Sensorineural hearing loss (SNHL) is the most common sensory deficit globally. Acquired SNHL results from ototoxic damage to cochlear hair cells (HCs) and is typically irreversible due to their limited regenerative capac...Sensorineural hearing loss (SNHL) is the most common sensory deficit globally. Acquired SNHL results from ototoxic damage to cochlear hair cells (HCs) and is typically irreversible due to their limited regenerative capacity. While no cure currently exists, targeting the underlying pathology offers potential. Preclinical studies have investigated transcription factors like ATOH1, which can induce non-sensory cells to transdifferentiate into HCs. Gene therapy using viral vectors to deliver is emerging as a promising regenerative approach. PubMed, Web of Science, and Embase were systematically searched. The review was conducted following the Systematic Review Center for Laboratory Animal Experimentation guidelines. Random-effects meta-analysis was conducted using R's "meta" and "metafor" packages. To corroborate our findings, differential gene expression (DEG) analysis was performed on the GEO dataset GSE127683 using DESeq2. K-means clustering and gene set enrichment analysis (GSEA) were conducted using iDEP 2.0 and Enrichr, respectively. Four studies including 52 rodents were included. gene therapy significantly reduced Auditory Brainstem Response thresholds ( dB SPL, CI: [-40.19; -2.54], = 0.027), indicating improved hearing. DEG analysis showed upregulation of genes crucial for hair cell differentiation and functioning, including GFI1, PTPRQ, OTOF, USH2A, and POU4F3. GSEA highlighted key upregulated pathways related to inner ear development, auditory receptor cell differentiation and sensory perception of sound. gene therapy shows promise for treating acquired SNHL. However, further clinical trials are essential to confirm these preclinical findings and advance towards a potential cure.
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...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.
Chronic kidney disease (CKD) is a major global health problem characterized by renal fibrosis, for which effective therapeutic options are still lacking. Mesenchymal stem cells (MSCs) have emerged as potential candidates...Chronic kidney disease (CKD) is a major global health problem characterized by renal fibrosis, for which effective therapeutic options are still lacking. Mesenchymal stem cells (MSCs) have emerged as potential candidates for treating fibrosis due to their paracrine effects. This study first compared the antifibrotic capacities of umbilical cord-derived MSCs (UCMSCs) and dental pulp stem cells (DPSCs). The results showed that DPSCs exhibited superior effects in suppressing fibrosis markers and improving the fibrotic microenvironment. Thus, subsequent studies focused on DPSC and their hepatocyte growth factor (HGF)-modified counterpart (HGF-DPSC). Using an unilateral ureteral obstruction (UUO) mouse model and an Transforming Growth Factor-Beta 1(TGF-β1)-induced Human Renal Proximal Tubule Epithelial Cell (HK-2 cell) model, this study systematically evaluated the promising antifibrotic effects and mechanisms of DPSC. The results demonstrated that HGF-DPSC significantly improved the fibrotic microenvironment by regulating the Phosphoinositide 3-Kinase/Protein Kinase B/Glycogen Synthase Kinase 3 Beta (PI3K/AKT/GSK3β) signaling pathway and suppressing β-catenin activation. We confirmed direct protein-protein interaction between HGF and Iodothyronine Deiodinase 2 (DIO2) through co-immunoprecipitation (Co-IP), which suggested a novel molecular mechanism by which HGF-DPSC exerts its antifibrotic effects. These findings highlight the multitarget mechanism of HGF-DPSC in the treatment of renal fibrosis and provide new insights and possibilities for the treatment of CKD.
The low-density lipoprotein receptor (LDLR) plays a crucial role in cholesterol regulation and lipoprotein transport. Variations in the gene can cause familial hypercholesterolemia (FH), with homozygous familial hyperch...The low-density lipoprotein receptor (LDLR) plays a crucial role in cholesterol regulation and lipoprotein transport. Variations in the 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 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 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, -KO, and homozygous 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- gene therapy were assessed through Western blot analysis, lipid profiling, and liver pathology. AAV8-mediated delivery effectively improved lipid levels in both -KO and homozygous 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- 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.
Duchenne muscular dystrophy (DMD) is a severe, progressive genetic disorder primarily affecting boys, characterized by muscle degeneration due to mutations in the DMD gene encoding dystrophin, a crucial protein for muscl...Duchenne muscular dystrophy (DMD) is a severe, progressive genetic disorder primarily affecting boys, characterized by muscle degeneration due to mutations in the DMD gene encoding dystrophin, a crucial protein for muscle fiber integrity. The disease leads to significant muscle weakness and eventually to loss of ambulation. Adeno-associated viral (AAV)-microdystrophin (MD) gene therapy shows promise in preclinical and clinical settings. However, muscle fibrosis, a consequence of chronic inflammation and extracellular matrix remodeling, exacerbates disease progression and may hinder therapeutic efficacy. Periostin, a matricellular protein involved in fibrosis, is upregulated in DMD rodent models and correlates with collagen deposition. We previously developed an antisense oligonucleotide strategy to induce exon 17 skipping and so reduce periostin expression and collagen accumulation in the fibrotic D2. mouse model of DMD. Here, we investigated the combined effects of periostin modulation and AAV-MD1 treatment. We found that systemic periostin splicing modulation significantly improved muscle function, assessed by forelimb grip strength and treadmill performance. Importantly, periostin exon skipping increased the MD protein expression. These findings suggest that targeting periostin in conjunction with MD therapy could represent a valid therapeutic strategy for DMD.
López-Manzaneda S, Mencía Á, Bonafont J
… +13 more, Bassons-Bascuñana A, García M, Nyström A, Duarte B, Llames S, Murillas R, Modamio-Hoybjor S, Morín M, Soletto L, Escamez MJ, Moreno-Pelayo MA, Rio MD, Larcher F
Mutations leading to premature termination codons in are commonly associated with severe generalized recessive dystrophic epidermolysis bullosa (RDEB). Previous research, including our own, has indicated that removing m...Mutations leading to premature termination codons in are commonly associated with severe generalized recessive dystrophic epidermolysis bullosa (RDEB). Previous research, including our own, has indicated that removing mutated exons along with the consequent reframing of may not pose noticeable impact on protein function, offering a potential therapeutic strategy. However, investigations into the long-term effects of genome editing-mediated removal of mutant exons have only focused on the small exon 80 thus far. Hence, this study focuses on exons 73 and 105 of to explore whether targeted exon removal, through a CRISPR/Cas9-assisted, Non-homologous end joining (NHEJ)-mediated approach, could be extended to other larger exons. Introducing ribonucleoprotein complexes carrying Cas9 and optimized sgRNA guide pairs for each exon (73 and 105) through electroporation efficiently led to their removal, consequently restoring type VII collagen (C7) synthesis in RDEB primary patient cells carrying frameshift mutations in these exons. tests indicated the normal stability of the resulting C7 variants expressed at physiological levels, while analyses of regenerated skin grafted onto immunodeficient mice using E73 or E105 RDEB edited cells demonstrated the proper deposition of C7 at the basement membrane zone, thereby restoring normal dermo-epidermal adherence. This study enhances the broader potential of the exon deletion approach in the treatment of RDEB.
Recombinant adeno-associated virus (rAAV) has emerged as a leading vehicle for human gene therapy. An accurate and precise infectious titer assay is critical for assessing rAAV quality, potency, and product stability. Th...Recombinant adeno-associated virus (rAAV) has emerged as a leading vehicle for human gene therapy. An accurate and precise infectious titer assay is critical for assessing rAAV quality, potency, and product stability. The current gold standard for measuring rAAV infectivity is the median tissue culture infectivity dose (TCID50) method, which is laborious and highly variable. In the past several years, the droplet digital PCR (ddPCR) technology has made profound impacts on gene therapy analytics as it provides absolute DNA copy quantitation and is more accurate and precise than qPCR. In this article, we leveraged the ddPCR technology and developed a method to quantify rAAV cellular uptake . The results demonstrated that our method is consistent with TCID50 but is significantly more precise. Utilizing a stable AAV receptor (AAVR) cell line, this method can be implemented as a platform approach for various AAV serotypes and target genes. Moreover, the method is stability indicating, as desired for a potency assay. In conclusion, a novel rAAV uptake assay has been developed which reflects the mechanism of action of rAAV, and is accurate, precise and sensitive to product quality; thus overcoming many of the challenges of the traditional TCID50 method. It is particularly useful for initial rAAV product quality assessment and can contribute to a robust assay matrix with other product-specific potency assays for late-stage programs.
-gene therapy for profound deafness in children has entered clinical trials. Given that there is an approved alternative therapy with cochlear implants, it is imperative to scrutinize the risks, while also highlighting t...-gene therapy for profound deafness in children has entered clinical trials. Given that there is an approved alternative therapy with cochlear implants, it is imperative to scrutinize the risks, while also highlighting the novel benefits, of this experimental gene therapy. Since the untreated inner ear subsequently degenerates in this form of inherited deafness, the -gene therapy will be most effective in young children. Moreover, the best outcome in terms of hearing and speech comprehension is expected when the gene therapy is applied before the age of 3 years. Given such "earlier the better" considerations, the optimal time for these clinical trials and this particular therapy is at an age when children are too young to give informed consent. Enrolling children, which are a vulnerable category of persons, in clinical trials where the balance of benefits and risks is uncertain, raises a series of ethical considerations. In this article, we outline how this research can be pursued in an ethically responsible manner.
Corneal neovascularization (CoNV) is both a sight-threatening condition in and of itself and a major risk factor associated with corneal graft failure. Here, we determine the effectiveness of an adeno-associated viral ve...Corneal neovascularization (CoNV) is both a sight-threatening condition in and of itself and a major risk factor associated with corneal graft failure. Here, we determine the effectiveness of an adeno-associated viral vector (AAV)-based gene therapy targeting both hematic and lymphatic neovascularization in a murine model of severe CoNV. We first assessed the profile of transgene expression mediated by intrastromal injection of AAV2/8[Y733F] via longitudinal visualization of an enhanced Green Fluorescent Protein (eGFP) transgene and found that this serotype mediates a temporary (∼18 day) transduction of the corneal epithelium and sustained (≥148 day) transduction within the stroma. Constitutively expressed or were prophylactically delivered via intrastromal injection of AAV2/8[Y733F] vector at various intervals prior to aggressive induction of CoNV in a murine model. The extent of CoNV induced was quantified by fluorescein angiography and immunohistochemistry 17 days after induction. AAV2/8[Y733F]-CMV-sFlt1 was highly effective in the prevention of hemangiogenesis (HA) induced at 3, 28, and 210 days after intrastromal injection, but ineffective in the prevention of lymphangiogenesis. Two variants of AAV2/8[Y733F]-CMV-sFlt4 were ineffective in the prevention of angiogenesis when delivered alone, but combined delivery of AAV2/8[Y733F]-CMV-sFlt1 and AAV2/8[Y733F]-CMV-sFlt4 suggested a synergistic effect. Our results show that a single intrastromal injection of AAV2/8[Y733F]-CMV-sFlt1 is sufficient to protect against a robust stimulus for corneal HA over the long term. This technique could also be applied to reduce the risk of failure in cases of "high-risk" corneal transplantation.
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,...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.
Recombinant adeno-associated virus (rAAV) has emerged as one of the most important gene delivery vectors in the field of gene therapy due to its unique advantages and characteristics. The empty and full ratio is a critic...Recombinant adeno-associated virus (rAAV) has emerged as one of the most important gene delivery vectors in the field of gene therapy due to its unique advantages and characteristics. The empty and full ratio is a critical quality attribute in the quality control (QC) of rAAV, and its accurate evaluation is crucial for ensuring the safety, effectiveness, and consistency of gene therapy products. Analytical ultracentrifugation (AUC) technology, with its high resolution and accuracy, is widely recognized by the industry as the gold standard for identifying the empty and full ratio of rAAV. However, the conventional sedimentation velocity analytical ultracentrifugation (SV-AUC) method has limited throughput, failing to meet the large-scale detection needs of rAAV in process development and QC. This study aims to develop a single-sector higher throughput SV-AUC method without the need for a reference sector for blank control in order to improve the throughput of detecting the empty and full ratio of rAAV vectors. We optimized the traditional double-sector SV-AUC method, which requires a reference sector for blank control in the cell. By converting the light intensity data of AUC into pseudo-absorbance data, we significantly improve the analytical throughput. By tracking the variation of light intensity data with radius, we could clearly observe the sedimentation process of the rAAV sample. Despite a difference in the absolute value of pseudo-absorbance, the accurately fitted relative absorbance value and the traditional SV-AUC absorbance value with blank control were comparable, further verifying the applicability of this upgraded rAAV analytical method. The detailed comparison and verification between the upgraded method and the traditional SV-AUC method showed that the consistency and repeatability of the percentage and sedimentation coefficient were excellent both within the same cell and across different cells. The analysis results of samples from seven independent cells with a total of 14 sectors showed that the overall data exhibited good repeatability. The consistency of the high percentage empty capsid (HE) samples repeatability results was good, and the overlay of the C(s) distribution diagram also showed good pattern consistency. The relative standard deviation of the average percentage of empty, partial, and full capsids was maintained within 5%. The upgraded method demonstrated excellent consistency and repeatability in the analysis of rAAV samples with different empty and full ratios, aligning closely with the data obtained with the traditional SV-AUC method, the gold standard. Linear correlation analysis between the titers of HE samples and the overall absorbance (A value) of AUC, as well as the absorbance of empty, partial, and full capsids, revealed a good linear relationship, further confirming the applicability and reliability of the upgraded AUC method for evaluating rAAV samples with different titers. We also preliminarily explored the robustness of this method and found that even in the presence of slight fluctuations in sample volume, the test results remained stable, effectively alleviating concerns about the impact of inaccurate sample volume on the results. By dropping ink to simulate window contamination or wear, it was found that although the peak shape of the C(s) distribution was affected, the ratio results were consistent with those of the traditional SV-AUC method, proving that the new method exhibits good anti-interference ability under varying testing conditions. We conducted a comparability study on rAAV samples containing different proportions of empty, partial, and full capsids. rAAV samples with different proportions of empty and full showed high consistency and repeatability in the results obtained from both methods. In summary, the single-sector higher throughput SV-AUC method without a reference sector for blank control proposed in this study not only improves the analysis efficiency of rAAV samples but also ensures the accuracy and precision of the results, providing a new reliable analysis tool with higher throughput for gene therapy. This technology is expected to accelerate the development and evaluation process of gene therapy products.
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 osteop...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 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.
Park SY, Feng Z, Choi SH
… +8 more, Zhang X, Tang Y, Gasser GN, Richart D, Yuan F, Qiu J, Engelhardt JF, Yan Z
Hum Gene Ther
· 2025 Aug · PMID 40359132
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Cystic fibrosis (CF) is caused by mutations in the () gene. While CRISPR-based editing approaches have shown proof-of-concept for functional rescue in primary airway basal cells, induced pluripotent stem cells, and org...Cystic fibrosis (CF) is caused by mutations in the () gene. While CRISPR-based editing approaches have shown proof-of-concept for functional rescue in primary airway basal cells, induced pluripotent stem cells, and organoid cultures derived from patients with CF, their efficacy remains suboptimal. Here, we developed the CuFi reporter system by integrating spCas9 and a non-fluorescent Y66S eGFP mutant into CuFi-8 cells, an immortalized human airway epithelial cell line derived from a patient with CF with homozygous F508del mutations. These cells retain the basal cell phenotype in proliferating cultures and can differentiate into polarized airway epithelium at an air-liquid interface (ALI), enabling both visualized detection of gene editing and electrophysiological assessment of functional restoration. Using this system, recombinant adeno-associated virus (rAAV)-mediated homology-directed repair (HDR) was evaluated in proliferating cultures. A correction rate of 13.5 ± 0.8% was achieved in a population where 82.3 ± 5.6% of cells were productively transduced by AAV.eGFP630g2-CMVmCh, an rAAV editing vector with an mCherry reporter. Dual-editing of F508del and Y66S was explored using AAV.HR-eGFP630-F508(g03) to deliver two templates and single guide RNAs. eGFP (Y66S-corrected) cells and eGFP (non-corrected) cells were sorted via fluorescence-activated cell sorting and differentiated at an ALI to assess the recovery of CFTR function. Despite a low F508 correction rate of 2.8%, ALI cultures derived from the eGFP population exhibited 25.2% of the CFTR-specific transepithelial Cl transport observed in CuFi-ALI cultures treated with CFTR modulators. Next-generation sequencing revealed frequent co-editing at both genomic loci, with sixfold higher F508 correction rate in the eGFP cells than eGFP cells. In both populations, non-homology end joining predominated over HDR. This reporter system provides a valuable platform for optimizing editing efficiencies in proliferating airway basal cells, particularly for development of strategies to enhance HDR through modulation of DNA repair pathways.
Tavora R, Zhang L, Tran MH
… +12 more, Li H, O'Hagan D, Pan A, Barrett L, Jablonski JA, Mediouni S, Lopez A, Comella Z, Bailey C, Choe H, Farzan M, Valente ST
Hum Gene Ther
· 2025 Jun · PMID 40356311
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Recombinant adeno-associated virus (rAAV) vectors are increasingly preferred for gene therapy due to their broad tropism, low immunogenicity, and sustained transgene expression. Nevertheless, in cases of adverse reactio...Recombinant adeno-associated virus (rAAV) vectors are increasingly preferred for 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 . 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...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.
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 microenvir...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 and interleukin 15 () 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 CD45CD3 T and CD8 T cells in immunocompetent mice. Moreover, Ad-CXCL9-IL15 treatment decreased Treg cells in tumor mass and increased CD44CD62L T cells in spleen. Indicating that Ad-CXCL9-IL15 modified the TME and augmented antitumor immune responses . 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.