Hunter syndrome, also known as mucopolysaccharidosis type II, is an X-linked lysosomal storage disease caused by the deficiency of functional iduronate-2-sulfatase (I2S) enzyme, leading to the accumulation of lysosomal g...Hunter syndrome, also known as mucopolysaccharidosis type II, is an X-linked lysosomal storage disease caused by the deficiency of functional iduronate-2-sulfatase (I2S) enzyme, leading to the accumulation of lysosomal glycosaminoglycans (GAGs) affecting multiple organs. Two-thirds of patients have central nervous system (CNS) manifestations. The current standard of care, enzyme replacement therapy (ERT) via weekly intravenous delivery of recombinant human I2S (rhI2S), does not address the neuropathy in the CNS due to its inability to cross the blood-brain barrier (BBB). Next-generation ERTs consisting of systemically administered rhI2S linked to antibodies that target the transferrin receptor (TfR) have shown clinical efficacy in addressing CNS and peripheral manifestations of disease. We demonstrate here that systemic administration of recombinant AAV9 gene therapy vectors encoding human I2S fusion protein with a TfR1-targeted Variable Heavy chain domain of Heavy chain (VHH) nanobody at the N-terminus normalized brain and cerebrospinal fluid GAGs in symptomatic knockout ( KO) mice. This ability to correct toxic substrate accumulation in the CNS was superior to gene therapy vectors expressing I2S with a C-terminal VHH tag or untagged I2S control. The VHH-I2S transgene product demonstrated a broader distribution in the brain parenchyma, coincident with a significant reduction of lysosomal-associated membrane protein 1 immunoreactivity, unlike untagged I2S and I2S-VHH transgene products. These data illuminate strategies to enhance AAV gene therapy vector design and leverage receptor-mediated transcytosis to strategize BBB-penetrating gene therapy for addressing the unmet medical needs of neuronopathic Hunter syndrome.
Liu W, Geard AF, Massaro G
… +17 more, Hughes MP, Aristorena M, Coombe-Tennant O, Xu L, Semenyuk O, Bush R, Te Vruchte D, Priestman D, Laban R, Veleva E, Heslegrave AJ, Zetterberg H, Platt FM, Smith AJ, Mole SE, Ali RR, Rahim AA
CLN5 disease, caused by mutations in the gene, is a form of neuronal ceroid lipofuscinoses (Batten disease). Patients suffer progressive motor dysfunction, vision loss, seizures, and dementia, leading to premature death...CLN5 disease, caused by mutations in the gene, is a form of neuronal ceroid lipofuscinoses (Batten disease). Patients suffer progressive motor dysfunction, vision loss, seizures, and dementia, leading to premature death. Here, we report a preclinical study of AAV9-mediated gene therapy in a mouse model. Single-dose AAV9 carrying human driven by the CAG or human synapsin 1 promoter (hSYN) was administered via intracerebroventricular injection into neonatal and juvenile mice. Treatment efficacy was evaluated by assessment of neurodegeneration, neuroinflammation, locomotor function, and survival. AAV9 expressing driven by the hSYN promoter significantly alleviated neurodegeneration, improved biochemical and glycosphingolipid profiles, neuropathological and locomotor function, and extended lifespan of the mice. However, gene transfer employing the CAG promoter demonstrated limited therapeutic efficacy. Furthermore, delayed intervention in juveniles provided superior therapeutic response compared with early neonatal intervention and normalized lifespan. Finally, blood plasma neurofilament light that is significantly elevated in the mice is restored to normal wildtype levels following treatment. These results indicate that brain-directed adeno-associated virus (AAV) gene therapy could be a promising treatment strategy for CLN5 disease and efficacy might be monitored using a noninvasive blood plasma biomarker.
Common variants of the apolipoprotein E (APOE) gene have a major impact on the risk of developing Alzheimer's disease (AD). Relative to homozygotes with the common E3 allele, the APOE4 variant (C112R) increases risk by 3...Common variants of the apolipoprotein E (APOE) gene have a major impact on the risk of developing Alzheimer's disease (AD). Relative to homozygotes with the common E3 allele, the APOE4 variant (C112R) increases risk by 3.5-fold in E3/E4 heterozygotes and 15-fold in E4 homozygotes. Since the E3 and E4 alleles differ only by a single nucleotide, gene editing of E4 to E3 is a potential strategy to reduce AD risk in E4 homozygotes. Because the APOE pool in the brain is separate from systemic APOE, editing to treat AD would ideally be directed to the brain. Following optimization of prime editing guide RNAs, efficient prime editing expression cassettes were inserted into the adeno-associated virus (AAV) split-intein system and packaged into pairs of AAV vectors for editing. The AAV vectors were administered to human homozygous APOE4-targeted replacement mice (TRE4), and APOE4 to APOE3 editing efficiency was assessed after 4 weeks. The prime editing construct designated APOE3/4-3_10 was the most efficient at APOE4 to APOE3 conversion, both in liver following intravenous delivery and in brain following intrahippocampal delivery. To assess brain-wide editing, two AAV capsids were compared, including AAVrh.10 with administration either directly to the hippocampus or to the cerebrospinal fluid via the cisterna magna and AAV-CAP.B10 administered intravenously. Other than minor differences in APOE4/3-3_10 mediated E4 to E3 editing in the cerebellum, the different capsids and routes yielded similar editing efficacy throughout the brain. This may represent a candidate treatment to reduce the risk of AD.
Epigenome editing technology holds great promise for treating diverse genetic disorders. In this study, we demonstrate epigenetic activation of the gene for the treatment of -CMD, a severe congenital muscle dystrophy (C...Epigenome editing technology holds great promise for treating diverse genetic disorders. In this study, we demonstrate epigenetic activation of the gene for the treatment of -CMD, a severe congenital muscle dystrophy (CMD) caused by biallelic mutations in the gene. is a sister homolog that is known to compensate for the function of . However, supplementing or gene via viral platform is not feasible due to the large size of their coding sequences. Through a single administration of our adeno-associated virus (AAV) vector encoding all the necessary elements for epigenetic activation, we observed significant gene upregulation and phenotype improvements in mouse disease models. The muscle-tropic AAV capsid exhibited desired vector biodistribution and promising pharmacodynamics with good safety profiles in 2-year-old juvenile nonhuman primates (NHPs). Moreover, administration to 8-month-old infant NHPs demonstrated superior pharmacodynamics compared with 2-year-old juveniles, even at half the dose. Our approach holds broad applicability for a range of loss-of-function genetic disorders and could offer a therapeutic breakthrough where active epigenome offers clinical benefit.
Cell and gene therapies present unique challenges for long-term follow-up as they may lead to adverse events that could emerge years after treatment. Long-term follow-up helps identify potential delayed adverse events, s...Cell and gene therapies present unique challenges for long-term follow-up as they may lead to adverse events that could emerge years after treatment. Long-term follow-up helps identify potential delayed adverse events, such as oncogenesis or immunogenicity, which might not manifest immediately after treatment. Current regulatory guidelines emphasize a risk-based approach, recommending follow-up durations based on the therapy's mechanism of action between 5 and 15 years. To facilitate long-term monitoring, regulatory authorities recommend the establishment of long-term follow-up protocols, often involving patient registries and supported by real-world data sources to systematically capture and track data from treated patients. These long-term follow-ups are instrumental in both post-approval safety studies and reimbursement decisions, where payers may link payments to treatment outcomes. As the field of cell and gene therapy evolves, regulatory frameworks continue to adapt, balancing the need for comprehensive long-term follow-up with the feasibility of implementation to ensure that therapies are adequately monitored, ensuring patient safety and therapeutic effectiveness over time. However, maintaining patient engagement over extended periods, ensuring high-quality data collection, and addressing privacy concerns present significant challenges. Innovative solutions such as decentralized data collection, digital health technologies, and data linkage with electronic health records aim to alleviate patient burden and improve data reliability.