Human Gene Therapy

Journal Information
ISSN / EISSN: 10430342 / 15577422
Total articles ≅ 5,512

Latest articles in this journal

Karen Anthony, Pierpaolo Ala, Francesco Catapano, Jinhong Meng, Joana Domingos, Mark Perry, Valeria Ricotti, Kate Maresh, Lauren Phillips, Laurent Servais, et al.
Published: 1 December 2022
Abstract:
Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin, but many patients have rare revertant fibres that express dystrophin. The skeletal muscle pathology of DMD patients includes immune cell infiltration and inflammatory cascades. There are several strategies to restore dystrophin in skeletal muscles of patients, including exon skipping and gene therapy. There is some evidence that dystrophin restoration leads to a reduction in immune cells, but dystrophin epitopes expressed in revertant fibres or following genome editing, cell therapy or microdystrophin delivery after AAV gene therapy may elicit T cell production in patients. This may affect the efficacy of the therapeutic intervention, and potentially lead to serious adverse events. To confirm and extend previous studies, we performed annual Enzyme- Linked Immunospot interferon-gamma assays on peripheral blood mononuclear cells from 77 paediatric boys with DMD recruited into a natural history study, 69 of whom (89.6%) were treated with corticosteroids. T cell responses to dystrophin were quantified using a total of 368 peptides spanning the entire dystrophin protein, organized into nine peptide pools. Peptide mapping pools were used to further localize the immune response in one positive patient. Six (7.8%) patients had a T cell-mediated immune response to dystrophin at at least one timepoint. All patients that had a positive result had been treated with corticosteroids, either prednisolone or prednisone. Our results show that ~8% of DMD individuals in our cohort have a pre-existing T cell-mediated immune response to dystrophin despite steroid treatment. Although these responses are relatively low-level, this information should be considered as a useful immunological baseline before undertaking clinical trials and future DMD studies. We further highlight the importance for a robust, reproducible standard operating procedure for collecting, storing and shipping samples from multiple centres to minimise the number of inconclusive data.
Haozheng Wang, Ms. Yangmin Wang, Zhongtao Luo, Xinjian Lin, Ms. Meilin Liu, Fenglin Wu, Hongwei Shao, Wenfeng Zhang
Published: 1 December 2022
Abstract:
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based genome editing system exhibits marked potential for both gene editing and gene therapy, and its continuous improvement contributes to its great clinical potential. However, the largest hindrance to its application in clinical practice is the presence of off-target effects (OTEs). Thus, in addition to continuous optimization of the CRISPR system to reduce and eventually eliminate OTEs, further development of unbiased genome-wide detection of OTEs is key for its successful clinical application. This paper summarizes detection strategies for off-target effects of different CRISPR systems, in order to provide detailed guidance for the detection of OTEs in CRISPR-based genome editing.
Ms. Tong Wu, Chi Huang, Ms. Yiran Yao, Zhaolin Du, Zhijun Liu
Published: 23 November 2022
Abstract:
The treatment of malignant tumors has always been one of the challenges that have plagued researchers and clinicians. The ideal status in cancer treatment is to eliminate tumor cells while avoiding damage to normal tissues. Different approaches have been investigated to achieve such a goal, and suicide gene therapy has emerged as a novel mode of cancer treatment. This approach involves the delivery of genes encoding enzymes that activate non-toxic prodrugs into cytotoxic metabolites that cause the death of transfected cancer cells. Despite promising results obtained both in vitro and in vivo, this innovative approach has long been stalled in the clinic due to the lack of a suitable delivery system to introduce the suicide gene into cancer cells. Ultrasound-targeted microbubble destruction (UTMD) represents a valuable non-viral vector system for site-specific and noninvasive gene therapy. Ultrasound promotes intracellular uptake of therapeutic agents by increasing vascular and cell membrane permeability, especially in the presence of microbubbles. In this scenario, the true potential of suicide genes can be translated into clinically valuable treatments for patients. This review provides background information on suicide gene therapy and UTMD technology, summarizes the current state of knowledge about UTMD-mediated suicide gene delivery in cancer treatment, and presents an outlook on its future development.
Huiren Zhao, Hans Meisen, Songli Wang, Ki Jeong Lee
Published: 18 November 2022
Abstract:
Optimization of recombinant adeno-associated virus (rAAV) production has important clinical implications, as manufacturing is one of the major challenges for rAAV gene therapy. In this study, we optimized upstream and downstream processing of the rAAV production platform created by an earlier Design-of-Experiment approach. Our results showed that adding peptones, yeastolate, Trypton N1 or both increased production yield by 2.8-3.4 folds. For downstream processing, a variety of wash buffers using an affinity resin, POROSTM CaptureSelectTM (PCS)-AAVX, were tested for their effects on rAAV8 purity, including NaCl, MgCl2, arginine, Triton X-100, CHAPS, Tween 20, octyl β-D-1-thioglucopyranoside (OTG), and low pH. The results showed that the OTG wash significantly improved the rAAV purity to 97% and reduced endotoxins to an undetectable level (<0.5 EU/mL), while retaining the yield at 87% of the PBS wash. The OTG wash was successfully applied to purifications of rAAV1, rAAV2 and rAAV5 using PCS-AAVX and rAAV9 using PCS-AAV9. rAAV8 purified with OTG wash showed comparable transduction efficiency in HEK293T cells to the rAAV8 purified with PBS wash. The optimized rAAV production process yielded 5.5-6.0x1014 and 7.6x1014 vector genome per liter of HEK 293T cells for purified rAAV8- and rAAV5-EF1-EGFP, respectively. The platform described here is simple with high yields and purity, which will be beneficial to both research and clinical gene therapy.
Qian Xu, Feimei Zhu, Ms. Yixuan Pan, Ms. Yanlin Ren, Jingyu Li, Ning Huang, Keyun Liu, Yi Wang
Published: 14 November 2022
Abstract:
Histone H3 is a nucleosome scaffold protein that is involved in a variety of intracellular processes. Aberrant modification of H3 is important in carcinogenesis. In contrast, free histones in cells can act as stimuli to trigger cellular immune responses and cell death. In this study, we linked cell-penetrating peptide HIV Tat to a histone H3 fragment to achieve intracellular delivery in tumor cells. We found that Tat-conjugated histone polypeptides localized to nuclei of lung and breast cancer cells and caused cell death. A trans-configured Tat sequence displayed dramatically improved peptide half-life and cytotoxicity. Mechanistic studies demonstrated that treatment with the peptides significantly elevated MAPK signaling, ROS production, as well as levels of stress-inducible transcription factor ATF3 and AP-1. Cytotoxicity of the peptide was significantly reduced by inhibition of AP-1 activity and ROS production. These results suggest the potential of Tat-conjugated H3 peptides as antitumor agents to induce cell death via increased cellular stress response by activating p38-MAPK signaling and intracellular ROS production.
Sumit Verma, Stella N. Nwosu, Raj Razdan, Saila R. Upadhyayula, Han C. Phan, Abubakarr A. Koroma, Isai Leguizamo, Ms. Natalie S. Correa, Michael Kuipa, David Lee, et al.
Published: 2 November 2022
Abstract:
Adeno-associated virus (AAV) based gene therapies are emerging strategies in Duchenne muscular dystrophy (DMD) treatment. Exposure to wild-type AAV can lead to development of neutralizing antibodies (NAb) and block AAV transduction, thereby limiting the delivery of AAV vector-based gene therapy. Therefore, it is imperative to check for the presence of AAV NAbs in a patient who is a candidate for gene therapy. We prospectively enrolled 101 genetically confirmed males with DMD (median age 11 years, 48% ambulatory, 59% on steroids) and performed AAV neutralization assays against AAV2, AAV8, AAV9, and AAVrh74 serotypes. Serotype analysis showed AAV9 (36%) and AAVrh74 (32%) seroprevalence was lower compared to AAV2 (56%) and AAV8 (47%). Interestingly, age was not correlated with NAb titer for any of the capsids. NAb responses were observed at a higher frequency in African American participants and at a lower frequency for Caucasian participants for all four serotypes. Further analysis showed no significant differences in NAb titers depending, regardless of serotype, on whether participants were taking steroids. Finally, we observed higher AAV8, AAV9, and AAVrh74 seroprevalence and significantly higher AAV2 and AAV8 NAb titers in participants that were ambulatory compared to the non-ambulatory participants. Overall, these data identify AAV9 and AAVrh74 as the two serotypes with lower preexisting NAbs in this study’s cohort of 101 males with DMD, possibly showing their utility for future gene therapy applications to treat this cohort men with DMD.
Jonas Weinmann, Julia Söllner, Ms. Sarah Abele, Ms. Gudrun Zimmermann, Kai Zuckschwerdt, Ms. Christine Mayer, Ms. Jenny Danner-Liskus, , Michael Schuler, Thorsten Lamla, et al.
Published: 2 November 2022
Abstract:
Adeno-associated viruses (AAV) represent highly attractive gene therapy vectors and potent research tools for the modulation of gene expression in animal models or difficult-to-transfect cell cultures. Engineered variants, comprising chimeric, mutated or peptide-inserted capsids, have strongly broadened the utility of AAVs by altering cellular tropism, enabling immune evasion, or increasing transduction efficiency. In this work, the performance of 50 of the most used, predominantly published, AAVs was compared on several primary cells, cell lines and iPSC-derived models from different organs, including adipose tissue, liver, lung, brain, and eyes. To identify the most efficient capsids for each cell type, self-complementary AAVs were standardized by digital PCR, arrayed on 96-well plates, and screened using high-content imaging. To enable best use of the data, all results are also provided in a web app. The utility of one selected AAV variant is further exemplified in a liver fibrosis assay based on primary hepatic stellate cells, where it successfully reversed an siRNA-induced phenotype. Most importantly, our comparative analysis revealed that a sub-selection of only five AAV variants (AAV2.NN, AAV9-SLRSPPS, AAV6.2, AAV6TM and AAV1P5) enabled efficient transduction of all tested cell types and markedly outperformed other well-established capsids, such as AAV2-7m8. These findings suggest that a core panel comprising these five capsid variants is a universally applicable and sufficient tool to identify potent AAVs for gene expression modulation in cellular systems.
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