Human Gene Therapy[JOURNAL]
Sun
200 papers
Genome Editing for Familial Hemophagocytic Lymphohistiocytosis: Design Principles, Challenges, and Translational Perspectives.
Minaiyan G, Aussel C, Ammann S … +1 more , Cathomen T
Hum Gene Ther · 2026 Jul · PMID 42389905 · Publisher ↗
Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome caused by genetic defects in cytotoxic lymphocyte function. Current therapies can control disease activity, but transplantation of...
Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome caused by genetic defects in cytotoxic lymphocyte function. Current therapies can control disease activity, but transplantation of allogeneic hematopoietic stem and progenitor cells (HSPCs) remains the only curative option and is associated with substantial risks. These limitations have accelerated development of genome editing approaches enabling precise correction of disease-causing mutations in autologous cells. Familial HLH (FHL) represents a compelling target for genome editing, but successful and safe clinical translation has remained challenging. Preclinical studies demonstrate that targeted editing of key genes, such as and , can restore cytotoxic function in HSPCs and T cells. Translation to the clinic, however, depends on multiple factors, including the choice of target cell population, the level of functional correction required, and gene-specific constraints such as locus complexity and regulation of gene expression. In this review, we synthesize current progress in genome editing for FHL and highlight critical biological and technical barriers to clinical implementation. We propose a conceptual framework for designing genome editing strategies tailored to FHL, emphasizing the alignment of editing platform, gene architecture, and cellular context to enable effective and clinically translatable therapies.
Lilly, Ascidian Launch Up-to-$1.9B RNA Exon Editor Collaboration.
Philippidis A
Hum Gene Ther · 2026 Jul · PMID 42388068 · Publisher ↗
Abstract loading — click title to view on PubMed.
Hyperthermia-Enhanced Adeno-Associated Virus Gene Therapy: Mechanisms, Delivery Technologies, and Translational Potential.
Lin NH, Mavropoulos SA, Ishikawa K
Hum Gene Ther · 2026 Jun · PMID 42333600 · Publisher ↗
Adeno-associated virus (AAV) gene therapy is a promising therapeutic approach for both inherited and acquired disorders, yet its effectiveness is often limited by barriers that reduce target cell uptake and transgene exp...
Adeno-associated virus (AAV) gene therapy is a promising therapeutic approach for both inherited and acquired disorders, yet its effectiveness is often limited by barriers that reduce target cell uptake and transgene expression. While body temperature is known to have a significant impact on viral infection, its controlled regulation for AAV gene therapy has been little explored. This article reviews therapeutic hyperthermia, carefully controlled mild heating, as a complementary strategy to improve delivery and expression. Evidence from cell and animal studies suggests that transient mild heating can increase blood flow and vessel permeability, promoting viral entry from the bloodstream into tissues. It can also trigger cellular stress responses that facilitate entry and downstream processing. Heating can also serve as an external switch when combined with heat-responsive genetic control systems, allowing gene expression to be activated only in the heated region. We summarize clinically used methods for delivering local, regional, and whole-body hyperthermia and discuss how spatially controlled heating could be integrated with AAV gene therapy workflows. We also highlight opportunities for clinical translation, key risks, and open questions, including thermal dose control, potential loss of viral particle stability with excessive heating, and the possibility of increased inflammatory or immune side effects.
Adeno-Associated Virus Gene Therapy Translation: Lessons from Early Regulatory Meetings.
Stan R, Lomash RM, Shchelochkov OA … +15 more , Manoli I, Galarreta Aima CI, Mangalampalli V, Choi EY, Chandler RJ, Li L, Sloan JL, Terse P, Xu X, PaVe-GT Team, Dehdashti J, Brooks PJ, Bönnemann CG, Venditti CP, Ottinger EA
Hum Gene Ther · 2026 Jun · PMID 42321968 · Publisher ↗
The Platform Vector-Gene Therapy (PaVe-GT) program is a National Institutes of Health (NIH) initiative that aims to develop adeno-associated virus (AAV) gene therapies for four monogenic rare diseases, two organic acidem...
The Platform Vector-Gene Therapy (PaVe-GT) program is a National Institutes of Health (NIH) initiative that aims to develop adeno-associated virus (AAV) gene therapies for four monogenic rare diseases, two organic acidemias and two congenital myasthenic syndromes. PaVe-GT's platform-based approach identifies and diminishes redundancies and applies efficiencies in preclinical, clinical, and regulatory activities. The program's hypothesis is that implementing these efficiencies can accelerate clinical trial initiation. Based on its platform-centric experience and public-serving mission, the PaVe-GT program actively shares its scientific and regulatory learnings with the public to benefit the development of similar gene therapy products for rare diseases. PaVe-GT's first investigational AAV gene therapy candidate is AAV serotype 9 human propionyl-CoA carboxylase alpha subunit (AAV9-hPCCA) for propionic acidemia caused by PCCA deficiency, which received initial feedback from the Food and Drug Administration (FDA) in an itial argeted ngagement for egulatory dvice on BER/Center for Drug Evaluation and Research (CDER) Producs (INTERACT) meeting. Upon further product development that took into consideration the FDA's initial advice, the program obtained the Agency's feedback in pre-investigational new drug (IND) (Type B) and Type C meetings. Here, we share our experience from these meetings, including strategy, preparation, pre- and post-meeting feedback from the FDA, and lessons learned during the AAV9-hPCCA regulatory process, which the program plans to apply across the PaVe-GT platform. Topics discussed in the regulatory meetings included animal model and efficacy studies, toxicology study plans, manufacturing of the investigational AAV product, and clinical trial design. The main lessons learned from the pre-IND and Type C meetings for AAV9-hPCCA are: (1) Pharmacology/Toxicology studies in a single rodent species are sufficient for filing an initial IND; (2) FDA feedback guides product quality improvements and early development of a quantitative potency assay; (3) use of biomarkers as potential surrogate endpoints in a future efficacy trial benefits from collection of data in the natural history study and the first-in-human Phase 1/2 study; and (4) evidence from the Phase 1/2 clinical trial could be leveraged to support a license application. Lightly redacted regulatory documents and comprehensive templates developed by the PaVe-GT team are available on the PaVe-GT website.
Genome Editing Approach to Disrupt Hydroxyacid Oxidase 1 for the Treatment of Primary Hyperoxaluria Type 1.
Greig JA, Breton C, Smith MK … +4 more , Furmanak T, Bell P, Clark P, Wilson JM
Hum Gene Ther · 2026 Jun · PMID 42299121 · Publisher ↗
Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder that leads to kidney and liver failure. PH1 is caused by a mutation in the alanine glyoxylate aminotransferase () gene, which encodes a key metabo...
Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder that leads to kidney and liver failure. PH1 is caused by a mutation in the alanine glyoxylate aminotransferase () gene, which encodes a key metabolic enzyme that converts glyoxylate to glycine in the liver. Inability to metabolize glyoxylate leads to oxalate overproduction, yielding insoluble calcium oxalate crystals; accumulation of these crystals leads to progressive organ failure. Here, we used a novel, minimally disruptive genome-editing approach to disrupt the mechanism of action of hydroxyacid oxidase 1 (HAO1), an upstream enzyme in the glyoxylate metabolic pathway. Successful gene editing and disruption of the gene is expected to increase levels of glycolate, a harmless intermediate of the glycine metabolic pathway, thereby preventing the formation of calcium oxalate crystals. We intravenously administered an adeno-associated virus (AAV) vector expressing the M1HAO1 meganuclease to both wild-type and mice, a mouse model of PH1. We observed >30% editing of in mice, correlating with a dose-dependent increase in serum glycolate levels. At the highest dose tested, urine glycolate levels increased by 79%, with a concomitant 75% decrease in urine oxalate levels. We also evaluated targeting in rhesus macaques injected with AAV expressing two different versions of the HAO1 meganuclease. Dose-dependent editing of hepatic DNA and RNA was achieved, and serum glycolate levels changed in a manner consistent with successful liver editing; additionally, the treatment was well tolerated. Our results indicate that AAV-delivered meganucleases can effectively target in mice and nonhuman primates to achieve high levels of gene editing. Moreover, increased glycolate levels in serum indicate that this intervention significantly impacts the HAO1-mediated glycolate-to-glyoxylate pathway. These data suggest that this approach may represent an effective treatment for PH1.
"Enhancing AAV9-UFμDys1 Gene Therapy Efficacy Through Immunosuppression in Mice with Pre-Existing Immunity and Enabling Redosing Strategies for Duchenne Muscular Dystrophy".
Hum Gene Ther · 2026 Jun · PMID 42290100 · Publisher ↗
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Resolving the Unresolvable: Nanopore Sequencing as a Comprehensive Quality Control Platform for Gene Therapy Vectors.
Xu Y, Zhang XB, Zhang JP
Hum Gene Ther · 2026 Jun · PMID 42286899 · Publisher ↗
Cell and gene therapy (CGT) manufacturing has outpaced traditional chemistry, manufacturing, and controls frameworks, leaving a "black box" in vector quality control (QC). Legacy assays such as Sanger and short-read next...
Cell and gene therapy (CGT) manufacturing has outpaced traditional chemistry, manufacturing, and controls frameworks, leaving a "black box" in vector quality control (QC). Legacy assays such as Sanger and short-read next-generation sequencing often fail to resolve complex structures including adeno-associated virus (AAV) inverted terminal repeats, lentiviral recombination, and mRNA poly(A) tails. Oxford Nanopore Technologies enables long-read, native single-molecule sequencing to access these attributes directly. This review summarizes nanopore sequencing across the CGT lifecycle. For plasmid DNA, it confirms full-length circular identity and reveals structural heterogeneity missed by restriction mapping. For viral vectors (AAV and lentivirus), it functions as an integrity assay to distinguish full genomes from truncations and to detect sequence-resolved impurities, including reverse-packaged plasmid backbones. For mRNA therapeutics, direct RNA sequencing profiles poly(A) tail length distributions and base modifications ( mΨ) in a single assay. We also discuss adaptive sampling for impurity enrichment and native epigenetic profiling of bacterial methylation. Finally, we assess limitations in accuracy and compliance and outline the regulatory path toward moving long-read sequencing from an orthogonal tool to a validated lot-release method. Overall, nanopore sequencing supports risk-based, high-resolution QC while reducing analytical turnaround time.
AviNP-Seq: A Blindspot-Free Single-Molecule Framework for Unmasking AAV Genome Heterogeneity and Determining Packaging Limits.
Li GH, Xu Y, Liu T … +6 more , Ma XY, Hou MT, Pang SC, Zhang JP, Cheng T, Zhang XB
Hum Gene Ther · 2026 Jun · PMID 42283432 · Publisher ↗
Comprehensive recombinant adeno-associated virus characterization is essential for establishing the knowledge base required to ensure clinical safety and efficacy, yet current long-read methods suffer from library prepar...
Comprehensive recombinant adeno-associated virus characterization is essential for establishing the knowledge base required to ensure clinical safety and efficacy, yet current long-read methods suffer from library preparation biases that obscure genome integrity. We present AviNP-seq, a blindspot-free nanopore sequencing framework utilizing one-end-sufficient ligation and Cas9-ribonucleoprotein (RNP) linearization to minimize terminal selection. Applied to a 1.5-6.5 kb panel, AviNP-seq delineates a sharp packaging cliff at 5.0-5.2 kb and reveals that sequence structure modulates integrity by 2-5× at fixed lengths. It unmasks covalent head-to-tail tandems in sub-3 kb vectors, detecting them with significantly higher sensitivity than PacBio HiFi. The Cas9-RNP step boosts ligation yield ∼7-fold, providing an unbiased assessment of genome integrity (≥95% inverted terminal repeat [ITR]-to-ITR). In addition, the assay quantifies plasmid impurities down to 0.05% with linear response. By integrating integrity mapping, tandem detection, and impurity profiling into a rapid (<36 h), low-input workflow, AviNP-seq provides a robust analytical tool to guide vector design and de-risk early-stage process development.
Inflammation-Inducible Expression of Human Indoleamine 2,3-Dioxygenase Driven by the E-Selectin Promoter Ameliorates Collagen-Induced Arthritis.
Karaçay M, Yılmaz İ, Barazi Soydaş AÖ … +7 more , Güvenç-Bayram G, Ersoy F, Özgüden Akkoç CG, Yılmaztepe Oral A, Akkoç A, Yalçın M, Oral HB
Hum Gene Ther · 2026 Jun · PMID 42237653 · Publisher ↗
Rheumatoid arthritis is a chronic autoimmune disease characterized by persistent synovial inflammation and progressive joint destruction. Although biological disease-modifying anti-rheumatic drugs (DMARDs) have transform...
Rheumatoid arthritis is a chronic autoimmune disease characterized by persistent synovial inflammation and progressive joint destruction. Although biological disease-modifying anti-rheumatic drugs (DMARDs) have transformed treatment, their systemic immunosuppression, high cost, and incomplete efficacy in certain joints remain significant challenges. To address these limitations, we developed a localized gene therapy using the human indoleamine 2,3-dioxygenase () gene driven by the E-selectin (ELAM-1) promoter, which is responsive to inflammatory cytokines. We assessed ELAM-1 promoter responsiveness to interleukin-1β (IL-1β) stimulation mainly using the HeLa cell line, measuring expression and enzymatic activity by enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting and kynurenine assays. , we established a robust collagen-induced arthritis (CIA) model in BALB/c mice, which are normally resistant to CIA, and delivered pELAM-1pro/hIDO plasmids intra-articularly via liposomes. Therapeutic efficacy was evaluated by clinical scoring, ankle circumference, histopathology, immunohistochemical quantification of synovial CD4 T cells and CD68 macrophages, and endpoint immunohistochemical assessment of IDO expression. , the ELAM-1 promoter allowed rapid, high-level expression of hIDO within 2 h of IL-1β stimulation, without IDO-induced suppression of the promoter. , pELAM-1pro/hIDO treatment significantly reduced ankle swelling and clinical arthritis scores more rapidly than constitutive CMV-driven hIDO, achieving marked histological improvement on day 7. The treated joints exhibited substantial reductions in pathogenic CD4 T cells and CD68 macrophages, consistent with the IDO immunoregulatory mechanism. This study supports the feasibility of a localized gene therapy approach using an inflammation-responsive promoter and suggests localized immunomodulation at disease sites in a manner consistent with inflammation-associated promoter activity.
Capsid Engineering of Adeno-Associated Viruses for Targeted Gene Therapy in Kidney Diseases.
Ghaffarzadeh-Esfahani M, Gheisari Y
Hum Gene Ther · 2026 May · PMID 42210494 · Publisher ↗
The global burden of chronic and genetic kidney diseases poses a significant challenge to healthcare systems. Current therapies, including dialysis, transplantation, and supportive pharmacotherapies, cannot halt disease...
The global burden of chronic and genetic kidney diseases poses a significant challenge to healthcare systems. Current therapies, including dialysis, transplantation, and supportive pharmacotherapies, cannot halt disease progression or address root causes, especially in monogenic disorders like Alport syndrome and Fabry disease. Adeno-associated virus (AAV)-based gene therapy is promising, enabling targeted correction of underlying genetic defects. However, renal delivery faces challenges, including cellular heterogeneity, anatomical barriers, and pre-existing immunity. This review evaluates advances in AAV capsid engineering to overcome these obstacles, focusing on strategies to enhance kidney-specific tropism, transduction efficiency, and immune evasion. We outline the evolution from conventional serotype selection to precision engineering via rational design, directed evolution, and in silico approaches. Artificial intelligence (AI) has emerged as a pivotal accelerator, with machine learning models and generative frameworks enabling data-efficient capsid optimization despite limited datasets. Multimodal AI, reinforcement learning, and agentic systems can refine renal targeting by balancing glomerular penetration, cell specificity, and safety. Future progress relies on scaling high-quality datasets through collaborative consortia, lab-in-the-loop validation, and explainable AI. By combining capsid engineering with renal pathophysiology insights, this roadmap paves the way for curative AAV therapies that move beyond current suboptimal treatments to correct underlying pathogenic mechanisms.
Ablation of Cbl-b in ROBO1 CAR-NK92 Cells Enhances Their Antitumor Efficacy.
Hu J, Zhang J, Shao Y … +3 more , Li H, Yang Y, Zhang J
Hum Gene Ther · 2026 May · PMID 42204417 · Publisher ↗
Emerging evidence suggests CAR-NK cell therapy shows great promise in cancer treatment. ROBO1 is highly expressed in various cancer types, including glioblastoma, hepatocellular carcinoma, lung cancer, breast cancer, and...
Emerging evidence suggests CAR-NK cell therapy shows great promise in cancer treatment. ROBO1 is highly expressed in various cancer types, including glioblastoma, hepatocellular carcinoma, lung cancer, breast cancer, and uterine cancer. Our and other laboratories' studies have shown that ROBO1 CAR-NK cells exhibit promising tumor therapeutic effects. However, the results still have some limitations. Cbl-b, an E3 ubiquitin ligase, has been reported to negatively regulate NK cell activation, homeostasis, and antitumor immunity. Therefore, we attempted to further enhance the antitumor activity of ROBO1 CAR-NK92 cells by knocking out using CRISPR/Cas9 gene-editing technology. In this study, we conjugated Cbl-b sgRNA with Cas9 protein to form ribonucleoprotein complexes, which were then delivered into ROBO1 CAR-NK92 and NK-92 cells (control cells) via electroporation. Through fluorescence-activated cell sorting, limiting dilution, and sequencing, we obtained monoclonal Cbl-b-knock-out (KO) cell lines. Both cytotoxicity assays and tumor xenograft experiments were conducted to examine whether Cbl-b knockout enhances the target cell killing and tumor suppression capacities of ROBO1 CAR-NK92 cells. In this study, monoclonal cell lines of ROBO1 CAR-NK92-Cbl-b-KO and NK92-Cbl-b-KO were successfully established. , at an effector-to-target (E:T) ratio of 0.1:1, ROBO1 CAR-NK92-Cbl-b-KO (50.55%) cells exhibited significantly higher cytolytic activity against ROBO1-positive T47D target cells after 3 h of coculture than ROBO1 CAR-NK92 (34.10%), NK92-Cbl-b-KO (22.22%), and parental NK-92 cells (3.28%). , tumor volume and weight measurements demonstrated that mice treated with ROBO1 CAR-NK92-Cbl-b-KO cells developed significantly smaller tumors than all control groups, achieving a tumor growth inhibition (TGI) rate of 32.45%, indicating enhanced antitumor efficacy conferred by Cbl-b knockout. and data confirmed that Cbl-b knockout potentiates the antitumor efficacy of ROBO1 CAR-NK92 cells. The overall cytotoxic capability ranked as follows: ROBO1 CAR-NK92-Cbl-b-KO > ROBO1 CAR-NK92 > NK92-Cbl-b-KO > NK-92.
A Dual-Viral Delivery Platform Enables Efficient Site-Specific Integration of Therapeutic-Length Genes in Human Primary Stem Cells.
Gao ZY, Shen TL, Cheng CY … +4 more , Sun YD, Zhang LM, Zhang JP, Zhang XB
Hum Gene Ther · 2026 May · PMID 42204401 · Publisher ↗
Site-specific integration of large genes in human primary stem cells remains a significant challenge in gene therapy, particularly for treating multiallelic diseases. Gene editing efficiency in primary stem cells is heav...
Site-specific integration of large genes in human primary stem cells remains a significant challenge in gene therapy, particularly for treating multiallelic diseases. Gene editing efficiency in primary stem cells is heavily influenced by the delivery strategy, which often faces issues with programmability, efficiency, and specificity. Here, we developed a dual-viral delivery system, targeted integration via virus-like particles and integrase-deficient lentivirus (TIVID). This system combines virus-like Cas9 edit particles for delivering Cas9/sgRNA ribonucleoprotein complexes and integrase-deficient lentiviral vectors for delivering HDR donor templates. The TIVID system achieves a knock-in efficiency of 65% ± 5% in human induced pluripotent stem cells (iPSCs). In erythroid progenitor HUDEP2 cells, TIVID mediates precise integration of a 7.1 kb HBB-GFP cassette (from cut site to cut site) at the locus with 20% efficiency and stable expression. Crucially, we demonstrate that TIVID overcomes stringent packaging constraints to deliver an approximately 6 kb full-length therapeutic cassette into primary human CD34 hematopoietic stem and progenitor cells. This platform achieved 5-10% targeted integration efficiency and preserved robust lineage-specific differentiation capacity, demonstrating its potential for treating β-thalassemia and other multiallelic disorders. In head-to-head comparisons, TIVID outperformed lentivirus-derived nanoparticles (∼50% vs. <10% at in K562 with M3814) and plasmid-based eePASSIGE in iPSCs (∼20% vs. ∼1.5%). Compared with traditional electroporation delivery, TIVID offers lower early cytotoxicity, promotes predominantly mono-allelic integration, and exhibits enhanced compatibility with primary stem cells. By decoupling nuclease and donor delivery, TIVID circumvents the payload constraints of single-vector systems and the toxicity of physical transfection, providing a robust engineering platform for complex gene replacement therapies.
Regeneron Wins FDA Approval for First Neurosensory Gene Therapy.
Philippidis A
Hum Gene Ther · 2026 Jul · PMID 42178736 · Publisher ↗
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Optimization of Alpha-1 Antitrypsin Expression from Adeno-Associated Virus Vectors.
Tang Q, Pires-Ferreira D, Mueller C … +2 more , Gruntman AM, Flotte TR
Hum Gene Ther · 2026 Jul · PMID 42159147 · Publisher ↗
Alpha-1 antitrypsin deficiency (AATD) is an inherited disorder caused by mutations in that result in insufficient circulating alpha-1 antitrypsin (AAT) and progressive lung and liver diseases. Adeno-associated virus (AA...
Alpha-1 antitrypsin deficiency (AATD) is an inherited disorder caused by mutations in that result in insufficient circulating alpha-1 antitrypsin (AAT) and progressive lung and liver diseases. Adeno-associated virus (AAV)-mediated gene therapy offers the potential for durable AAT expression; however, achieving therapeutic serum concentrations (≥11 µM) at clinically acceptable vector doses remains a major challenge. Here, we evaluated multiple AAV vector design strategies to enhance AAT expression and increase vector potency, thereby reducing the required dose to levels below those associated with severe adverse events. Using AAV1- and AAV8-based platforms, we compared promoter and enhancer configurations, codon optimization of the transgene, single-stranded versus self-complementary vector genomes, alternative polyadenylation signals, and an engineered oxidation-resistant AAT variant. Across mouse and ferret models, the chicken β-actin expression cassette consistently produced higher AAT levels than a liver-specific promoter variant despite comparable vector biodistribution, reflecting superior intrinsic transcriptional activity or an important contribution. Codon optimization did not enhance expression and, in some cases, modestly reduced AAT levels. Self-complementary AAV vectors exhibited reduced overall expression due to required promoter truncation, yielding lower transgene output than full-length single-stranded constructs. Modifications to polyadenylation signals or enhancer combinations did not improve expression. An oxidation-resistant AAT variant resulted in lower circulating levels but may retain therapeutic potential through enhanced functional stability. Collectively, these findings demonstrate that promoter strength and cassette architecture are dominant determinants of AAV-AAT potency and that full-length, single-stranded vectors with robust regulatory elements provide the highest expression. This work defines key parameters governing AAT expression and provides a foundation for next-generation AAV designs aimed at achieving therapeutic efficacy at safer, lower vector doses for the treatment of AATD.
The Application of Cell and Gene-Modified Cell Therapy in the Treatment of Osteopetrosis.
Najdanović JG, Živković JM, Najman SJ
Hum Gene Ther · 2026 Jul · PMID 42131975 · Publisher ↗
Bone tissue represents a dynamic tissue that undergoes continuous renewal and remodeling through the coupled actions of osteoblast-driven bone formation and osteoclast-mediated bone resorption. Proper coupling between th...
Bone tissue represents a dynamic tissue that undergoes continuous renewal and remodeling through the coupled actions of osteoblast-driven bone formation and osteoclast-mediated bone resorption. Proper coupling between these two processes is essential for maintaining bone homeostasis, whereas its disruption leads to skeletal pathology. Excessive osteoclast activity underlies disorders such as osteoporosis and Paget's disease, while osteoclast deficiency, either quantitative or functional, results in osteopetrosis. Osteopetrosis comprises a group of genetically heterogeneous and clinically variable rare metabolic bone diseases characterized by increased bone mass. Clinically, osteopetrosis is commonly classified into severe autosomal recessive osteopetrosis (ARO), also known as infantile malignant osteopetrosis, intermediate ARO, milder autosomal dominant osteopetrosis, and X-linked recessive osteopetrosis. Effective treatment options for both severe and milder forms of osteopetrosis remain an unresolved clinical challenge. This review sheds light on the potential of cell-based and gene-modified cell therapies as emerging strategies for the treatment of osteopetrosis. Current cell-based therapy approaches increasingly focus on induced pluripotent stem cells, while recent advances in gene therapy enable the correction of causative genetic defects through viral vector-mediated gene transfer, CRISPR/Cas9-based genome editing, or RNA interference-mediated gene silencing. In addition, the therapeutic potentials of immunological approaches based on recombinant human interferon gamma-1b is discussed. Despite significant progress, sustained research efforts are still required to translate cell and gene-modified cell therapy into effective and personalized clinical treatments for osteopetrosis.
AAV-Mediated Base Editing for Correction of Mutations in Primary Ciliary Dyskinesia: A Proof-of-Concept Study.
De Carli A, Pastore S, Maj D … +13 more , Filippini F, Matteucci M, Di Cicco ME, Peroni D, Donzelli G, Crucitta S, Zentilin L, Michelucci A, Gabellini C, Freer G, Lai M, Pifferi M, Pistello M
Hum Gene Ther · 2026 Jul · PMID 42050778 · Publisher ↗
Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous disorder, with abnormal ciliary motility, usually due to an ultrastructural defect, with chronic airway infections. Currently, no curative therapy exi...
Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous disorder, with abnormal ciliary motility, usually due to an ultrastructural defect, with chronic airway infections. Currently, no curative therapy exists for PCD. Given the prevalence of single nucleotide variants (SNVs) among causative mutations, we evaluated a novel base-editing approach. Specifically, we used a nickase Cas9 fused to adenosine deaminase to correct mutations in the radial spoke head component 4 A () gene, causing PCD. We selected two PCD patients sharing the same SNV in , one with compound heterozygosity (child, patient 1) and one with homozygosity (adult, patient 2). After designing gRNAs, HEK293T cells with or without a DNA fragment containing the SNV in , were co-transfected with base editor plasmids. Complex formation and editing efficiency were validated by Western blot and digital PCR. We then treated patient cells with AAV containing the base editors and assessed ciliary beat frequency and motion pattern using high-speed video and confocal microscopy to evaluate delivery.Base editor complexes formed efficiently . AAV-mediated delivery in patient 1 cells led to an approximately 30.4% increase in normal motion pattern, with a corresponding reduction in circular motions ( < 0.001) compared with pre-treatment, and a 20% of editing efficiency detected by dPCR in transduced cells. Our data indicate that this limited editing efficiency is due to reduced AAV penetration in the lower layers of cells.This proof-of-concept study demonstrates the therapeutic potential of base editing for PCD, though current limitations include low editing efficiency and restricted delivery to inner cell layers in our experimental model. Future work should focus on optimizing base editors and testing novel delivery strategies to target progenitor cells, thereby enhancing the prospects for personalized gene therapy in PCD.
Strategies and Advances in Site-Specific Integration of Exogenous Large Genes.
Wu Z, Chen J, Huang M … +4 more , Hu W, Liu Y, Shao H, Zhang W
Hum Gene Ther · 2026 Jul · PMID 42026979 · Publisher ↗
Large genomic deletions (≥1 kb) are a recurrent class of disease-causing lesions in monogenic disorders, frequently leading to complete gene inactivation or the loss of critical cis-regulatory elements. Addressing these...
Large genomic deletions (≥1 kb) are a recurrent class of disease-causing lesions in monogenic disorders, frequently leading to complete gene inactivation or the loss of critical cis-regulatory elements. Addressing these defects in a therapeutically relevant manner requires integration modalities capable of delivering and stably installing large exogenous DNA sequences at predefined genomic loci with an improved safety profile. By contrast, legacy approaches-including viral-vector delivery, recombinase-based strategies, and transposon-mediated insertion-typically achieve integration through random or semi-random mechanisms, which, despite their practicality and often favorable efficiencies, limit control over insertion site and copy number and may increase the risk of insertional mutagenesis and position-dependent variability in transgene expression. The past few years have witnessed rapid methodological diversification driven by genome editing, resulting in a growing repertoire of locus-specific strategies for large-fragment DNA insertion that are reshaping both disease-model construction and genetic therapeutics. In this Review, we synthesize the main classes of targeted large-fragment integration technologies reported to date. We begin with homology-directed repair (HDR)-dependent CRISPR-Cas9 knock-in strategies and discuss how donor architecture and local donor recruitment can be leveraged to improve integration outcomes for kilobase-scale payloads. We then examine approaches centered on prime editing, particularly those that couple prime editing with engineered serine/tyrosine recombinases to support programmable insertion of large DNA cargos. We close by surveying emerging HDR-independent systems based on CRISPR-guided transposition and retrotransposition, and we provide a comparative perspective on their performance envelopes, constraints, and trajectories toward broader biomedical applications.
AVLAYAH and KRESLADI Win FDA Accelerated Approvals.
Philippidis A
Hum Gene Ther · 2026 Jun · PMID 42010752 · Publisher ↗
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Harvest Process and Affinity Resin Selection Impacts on Adeno-Associated Virus Residual Host Cell Protein Retention.
Leibiger TM, Min L, Lee KH
Hum Gene Ther · 2026 Jul · PMID 41992847 · Publisher ↗
Scalable purification platforms have been developed for adeno-associated virus (AAV) processing to support large-scale vector manufacturing. The ability of column chromatography to recover packaged vectors and remove emp...
Scalable purification platforms have been developed for adeno-associated virus (AAV) processing to support large-scale vector manufacturing. The ability of column chromatography to recover packaged vectors and remove empty capsids has been well-established, but knowledge gaps remain for understanding process parameter impacts on impurity retention. In this work, we examine the impacts of two key process parameters-the harvest method and affinity resin selection-on residual host cell protein (HCP) retention. Sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) proteomics is applied to comprehensively profile residual HCPs in affinity chromatography (AC) elution pools from four AAV serotypes (AAV2, -5, -8, and -9) produced by suspension HEK293 cells. Vectors were purified from cell culture lysates and from supernatants using POROS CaptureSelect AAVX (AAVX) and one additional serotype-specific affinity resin-Capto AVB (AVB), POROS CaptureSelect AAV8 (PAAV8), or POROS CaptureSelect AAV9 (PAAV9). Significant divergence in residual HCP profiles was observed with the use of different affinity resins, with AVB and PAAV9 showing reduced residual HCP content in elution pools compared with AAVX and PAAV8. Processing of null culture lysates with fresh resins and resins with digested single-domain antibody fragments (sdAbs) shows that differences in resin performance are driven by variable nonspecific sdAb association with cellular impurities. Proteomic analysis of vector preparations from lysates compared with supernatants demonstrates product quality advantages of designing a media-only harvest process, specifically for AAV8, which was measured to contain an average of 66% of total vector genome content in the cell culture media. This work highlights the importance of serotype-specific tailoring of AAV downstream process design for improved product quality attributes to support clinical manufacture and small-scale analytics workflows.
Monitoring Fetal Somatic Cell Genome Editing with Total-Body Positron Emission Tomography: Studies in Rhesus Macaques ().
Tarantal AF, Martinez ML, Sanz L … +5 more , Lee CI, Yang H, O'Geen H, Hartigan-O'Connor DJ, Segal DJ
Hum Gene Ther · 2026 Jul · PMID 41981831 · Full text
Translational development of somatic cell genome editing requires monitoring the extent of editing in the body at a given time, the specificity of editing, durability, and the potential for adverse events. A noninvasive...
Translational development of somatic cell genome editing requires monitoring the extent of editing in the body at a given time, the specificity of editing, durability, and the potential for adverse events. A noninvasive approach that can identify edited cells is beneficial for addressing these and related questions. These studies used total-body positron emission tomography (PET) to identify somatic cell gene editing in fetal rhesus macaques. Rhesus dams were screened to confirm they were seronegative for AAV serotype 9 and Cas9 antibodies, then selected for the study. Fetuses were administered a dual imaging vector (AAV9/Cas9 and AAV9/ gRNA/HSV-sr39TK) using an ultrasound-guided fetal intrahepatic approach in the second trimester (10 vector genomes/fetus). After maternal intravenous administration of 9-(4-(18)F-Fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG) (∼3 mCi/kg), PET imaging was performed in the second and third trimesters. PET imaging provided evidence of editing in the fetal liver, which was sustained. Appropriate insertion of the promoterless HSV-sr39TK reporter in frame with the gene was confirmed in the fetal liver near term using RNA sequencing, and correctly targeted insertions were observed. These studies have shown that total-body PET can provide insights into gene-edited somatic cells and without evidence of adverse effects.
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