Gene Therapy Research Articles

Membrane Filtration of Nanoscale Biomaterials: Model System and Membrane Performance Evaluation for AAV2 Viral Vector Clarification and Recovery

The growing demand for viral vectors as nanoscale therapeutic agents in gene therapy necessitates efficient and scalable purification methods. This study examined the role of nanoscale biomaterials in optimizing viral vector clarification through a model system mimicking real AAV2 crude harvest material. Using lysed HEK293 cells and silica nanoparticles (20 nm) as surrogates for AAV2 crude harvest, we evaluated primary (depth filters) and secondary (membrane-based) filtration processes under different process parameters and solution conditions. These filtration systems were then assessed for their ability to recover nanoscale viral vectors while reducing DNA (without the need for endonuclease treatment), protein, and turbidity. Primary clarification demonstrated that high flux rates (600 LMH) reduced the depth filter’s ability to leverage adsorptive and electrostatic interactions, resulting in a lower DNA removal. Conversely, lower flux rates (150 LMH) enabled >90% DNA reduction by maintaining these interactions. Solution conductivity significantly influenced performance, with high conductivity screening electrostatic interactions, and the model system closely matching real system outcomes under these conditions. Secondary clarification highlighted material-dependent trade-offs. The PES membranes achieved exceptional AAV2 recovery rates exceeding 90%, while RC membranes excelled in DNA reduction (>80%) due to their respective surface charge and hydrophilic properties. The integration of the primary clarification step dramatically improved PES membrane performance, increasing the final flux from ~60 LMH to ~600 LMH. Fouling analysis revealed that real AAV2 systems experienced more severe and complex fouling compared to the model system, transitioning from intermediate blocking to irreversible cake layer formation, which was exacerbated by nanoscale impurities (~10–600 nm). This work bridges nanomaterial science and biomanufacturing, advancing scalable viral vector purification for gene therapy.

Analysis of Hepatic Lentiviral Vector Transduction: Implications for Preclinical Studies and Clinical Gene Therapy Protocols

Lentiviral vector-transduced T cells were approved by the FDA as gene therapy anti-cancer medications. Little is known about the effects of host genetic variation on the safety and efficacy of the lentiviral vector gene delivery system. To narrow this knowledge gap, we characterized hepatic gene delivery by lentiviral vectors across the Collaborative Cross (CC) mouse genetic reference population. For 24 weeks, we periodically measured hepatic luciferase expression from lentiviral vectors in 41 CC mouse strains. Hepatic and splenic vector copy numbers were determined. We report that the CC mouse strains showed highly diverse outcomes following lentiviral gene delivery. For the first time, a moderate correlation between mouse-strain-specific sleeping patterns and transduction efficiency was observed. We associated two quantitative trait loci (QTLs) with intrastrain variations in transduction phenotypes, which mechanistically relates to the phenomenon of metastable epialleles. An additional QTL was associated with the kinetics of hepatic transgene expression. Genes found in the above QTLs are potential targets for personalized gene therapy protocols. Importantly, we identified two mouse strains that open new directions for characterizing continuous viral vector silencing and HIV latency. Our findings suggest that wide-range patient-specific outcomes of viral vector-based gene therapy should be expected. Thus, novel clinical protocols should be considered for non-fatal diseases.

Supramolecular Polymers for Drug Delivery

AbstractSupramolecular polymers are constructed through highly reversible and directionally specific non‐covalent interactions between monomer units. This unique feature enables supramolecular polymers to undergo controlled structural reconfiguration and functional transformation in response to external stimuli, imparting them with high environmental responsiveness and self‐healing properties. In particular, supramolecular polymers exhibit several specific advantages compared to conventional polymers, such as inherent degradability, the ease of preparation and the incorporation of functional units, and smart responsiveness to various biological stimuli. These characters make supramolecular polymers promising candidates for intelligent drug delivery systems in complex biological environments. In this review, we comprehensively summarize the latest developments and representative achievements of supramolecular polymers in drug delivery fields, focusing primarily on the design and synthesis, the properties and functionalities, and the practical applications of supramolecular polymers in small molecule drug delivery, gene therapy, and protein delivery. Finally, we highlight future research directions, focusing on multifunctionality, adaptability, and personalized therapy. We focus on recent studies that address key challenges in the field, providing rational polymer design, important properties, functionality, and understanding delivery strategies. These developments are expected to advance supramolecular polymers as new platforms of intelligent drug delivery systems, offering innovative solutions for the treatment of complex diseases.

Controlled release of MIF siRNA and GDNF protein from a photocurable scaffold efficiently repairs spinal cord injury

AbstractCompared with traditional treatment strategies, siRNA‐based gene therapy combines with protein therapy to offer a new strategy for spinal cord injury (SCI). The siRNA and protein therapy are limited by the large and deep lesion site and local co‐delivery vectors. However, the photocurable scaffold has the properties of injectable, flexible, and biodegradable, which provide a potential formulation for siRNA and protein combined therapy. Here, a photocurable lipid nanoparticle gel (PLNG) scaffold is designed for efficiently sustained and controlled release of the macrophage migration‐inhibitory factor (MIF) targeted siRNA and co‐delivery of GDNF protein for SCI. The GDNF is chemically modified in the scaffold and the prepared GDNF‐PLNG/siRNA scaffold is injectable with easily photocured. This formulation can inhibit inflammation by promoting macrophage M2 polarization and effectively promote primary neuron axon growth. After locally administered with GDNF‐PLNG/siMIF scaffold to SCI mice, the scaffold promoted neuron regeneration by upregulation of neuron cytokine production and inhibited inflammation through the downregulation immune pathway. With the interaction mechanism of GDNF and MIF siRNA, GDNF‐PLNG/siMIF scaffold increases the collagen and integrin expression to promote spinal cord repairing and significantly improve motor function, so that scaffold is a potential candidate gene formulation applied to clinical SCI treatment.

Peptides rapidly transport antibiotic across the intact tympanic membrane to treat a middle ear infection

The tympanic membrane (TM) forms an impenetrable barrier to medical therapies for middle ear (ME) diseases like otitis media. By screening a phage-displayed peptide library, we have previously discovered rare peptides that mediate the active transport of cargo across the intact membrane of animals and humans. Since the M13 filamentous bacteriophage on which the peptides are expressed are large (nearly 1 µm in length), this offers the possibility of noninvasively delivering drugs, large drug packages, or gene therapy to the ME. To evaluate this possibility, EDC chemistry was employed to covalently attach amoxicillin, or neomycin molecules to phage bearing a trans-TM peptide, as a model for large drug packages. Eight hours after application of antibiotic-phage to the TM of infected rats, ME bacterial titers were substantially reduced compared to untreated animals. As a control, antibiotic was linked to wild-type phage, not bearing any peptide, and application to the TM did not affect ME bacteria. The results support the ability of rare peptides to actively deliver pharmacologically relevant amounts of drugs through the intact TM and into the ME. Moreover, since bacteriophage engineered to express peptides are viral vectors, the trans-TM peptides could also transport other viral vectors into the ME.

Insights from Computational Studies of Polymeric Systems for Nucleic Acid Delivery.

The safe and efficient delivery of nucleic acids into cells is a critical step in the success of gene and cell therapies. Although viral vectors are the predominant tools in current gene and cell therapy practices, they present significant challenges including high costs and safety concerns. Nonviral delivery systems for nucleic acids show immense potential for future medicine, particularly as nucleic acid therapeutics continue to be developed for the treatment of a wide range of diseases, including cancer. Significant research efforts, both experimental and computational, have been devoted to the development, characterization, and understanding of nonviral delivery processes. While numerous reviews have documented these research advancements, few have specifically addressed the contributions from computational studies. In this review, we provide an overview of the insights gained from computational and theoretical studies of polymeric systems for nucleic acid delivery. We also highlight future directions where computational and experimental approaches could synergize to advance the field.

Prelimbic cortex is involved in the regulation of morphine-induced conditioned place preference in both resistant and sensitive mice

A primary behavioral pathology in drug addiction is the overpowering motivational strength and decreased ability to control the desire to obtain drugs, which shows some variation between different individuals. Here, using a morphine-induced conditioned place preference (CPP) model with footshock, we found that mice exhibited significant individual differences in morphine-induced addiction. Despite the consequences of footshock, a small percentage of mice (24%) still showed stable morphine preference, demonstrating resistant to punishment. The majority of mice (76%) were relatively sensitive to punishment and showed termination of morphine preference. As a region of advanced cognitive function in the mammalian brain, the medial prefrontal cortex (mPFC) is involved in regulating drug-induced addictive behaviors. We found that activating the pyramidal neurons in the prelimbic cortex (PrL) could effectively reverse morphine-induced CPP in resistant mice, and inhibiting pyramidal neurons in the PrL could promote morphine-induced CPP in sensitive mice. To further explore the differences between resistant and sensitive mice, we analyzed the differences in gene expression in their PrL regions through RNA-seq analysis. The results showed that compared to sensitive mice, the significantly downregulated differentially expressed genes (DEGs), such as Panx2, Tcf7l2, Htr2c, Htr5a, Orai3, Slc24a4 and Cacnb2, in resistant mice were mainly involved in synaptic formation and neurodevelopment. We speculated that there may be defects in the neuronal system of resistant mice, and caused they are more prone to morphine-induced CPP. These findings are likely to contribute to research in gene therapy, and they may also serve as potential therapeutic targets for drug addiction.

Gene therapy with feline anti-Müllerian hormone analogs disrupts folliculogenesis and induces pregnancy loss in female domestic cats

For female domestic cats, ovariohysterectomy is the only method of inducing permanent infertility. However, hundreds-of-millions of free-roaming cats globally highlight the necessity for alternative contraceptive approaches. One strategy involves a single injection of vector delivering a fertility-inhibiting protein for lifetime contraception. Recent studies in mice and cats have identified anti-Müllerian hormone as an excellent candidate for this type of contraception. Here, we leverage our recent characterization of the molecular mechanisms underlying human anti-Müllerian hormone synthesis and activity, to generate potent feline anti-Müllerian hormone analogs. Single intramuscular delivery of these analogs to female cats using an adeno-associated viral vector leads to a greater than 1000-fold increase in feline anti-Müllerian hormone levels, which are sustained for 9 months. High serum anti-Müllerian hormone is associated with abnormal estrus cyclicity, non-follicular ovarian cyst formation, and a progressive decline in antral follicle numbers, however, the few surviving large follicles continue to ovulate. Unlike previous studies, supraphysiologic levels of anti-Müllerian hormone do not block conception, although they are incompatible with the maintenance of pregnancy. Our findings highlight the complexity of the effects of anti-Müllerian hormone on ovarian physiology but confirm that this growth factor is a candidate for fertility control in free-roaming cats.

Pilocytic Astrocytoma in a Child with Spinal Muscular Atrophy Treated with Onasemnogene Abeparvovec.

Spinal muscular atrophy (SMA) is a severe neuromuscular disease, leading to progressive muscle weakness and potentially early mortality if untreated. Onasemnogene abeparvovec is a recombinant adeno-associated virus serotype 9 (rAAV9)-based gene therapy that has demonstrated improvements in survival and motor function for SMA patients. Here, we present a case of a patient diagnosed with a grade 1 pilocytic astrocytoma at the age of 2 years, approximately 8 months after onasemnogene abeparvovec treatment. Although vector genomes delivered by rAAVs persist primarily as episomes, rare integration events have been linked to tumor formation in neonate murine models. Therefore, we investigated the presence and possible integration of onasemnogene abeparvovec in formalin-fixed paraffin embedded (FFPE) and frozen tumor samples. In situ hybridization demonstrated variable transduction levels in individual tumor cells while droplet digital PCR measured an average vector copy number ranging from 0.7-4.9 vector genomes/diploid genome. Integration site analysis identified a low number of integration sites that were not conserved between technical replicates, nor between FFPE and frozen samples, indicating that cells hosting integrating vector genomes represent a minority in the overall cell population. Thus, molecular analysis of the tumor tissue suggests that tumorigenesis was causally independent of the administration of onasemnogene abeparvovec.

Microneedle Delivery of Protein and Peptides: Advances in Drug Delivery

Microneedles are the advances in the transdermal drug delivery system of proteins and peptide drugs which exerts its effect through the formation of the micro channels. Microneedles are of various types such as solid microneedles, dissolving microneedles, metallic microneedles, 3D printed microneedles; polymer based microneedles, Hydrogel microneedles and coated microneedles. Microneedle characterization is the most important part after the formulation. These dosage forms have both advantages and limitations. Stability enhancement, targeted drug delivery, low invasiveness, enhanced skin absorption etc. are some of the advantages associated with microneedles. Likewise shallow penetration, chances of skin irritation and injury, chances of degradation in different extreme temperature and pH etc. are some of the limitations. Needle integrity, uniform drug distribution, diffusion and degradation, sterility and contamination, immunogenicity and immune response, Activity preservation are the crucial parts that should be controlled during formulation. Improved stability of proteins, stabilization of inactive ingredients, utilization in the field of gene therapy and mRNA delivery, development of smart microneedles, development of multilayered microneedles, ligand targeting etc. are some of the advances in the microneedle delivery system. These delivery systems are widely recognized as the future of the drug delivery addressing the challenges associated with the patient compliance. Keywords: Microneedles, Needle Integrity, Immunogenicity, Patient compliance

An epigenetic regulator synergizes with alphavirus-mediated gene therapy via biomimetic delivery for enhanced cancer therapy.

Gene therapy is promising for treating genetic disorders, but faces challenges in treating cancer due to the intricate genetic and immunosuppressive landscape of this disease. Here, we describe a technology combining alphavirus-based gene therapy with an epigenetic regulator via pyroptosis and immune checkpoints to address these challenges. A filamentous actin-mimicking liposomal delivery system, with high fusion efficiency, was developed that encapsulates the Semliki Forest virus (pSFV) DNA vector to deliver p53 and PDL1 scFv DNA, bypassing traditional endocytic barriers to deliver genes with high efficiency via membrane fusion. To enhance this combined therapy, the DNA methyltransferase inhibitor decitabine (DAC) was used to increase Gasdermin E (GSDME) expression, converting apoptosis to pyroptosis. This approach kills apoptosis-resistant tumor cells, and also promotes T cell infiltration and activation, facilitating an anti-PDL1 therapy and the systemic antitumor immune response. This multifaceted therapeutic strategy combines gene therapy with epigenetic regulation to significantly improve immune checkpoint therapy (ICT) effectiveness, offering a robust potential as a transformative cancer treatment.

Nitroglycerin-responsive gene switch for the on-demand production of therapeutic proteins.

Gene therapies and cell therapies require precise, reversible and patient-friendly control over the production of therapeutic proteins. Here we present a fully human nitric-oxide-responsive gene-regulation system for the on-demand and localized release of therapeutic proteins through clinically licensed nitroglycerin patches. Designed for simplicity and robust human compatibility, the system incorporates human mitochondrial aldehyde dehydrogenase for converting nitroglycerin into nitric oxide, which then activates soluble guanylate cyclase to produce cyclic guanosine monophosphate, followed by protein kinase G to amplify the signal and to trigger target gene expression. In a proof-of-concept study, human cells expressing the nitroglycerin-responsive system were encapsulated and implanted subcutaneously in obese mice with type 2 diabetes. Transdermal nitroglycerin patches applied over the implant enabled the controlled and reversible production of glucagon-like peptide-1 throughout the 35-day experimental period, effectively restoring blood glucose levels in these mice without affecting heart rate or blood pressure. The approach may facilitate the development of safe, convenient and responsive implantable devices for the sustained delivery of biopharmaceuticals for the management of chronic diseases.

New Insights into Genetic Right Ventricular Cardiomyopathies.

Inherited right ventricular disease in the form of arrhythmogenic right ventricular cardiomyopathy (ARVC) was first described 40 years ago. The ARVC-causing genes have progressively been identified from the year 2000, accompanied by a robust journey of deep phenotyping. The explosion of genotype and phenotype data coupled with a collaborative spirit in the ARVC community has led to an immense advance in our understanding of the various faces of this disease, with a recent focus on gene specific phenotypes, risk assessment and mitigation. The modern cardiogenetic team has a wealth of information that informs the biology of the disease, its phenotypic expression and the processes of care to detect the presence and progression of disease. Gene-specific considerations will raise the bar in precision medicine applied to diagnosis, natural history, and potentially curative interventions with targeted small molecules and gene therapy. This is an exciting time for the ARVC collaborative community to usher in a new era in changing the course of ARVC for patients and their families.

A high-capacity combination of Pluronic L64-Cupping for intramuscular gene delivery.

Intramuscular injection of plasmid DNA (pDNA) is a promising approach for gene therapy, but its efficiency is hindered by both extracellular and intracellular barriers. The extracellular matrix (ECM), including collagens and nucleases, obstructs pDNA penetration, while intracellular challenges include crossing the plasma membrane, escaping endosomes, and reaching the nucleus. Though non-viral carriers like polymers and cationic lipids have been developed, they often fail to address both barriers simultaneously, leading to poor gene transfer in vivo. Physical methods exist but may damage tissues and cause patient discomfort. Here, we introduce a Pluronic L64-Cupping (L/C) gene delivery system that enhances pDNA delivery by sequentially overcoming ECM diffusion, membrane permeabilization, and intracellular transfection. After intramuscular injection of the pDNA-Pluronic L64 mixture, negative pressure is applied to the injection site, significantly boosting reporter gene expression and sustaining it for at least 42 days. Additionally, this system effectively induces HBsAb production in mice, offering a safe, efficient, and cost-effective platform for both laboratory and clinical gene therapy applications.

Brain-Wide Neuroregenerative Gene Therapy Improves Cognition in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive and irreversible brain disorder with extensive neuronal loss in the neocortex and hippocampus. Current therapeutic interventions focus on the early stage of AD but lack effective treatment for the late stage of AD, largely due to the inability to replenish the lost neurons and repair the broken neural circuits. In this study, by using engineered adeno-associated virus vectors that efficiently cross the blood-brain-barrier in the mouse brain, a brain-wide neuroregenerative gene therapy is developed to directly convert endogenous astrocytes into functional neurons in a mouse model of AD. It is found that ≈500000 new neurons are regenerated and widely distributed in the cerebral cortex and hippocampus. Importantly, it is demonstrated that the converted neurons can integrate into pre-existing neural networks and improve various cognitive performances in AD mice. Chemogenetic inhibition of the converted neurons abolishes memory enhancement in AD mice, suggesting a pivotal role for the newly converted neurons in cognitive restoration. Together, brain-wide neuroregenerative gene therapy may provide a viable strategy for the treatment of AD and other brain disorders associated with massive neuronal loss.

The liver-specific long noncoding RNA FAM99B inhibits ribosome biogenesis and cancer progression through cleavage of dead-box Helicase 21

Emerging evidence has demonstrated that long noncoding RNAs (lncRNAs) are promising targets or agents for the treatment of human cancers. Most liver-specific lncRNAs exhibit loss of expression and act as tumor suppressors in liver cancer. Modulating the expression of these liver-specific lncRNAs is a potential approach for lncRNA-based gene therapy for hepatocellular carcinoma (HCC). Here, we report that the expression of the liver-specific lncRNA FAM99B is significantly decreased in HCC tissues and that FAM99B suppresses HCC cell proliferation and metastasis both in vitro and in vivo. FAM99B promotes the nuclear export of DDX21 through XPO1, leading to further cleavage of DDX21 by caspase3/6 in the cytoplasm. FAM99B inhibits ribosome biogenesis by inhibiting ribosomal RNA (rRNA) processing and RPS29/RPL38 transcription, thereby reducing global protein synthesis through downregulation of DDX21 in HCC cells. Interestingly, the FAM99B65-146 truncation exhibits tumor-suppressive effects in vivo and in vitro. Moreover, GalNAc-conjugated FAM99B65-146 inhibits the growth and metastasis of orthotopic HCC xenografts, providing a new strategy for the treatment of HCC. This is the first report of the use of a lncRNA as an agent rather than a target in tumor treatment.Graphical illustration of the mechanism of FAM99B in HCC.

Comparative analysis of AAV serotypes for transduction of olfactory sensory neurons

Olfactory sensory neurons within the nasal epithelium detect volatile odorants and relay odor information to the central nervous system. Unlike other sensory inputs, olfactory sensory neurons interface with the external environment and project their axons directly into the central nervous system. The use of adeno-associated viruses to target these neurons has garnered interest for applications in gene therapy, probing olfactory sensory neuron biology, and modeling disease. To date, there is no consensus on the optimal AAV serotype for efficient and selective transduction of olfactory sensory neurons in vivo. Here we utilized serial confocal imaging and single-nucleus RNA sequencing to evaluate the efficacy of 11 different AAV serotypes in transducing murine olfactory sensory neurons via non-invasive nasal inoculation. Our results reveal that AAV1, while highly effective, exhibited broad tropism, whereas AAV-DJ/8 showed the greatest specificity for olfactory sensory neurons.

Challenges and Emerging Strategies of Immunotherapy for Glioblastoma.

Glioblastoma (GBM) is recognized as the most lethal primary malignant tumor of the central nervous system. Although traditional treatments can somewhat prolong patient survival, the overall prognosis remains grim. Immunotherapy has become an effective method for GBM treatment. Oncolytic virus, checkpoint inhibitors, CAR T cells and tumor vaccines have all been applied in this field. Moreover, the combining of immunotherapy with traditional radiotherapy, chemotherapy, or gene therapy can further improve the treatment outcome. This review systematically summarizes the features of GBM, the recent progress of immunotherapy in overcoming GBM.

Development of 2LTRZFP-expressing induced pluripotent stem cells as a potential anti-HIV-1 gene therapy against viral integration.

Highly active antiretroviral drug (HAART) is the standard treatment for HIV-1 infection to suppress the viral load. However, this treatment does not completely eradicate the virus; it simply decreases the viral load to undetectable levels. The development of a novel therapy to cure the disease is essential. Previously, we developed an engineered zinc finger protein (ZFP) that specifically binds to the 2-LTR-circle junction (2LTRZFP), the target site for viral integrase, preventing HIV-1 integration in human CD34+ hematopoietic stem/progenitor cells (HSPCs) and macrophages. Although the transduction efficiency of 2LTRZFP was approximately 50%, purifying and expanding the 2LTRZFP-expressing HSPCs proved difficult. In addition, the batch-to-batch variability in transduction efficiency could have a major impact on the therapeutic efficacy. In this study, we introduced the 2LTRZFP into human induced pluripotent stem cells (iPSCs) followed by clonal isolation and functional validation of the 2LTRZFP. Upon the HIV-1 challenge, the 2LTRZFP protein was found to inhibit the viral integration in iPSCs, iPSC-derived HSPCs, and macrophages. The engineered iPSC clone could be differentiated into functional macrophages, as evidenced by M1 and M2 polarization, and phagocytosis. Our finding revealed that the 2LTRZFP did not perturb the macrophage differentiation process. Therefore, the 2LTRZFP-expressing iPSCs could provide an unlimited supply of HIV-1-resistant HSPCs for transplantation, potentially leading to HIV-1-resistant blood cells. The knowledge obtained from this study will provide a cornerstone for HIV-1 gene therapy using HSPC transplantation as a sustainable HIV-1 treatment in the future.

Oligogenic structure of amyotrophic lateral sclerosis has genetic testing, counselling and therapeutic implications

BackgroundDespite several studies suggesting a potential oligogenic risk model in amyotrophic lateral sclerosis (ALS), case–control statistical evidence implicating oligogenicity with disease risk or clinical outcomes is limited. Considering its direct...