Human gene therapy最新文献

The low-density lipoprotein receptor (LDLR) plays a crucial role in cholesterol regulation and lipoprotein transport. Variations in the LDLR gene can cause familial hypercholesterolemia (FH), with homozygous familial hypercholesterolemia (HoFH) being the most severe form. HoFH is marked by elevated low-density lipoprotein cholesterol (LDL-C) levels and early onset of cardiovascular disease, often with a poor prognosis. Current treatment options for HoFH are limited by insufficient effectiveness and restricted availability. Gene therapy, which involves the delivery of functional LDLR genes, offers a promising and innovative approach that could significantly improve outcomes for patients with HoFH. In this study, the adeno-associated virus serotype 8 (AAV8) vector was used to deliver the LDLR gene specifically to hepatocytes. The vector was designed using the pAAV-TBG plasmid, incorporating a hepatocyte-specific thyroid hormone-binding globulin (TBG) promoter. Viral packaging was performed in HEK 293T cells, followed by virus collection, purification, and titration. Mice, including C57BL/6J, Ldlr-KO, and homozygous Ldlr p.W483X mice, were injected with low, medium, or high doses of the virus via the tail vein. The efficacy and safety of the AAV8-LDLR gene therapy were assessed through Western blot analysis, lipid profiling, and liver pathology. AAV8-mediated LDLR delivery effectively improved lipid levels in both Ldlr-KO and homozygous Ldlr p.W483X mice. LDL-C levels showed a sustained reduction over the 2-month observation period. Western blot analysis confirmed the expression of LDLR protein in the liver, while lipid profiling demonstrated significant reductions in total cholesterol, triglycerides, LDL-C, and high-density lipoprotein cholesterol levels. Liver histopathology revealed no significant differences in non-alcoholic fatty liver disease scores between groups, indicating a favorable safety profile, particularly at low and medium doses. AAV8-LDLR gene therapy shows considerable promise as an effective treatment for HoFH. Our results indicate that this therapy significantly reduces lipid levels while maintaining a favorable safety profile.

Recombinant adeno-associated viruses (AAVs) are clinically relevant vectors for gene therapy that persist largely as extrachromosomal episomes but also infrequently integrate into host genomes. Valoctocogene roxaparvovec is an approved AAV-based gene therapy for severe hemophilia A. We present a molecular characterization of the vector integration profiles in 5 human biopsy samples from valoctocogene roxaparvovec clinical trials as well as in samples from valoctocogene roxaparvovec-treated nonhuman primates (NHPs). The number of genomic integrations was substantially below the previously reported number of transgene-expressing cells, and integration profiles were similar between human and NHP samples. The integration profiles were polyclonal, similarly distributed across the genome, and demonstrated a small bias toward regions of open chromatin and actively transcribed genes, with no relative enrichment in cancer-associated genes. These observations were replicated between species and support the concept that preclinical assessment of AAV vector integration in NHPs is representative of outcomes in humans.

Gene therapy using adeno-associated virus (AAV) vectors is currently expanding to broad clinical applications. As the presence of a neutralizing antibody (NAb) against AAV capsids significantly restrains their efficacy, an accurate evaluation of NAb status is crucial for selecting appropriate candidates for gene therapy. Notably, cell-based NAb assays may not be sufficiently sensitive for detecting low-titer NAb, and few assays can evaluate multiple AAV serotypes using a commonly available cell. In this study, we developed a sensitive NAb assay against various AAV serotypes using commonly available HEK293 and Huh-7 cells. We found that adding glucose efficiently enhanced transgene expression across various AAV serotypes without causing cell damage. In addition, by combining a highly sensitive reporter gene, NanoLuc, the necessary dose of AAV vector was significantly reduced. The reduction of AAV dose resulted in the increased sensitivity of NAb detection as low as 100 vector genomes/cell. At the lower vector doses, sensitivity improvement was not observed regardless of serotypes, suggesting the limit of assay sensitivity of the cell-based NAb assay. These findings provide a highly sensitive methodology for assessing NAb titers and offer insights into conditions to attain maximal sensitivity in the cell-based NAb assay.

Chimeric antigen receptor T cell (CAR-T) therapy has achieved great success and progress for treatment of hematological malignancy, but it still cannot overcome the obstacles in solid tumors. The hostile tumor microenvironment (TME), such as dense extracellular matrix, hypoxia, low pH, and tumor-derived metabolites, largely impedes CAR-T function. Oncolytic virus, as a form of immunotherapy, provides a way to antagonize the TME and improve the efficacy of CAR-T cells in solid tumors. In this study, the chemokine CXCL9 and interleukin 15 (IL15) genes were genetically integrated into adenoviral vector to construct oncolytic adenovirus (OAV) Ad-CXCL9-IL15, which could infect tumor cells to express and secrete CXCL9 and IL15. Ad-CXCL9-IL15 showed potent antitumor activity in xenografted prostate cancer model and augmented the tumor infiltration of CD45⁺CD3⁺ T and CD8⁺ T cells in immunocompetent mice. Moreover, Ad-CXCL9-IL15 treatment decreased Treg cells in tumor mass and increased CD44⁺CD62L⁺ T cells in spleen. Indicating that Ad-CXCL9-IL15 modified the TME and augmented antitumor immune responses in vivo. Furthermore, administration of Ad-CXCL9-IL15 dramatically promoted infiltration and survival of B7H3-targeting CAR-T cells, improved the therapeutic efficacy, and prolonged the survival time of prostate cancer-bearing mice. Therefore, cytokine-armored OAV Ad-CXCL9-IL15 could be used as a bioenhancer to modify TME and boost immunotherapy for solid tumors.

Recombinant adeno-associated virus (rAAV) vectors are increasingly preferred for in vivo gene therapy due to their broad tropism, low immunogenicity, and sustained transgene expression. Nevertheless, in cases of adverse reactions to these expressions, a method to suppress or permanently halt rAAV transgene activity could significantly enhance the safety of these vectors. To address this need, we employed meganucleases-highly specific DNA endonucleases with long recognition sequences. By placing meganuclease target sites within rAAV transgenes, we created a system in which targeted cleavage leads to controlled disruption of transgene expression. Utilizing a luciferase assay, we screened various meganucleases and identified I-AniI-Y2, I-BmoI, and I-PpoI as prime candidates due to their high cleavage efficiencies. By strategically placing multiple meganuclease target sequences within introns, as well as in the 5' and 3' untranslated regions (UTRs) of transgenes, we significantly enhanced the cleavage efficiency of these meganucleases, ensuring robust and targeted suppression of transgene expression. Finally, we employed an mRNA-loaded lipid nanoparticledelivery system to demonstrate the ability of meganucleases to robustly inhibit rAAV-mediated transgene expression in vitro. Our findings underscore the potential of meganucleases as a viable safety mechanism in rAAV gene therapies, marking a significant advance toward safer long-term gene therapy approaches.

The advent of genome-editing technologies, particularly the RNA-guided the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) 9, which originates from prokaryotic CRISPR/Cas adaptive immune mechanisms, has revolutionized molecular biology. Renowned for its simplicity, cost-effectiveness, and capacity for multiplexed gene editing, CRISPR/Cas9 has emerged as the most versatile and widely adopted genome-editing platform. Its applications span fundamental research, biotechnology, medicine, and therapeutics. This review highlights recent advancements in CRISPR-based technologies, focusing on CRISPR/Cas9, CRISPR/Cas12a, and CRISPR/Cas12f. It emphasizes precision editing methods like base editing and prime editing, which enable targeted nucleotide changes without double-strand breaks. The specificity of these tools, including on-target accuracy and off-target risks, is critically evaluated. Additionally, recent preclinical and clinical efforts to treat diseases such as cancer and sickle cell disease using CRISPR are summarized. Finally, the challenges and future directions of CRISPR-mediated gene therapy are discussed, emphasizing its potential to integrate with other molecular approaches to address unmet medical needs.

Multiple myeloma (MM) is an incurable hematological malignancy of plasma cells. Myeloma cells interfere with hematopoietic activities of the bone marrow, often leading to anemia, and can cause the bones to develop osteoporotic and lytic lesions. Clinical experience with chimeric antigen receptor T-cell (CAR-T) therapy targeting B-cell maturation antigen (BCMA) has been promising, with good response rates, favorable safety profiles, and low incidences of severe cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. However, CAR-T therapy in MM is accompanied by several new challenges, including therapeutic failure and relapse, and much attention has been paid to the further development of B-cell maturation antigen-chimeric antigen receptor (BCMA-CAR). Although most of the reported benefits of BCMA-CAR have been discussed, whether cancer can be eliminated, as well as the efficacy of CAR-T therapy for anemia and bone lesions, both myeloma-defining events, have not yet been reported in any animal model. In this study, we designed and verified a novel BCMA-specific chimeric antigen receptor (CAR). Our BCMA-CAR demonstrated the fundamental properties of CAR-T cells, including target-specific cytotoxic activity, cytokine production, and in vivo antitumor effects. In addition, we evaluated the therapeutic effect of BCMA-CAR in mice by imaging bone lesions and conducting blood examinations. Tumor mouse models showed systemic progression of MM in the bone marrow, and mice treated with saline or nongene modified T cells showed continued tumor progression, progressive bone lesions, and prolonged anemia. In contrast, all mice treated with gene modified T cells achieved a complete response, improved anemia to the level observed in normal mice, and suppressed progression of bone lesions. We concluded that anemia was improved with BCMA-CAR-T cell therapy. However, novel strategies to support the recovery of bone lesions by enhancing CAR-T cell function must be developed.