Gene Therapy最新文献

Thoracic aortic disease poses a significant threat due to its high mortality rates and genetic. underpinnings. While gene therapy holds promise for cures, the challenge lies in achieving. effective and safe gene delivery to the thoracic aorta. Tail vein injection (TI), is hindered by. off-target effects and hepatotoxicity, and traditional blinded percutaneous left heart injection. (TLI) carries a heightened risk of bleeding and mortality. To address these limitations. ultrasound-guided techniques present a viable solution. Adeno-associated virus (AAV) vectors. were delivered into the thoracic aorta of mice through TI, TLI, and ultrasound-guided. percutaneous left heart injection(ULI). While all three injection methods can achieve gene transduction in the thoracic aorta, the ULI approach provides the optimal balance between. high transduction efficiency and safety. The ULI method represents an efficient and safe. strategy for targeted gene delivery to the mouse thoracic aorta, providing a powerful tool for preclinical aortic gene therapy research that warrants broader application.

Blood stem cell gene therapy to treat hemoglobinopathies is beginning to transform health for small numbers of patients in the U.S. and Europe, where these conditions qualify as rare diseases. Yet hemoglobinopathies are common globally, disproportionately affecting low- and middle-income countries (LMICs), creating an ethical imperative to ensure access where disease burden is greatest. Gene therapy could have blockbuster drug potential if distributable to these regions, but cost is a major barrier. Cost-effectiveness analysis (CEA) models are seldom adapted to low-income settings, where limited data and resources constrain efforts to contextualize high-income evidence. Here, we present a novel framework to evaluate high-income country authorized gene therapies in LMIC contexts. Uganda, where sickle cell disease (SCD) imposes a major burden and no curative therapies are available, is the test case. We evaluate cost-effectiveness of gene therapy for adolescents and adults with SCD in Uganda, adapting U.S. evidence to local economic conditions. Using a three-state Markov model to estimate lifetime costs of standard-of-care in Uganda, two U.S.-based CEA models were adapted using scaling factors and applied to two authorized gene therapies for SCD, Lyfgenia™ (lovo-cel) and Casgevy® (exa-cel), assuming biologically consistent efficacy across populations. Incremental cost-effectiveness ratios (ICERs) were calculated from healthcare and societal perspectives, with internationally accepted gross domestic product-based thresholds. This study demonstrates that Casgevy could be cost-effective in Uganda at a scaled cost when societal benefits are considered. This framework enables CEAs for emerging therapies where local clinical trial data are limited, supporting local decision-makers, global funders, and manufacturers in advancing equitable access to transformative therapies in LMICs.

The advancement of adeno-associated virus (AAV) gene therapy applications faces significant challenges, particularly in the separation of empty (E), full (F), and intermediate capsids during manufacturing, as well as scaling up downstream processing. This study demonstrates the effectiveness of monolithic chromatography in maintaining high flow rates without sacrificing resolution across scales, making it ideal for large-scale applications. The CIMmultus® HR columns ensure high reproducibility at various purification scales, achieving ± 3% isoconductivity elution of empty AAV2/8 capsids.In combination with the improved method, it enriches full AAV capsids to 88% ± 9% with genome recovery of 77% ± 3% within this study. The screening of polishing columns exhibited highest enrichment and recovery of full capsids with an improved method developed on QA HR preparative column. High-throughput screening (HTS) on CIM® QA HR Monolithic Well Plate and subsequent single-column experiments confirmed these findings, facilitating predictions for implementing step elution approaches. The high reproducibility of the columns enabled the development of step elution methods in both bind-elute and flow through modes, with both approaches yielding comparable recovery and purity of full AAV capsids. Ultimately, the optimized AAV polishing method was successfully scaled up from 1 to 8000 mL monolith using step elution approach.

Patients diagnosed with esophageal cancer (EC) currently rely on treatments given at specialist care centers (surgery, chemotherapy, radiation), which despite their low cure rates are extremely life-disruptive, cause severe pain, and have strong side effects. In particular, dysphagia is one of the most distressing and debilitating symptoms in patients with cancer-related esophageal obstruction. There is clearly an urgent need for new effective and accessible therapies for EC patients that allow patients to continue normal activity as much as possible. Here, we describe a drinkable methylcellulose/xanthan gum-based gene therapy foam that coats the esophagus and accumulates an apoptosis-inducing gene therapy drug (mRNA lipid nanoparticles encoding Pseudomonas exotoxin A) at the tumorous esophageal stricture. In an in vitro EC tissue model, we show that gene therapy foam induces 110-fold higher tumor regression compared to suspension treatment. We also establish that gene therapy foam given prior to radiotherapy strongly enhances anti-tumor effects. Once implemented in the clinic, this treatment, which can be administered orally by a local family doctor or at home by the patient or caregiver, could maximize the time EC patients can live normal lives outside of the hospital and allow them to maintain their ability to swallow and eat.

Neovascular age-related macular degeneration (nAMD) is a major cause of irreversible vision loss in the elderly, driven by choroidal neovascularization and dysregulated vascular endothelial growth factor (VEGF) signaling. While anti-VEGF injections have transformed management, their frequent administration imposes a substantial burden on patients and limits adherence. Adeno-associated virus (AAV)-based gene therapy offers sustained intraocular delivery of anti-angiogenic agents with a single treatment, potentially overcoming these limitations. This review summarizes the rationale for AAV use in ocular gene therapy, compares major delivery routes, and highlights leading clinical candidates, including RGX-314, ADVM-022, 4D-150, and NG101. Advances in vector engineering, promoter optimization, and immune modulation are discussed alongside key challenges such as preexisting immunity and inflammation. Future directions include next-generation capsids, combination regimens, and precision patient selection. Collectively, these developments position AAV-based gene therapy as a promising strategy to redefine the therapeutic landscape of nAMD.

While recombinant adeno-associated virus (AAV) holds significant promise for effective and durable gene delivery for gene therapy, a thorough understanding of the critical quality attributes (CQAs) along with the degradation pathways of AAV under the various stresses that may occur during manufacturing, storage, and handling remains limited. To address this gap, we performed a comprehensive forced degradation study to elucidate the degradation pathways of AAV8 under a series of stress conditions, such as oxidation, extreme pH, high temperature, freeze-thaw, and agitation. Our results show that, under these stress conditions, distinct post-translational modifications (PTM), including methionine oxidation, asparagine deamidation, and aspartic acid isomerization, along with multiple physical degradation pathways, including capsid aggregation, viral protein fragmentation, and genome DNA leakage, could occur. Alterations in AAV8 biological activity were frequently attributed to the combination effect from chemical and physical degradation mechanisms. The results from this study provide a valuable insight into the establishment of stability-indicating methods and the identification of CQAs for AAV. It will also support the development of robust manufacturing process as well as stable and efficacious AAV gene therapy drug products.

Previously, we confirmed that BaEV-LVs outperformed VSV-G-LVs for gene delivery or correction of human T cells, B cells, NK cells and HSPCs correlating with high expression of its receptors, ASCT-1 and ASCT-2 on these cells. Since HERV-W gp uses the same entry receptors, we compared transduction efficiencies for BaEV-LVs and HERV-W-LVs in hematopoietic cells. HERV-W LV transduction was efficient but inferior to BaEV-LV in TCR-stimulated T cells (40% versus 80%) and this low efficiency was even more pronounced in IL-7/IL-15 pre-stimulated T cells. BaEV-LVs were significantly superior over HERV-W-LVs for the transduction of B cells and NK cells. High HERV-W-LV mediated transduction levels were achieved for pre-stimulated hCD34+ cells, which remained though lower than for the BaEV-LVs. Additionally, BaEV-LVs reached over 80% of transduction in severe combined immunodeficiency (SCID) repopulating cells (SRC) in 6/6 engrafted NBSGW mice. HERV-W-LVs reached this transduction level in 1/5 mice, while 3/5 engrafted NBSGW mice reached significantly lower transduction levels (20-50%). For both vectors the transduction levels were equivalent in the lymphoid and myeloid lineages in all hematopoietic tissues, suggesting transduction of immature HSPCs. Summarizing, BaEV-LVs outperformed HERV-W-LVs for transduction of important gene therapy target cells such as NK, B, T cells and CD34+ HSPCs.

Efficient delivery of CRISPR ribonucleoproteins into primary hematopoietic stem and progenitor cells (HSPCs) is essential for durable gene editing therapies but remains challenging. Here, we advance a modular, benchtop-assembled gold-polymer hybrid nanoparticle (CRISPR-AuNP) platform that enables non-viral delivery of multiple CRISPR systems into HSPCs. Guided by a mechanistic understanding of Cas9's interaction with gold surfaces, we engineered the formulation by conjugating pre-formed RNP-polymer complexes, assembled using thiolated polyethyleneimine-polyethylene glycol, to gold nanoparticles. This system achieved efficient editing in primary CD34+ HSPCs for Cas9, Cas12a, and Cas12a-M29-1 without compromising cell viability. Notably, the nanoformulation can be assembled in under 2 h in a PCR tube for less than $70/million HSPCs treated. This work establishes a scalable, cost-effective, and accessible gene editing system with the potential to democratize CRISPR applications in HSPC research and therapy.

Immune responses against recombinant adeno-associated virus (rAAV) are one of the major obstacles in gene therapy. We investigated the potential of Programmed Death 1 ligands 1 and 2 (PD-L1/2) to protect AAV-transduced cells from immunological clearance. Ligand compatibility for co-delivery was first evaluated using two transgenes, VEGF-B186 and muSEAP, separated from PD-L1/2 by a self-cleaving P2A peptide. After proper cleavage and biological activity of the co-produced proteins were demonstrated in vitro, the effect of PD-L1/2 co-expression on muSEAP production and persistence was studied in naïve and vector pre-immunized mice. Vectors (rAAV6-muSEAP, rAAV6-muSEAP-PD-L1, or rAAV6-muSEAP-PD-L2) were injected into two sites of the gastrocnemius muscle at a total dose of 1×10¹⁰ vg. Co-delivery of PD-L1, particularly, significantly enhanced muSEAP secretion into the bloodstream up to 12 weeks despite elevated anti-AAV6 responses in pre-immunized mice. muSEAP secretion increased 33.3- and 31.4-fold with the co-delivery of PD-L1, while the increase was only 5.6- and 9.3-fold in the muSEAP control group at 5 and 12 weeks, respectively. Ligand-treated pre-immunized animals also had less T-cell infiltration into the treated muscle compared to naïve animals. In summary, co-delivery of PD-L1/2 alongside a transgene represents a promising strategy for achieving sustained gene expression in individuals pre-exposed to AAV.

Adoptive Cell Therapies based on cytokine-induced killer cells (CIKs) can address the heterogeneity of solid tumors due to their multiple mechanisms of cancer cell recognition. However, tumor trafficking is one of the main limitations. In this work, we describe that a high proportion of CIKs obtained from pancreatic ductal adenocarcinoma patients express the CXCR3 and CCR5 receptors, and they migrate towards their corresponding chemokines CXCL10 and CCL5 in vitro. Using an immune competent orthotopic PDAC mouse model, we have investigated the ability of different clinically compatible interventions to increase the expression of these chemokines. No significant elevation was obtained with chemotherapy (5-fluorouracil, irinotecan, oxaliplatin, paclitaxel, gemcitabine or temozolomide), tyrosine kinase inhibitors sorafenib and sunitinib, or the immunostimulatory agents polyinosinic:polycytidylic acid, Mycobacterium tuberculosis antigens and diphtheria/pertussis/tetanus vaccine. In contrast, CXCL10 and CCL5 expression was stimulated by local administration of an adenoviral vector equipped with a drug-inducible expression system for interleukin-12 (IL-12). Combination of the vector and CIKs obtained a strong antitumor effect in the PDAC model, although it was mainly due to vector-mediated recruitment of endogenous immune cells. We conclude that additional barriers beyond chemokine expression should be overcome in order to unleash the full potential of CIKs on solid tumors.