ABSTRACT
Objective:To investigate the effects of off-target isocenter plans with different off-target distances on the plan quality and delivery accuracy of stereotactic body radiotherapy (SBRT) for lung cancer, aiming to provide a reference for the clinical plan design of SBRT for lung cancer.Methods:For 10 lung cancer patients treated with SBRT, isocenter reference plans were designed by setting the plan isocenters at the mass centers of tumors and 60 off-target isocenter plans by setting the isocenters at distances of 1, 3, 5, 8, and 10 cm from the mass centers of tumors. The dosimetric differences between the off-target isocenter plans and the reference plans. Subsequently was analyzed, under different positional errors (0-5 mm). The gamma pass rates (GPRs) of these plans were measured using the Octavius 4D high-resolution dose verification system, and 240 verifications of these plans were completed. The robustness of the delivery accuracy of the reference plans and off-target isocenter plans were analyzed under different positional errors.Results:The off-target isocenter plans yielded slightly worse dose gradient indices than the isocenter reference plans, but there was no statistically significant differences. With an increase in the off-target distance, the mean lung dose (MLD), V20 of normal lungs, as well as the Dmax of bronchi, showed slight upward trends. Compared with the isocenter reference plans, the MLD of the off-target isocenter plans increased by 0.8%, 0.8%, and 1.9% at off-target distances of 1, 3, and 10 cm, respectively, with statistically significant differences ( z = -2.34 to -1.99, P < 0.05), and the V20 of the off-target isocenter plans increased by 2.0%, 2.5%, and 3.7% at off-target distances of 1, 5, and 10 cm, respectively, with statistically significant differences ( z =-2.11 to -1.99, P < 0.05). In the case of a positional error of up to 5 mm, the GPRs of plans with off-target distances of 5 cm and above decreased by more than 1.0% on average and up to a maximum of 3.5%, showing statistically significant differences ( z = 2.13-2.75, P < 0.05). Conclusions:Compared to the reference plans, the off-target isocenter plans showed slightly lower dosimetric quality and less robust delivery accuracy under different positional errors. Therefore, it is necessary to avoid the plans and treatment with too large off-target distances (≥ 5 cm) as far as possible for SBRT of lung cancer.
ABSTRACT
Objective:The respiratory waveform of lung cancer patients based on 4D-CT respiratory gating was analyzed to evaluate the accuracy of gating during radiotherapy, and to explore the off-target in the 4D-CT respiratory gating radiotherapy.Methods:Clinical data of 18 patients with lung cancer admitted to Radiotherapy Department of Jiangsu Cancer Hospital were collected to obtain the respiratory waveform data during 4D-CT respiratory gating radiotherapy. The waveform in each treatment working cycle was compared with the waveform in 4D-CT scan to study whether there was a possibility of the off-target in the treatment of lung cancer patients.Results:There were 154 treatment sessions and 20,790 treatment breathing cycles in 18 patients, among which the threshold of gated opening beam miss amplitude (Δm-en) was greater than 0 in 95 treatment breathing cycles in 7 patients, accounting for 0.46% of all breathing cycles, and the threshold of gated closing beam miss amplitude (Δm-dis) was greater than 0 in 1419 treatment breathing cycles in 13 patients, accounting for 6.83% of all cycles. Among the 13 patients withΔm-dis greater than 0, actual tumor range of motion (R G) was greater than the sum of the value of target margin (M) and the value of plan tumor range of motion (R T) in 7 patients, R G was more than 1.5 times of M+R T in 7 patients, and there were also 7 patients in the phase of rapid rise and fall of respiratory curve. The correlation efficients between R G-M-R T and the percentage of beam on miss phase (T en%) and the percentage of beam closing off phase (T dis%) were 0.41 and 0.57, respectively. Conclusion:When R G is more than 1.5 times of M+R T value and the gating beam on phase contains the phases in the rapid rise and fall of the respiratory curve, the possibility of the off-target during radiotherapy is significantly increased.
ABSTRACT
Chimeric antigen receptor T cell (CAR-T) is a kind of adoptive cell immunotherapy, in which T cells are genetically modified to exert targeted killing effect on tumors. CAR-T cell therapy has shown remarkable antitumor efficacy for the treatment of tumors, especially for hematological malignancies, but is less effective in solid tumors. Single-target CAR-T is prone to off-target effect during application, and there is a risk of relapse or more refractory treatment. The development of double-target or multi-target CAR-T is expected to extend the antigen coverage of target cells, effectively avoids antigen escape and prevents tumor recurrence, and prolongs the survival time of patients. This article reviews the advances of multi-target chimeric antigen receptor T cell, and discusses the prospect of its development.
ABSTRACT
@#[Abstract] Objective: To investigate whether AP1903, a small-molecule chemical inducer, can terminate the cytotoxicity of CD19CAR-T cells over-expressing iCasp9 suicide gene in vivo and in vitro. Methods: CD19CAR-T cells over-expressing iCasp9 (iCasp9-CD19CAR-T) were constructed and co-incubated with AP1903. Then, the cell phenotype and apoptosis were detected by Flow cytometry, and the iCasp9/CID suicide gene system was verified on K562 and T cells, respectively. The cytotoxicity of iCasp9-CD19CAR-T cells was detected in vivo (survival rate of NCG mice bearing Raji cell transplanted xenograft) and in vitro (cell killing function was detected by Flow cytometry) under the administration of AP1903. Results: Compared with CD19CAR-T cells, iCasp9-CD19CAR-T cells showed in significant difference in proliferation, phenotype and cytotoxicity both in vitro and in vivo (all P>0.05). At 2 h after AP1903 administration, the apoptosis rates of K562 and T cells co-expressing iCasp9 and CD19CAR were (33.8±0.9)% and (27.95±0.35)%, respectively; and at 24 h after AP1903 administration, the apoptosis rates reached 100% in both cell lines. The in vitro cytotoxicity of iCasp9-CD19CAR-T cells induced by AP1903 was significantly lower than that without AP1903 treatment (P<0.01); the 60-day survival rate of mice bearing Raji cell transplanted tumor treated with AP1903-induced iCasp9-CD19CAR-T cells was also significantly lower than those treated with iCasp9-CD19CAR-T cells alone (P<0.01). Conclusion: AP1903 can effectively terminate the cytotoxicity of CD19CAR-T cells over-expressing iCasp9 suicide gene in vitro and in vivo.
ABSTRACT
@# Due to the long-lasting, scalable, multi-targeting characteristics of T-cell immunity, T-cell-based tumor immunotherapy is considered to be the most likely means of bringing about tumor healing in addition to surgery. Especially in recent years, accumulating clinical data have confirmed the safety and effectiveness of cell therapy represented by chimeric antigen receptor modified T (CAR-T) cell. Among these, CAR-T therapy targeting CD19 has become a model therapy for genetic modified T cell therapy research in most institutions because of its remarkable effects. However, both practice and theory have suggested that CAR-T therapy faces more complicated problems in the treatment of solid tumors. How to use CAR-T cells to treat solid tumors reasonably and effectively still requires constant exploration and understanding. Here, we briefly summarize the current status of clinical practice of CAR-T cell therapy and its treatment of solid tumors, propose problems that need to be solved, and discuss the future research directions, in order to provide reference and research ideas for the treatment of solid tumors by CAR-T cells.
ABSTRACT
CRISPR/Cas9 gene-editing system has been broadly used in various fields of bioscience and medicine in recent years. The system can be guided by RNA to specific DNA site thus achieving targeted gene editing.Off-target effect and editing efficiency remain to be two crucial challeges to the system. Currently, a number of researches have been focused on the optimization of the system by reducing off-target effects and increasing editing efficiency, which may enhance its safety and expand its application.
ABSTRACT
Viral infections cause at least 10%-15% of all human carcinomas. Over the last century, the elucidation of viral oncogenic roles in many cancer types has provided fundamental knowledge on carcinogenetic mechanisms and established a basis for the early intervention of virus-related cancers. Meanwhile, rapidly evolving genome-editing techniques targeting viral DNA/RNA have emerged as novel therapeutic strategies for treating virus-related carcinogenesis and have begun showing promising results. This review discusses the recent advances of genome-editing tools for treating tumorigenic viruses and their corresponding cancers, the challenges that must be overcome before clinically applying such genome-editing technologies, and more importantly, the potential solutions to these challenges.
Subject(s)
Humans , Antiviral Agents , Therapeutic Uses , CRISPR-Cas Systems , Carcinoma , Genetics , Therapeutics , Virology , Gene Editing , Genetic Predisposition to Disease , Genetic Therapy , Methods , Tumor Virus InfectionsABSTRACT
Objective To establish a method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9, and analyze its off-target effect. Methods The gene editing site for MAD2L1 gene was designed by CHOP?CHOP, and the Cas9?MAD2L1 vector was constructed based on the designed editing site. Cas9?MAD2L1 was then transfected into NIH/3T3 cells and screened with puromycin, followed by observing GFP expression using fluorescence microscopy. The genomic DNA from transfected cells was extracted and a partial fragment of MAD2L1 gene was amplified by PCR. T7E1 analy?sis and Sangger sequencing were used for gene editing and off?target analysis. Results After Cas9?MAD2L1 transfection and puromycin screening, a large number of GFP?expressing cells were observed under the fluorescence microscope. Combined the PCR result with TE71 analysis, the amplified 228 bp PCR products can be digested into 166 bp and 62 bp fragments. The se?quencing result showed that the second exon of MAD2L1 gene was successfully edited, and the off?target effect was undetected in our system. Conclusions The method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9 is successfully established, and off?target effect of MAD2L1 gene is not detected.
ABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPR) is an acquired immune system existing in archaea and bacteria with the long-term process of evolutionary.CRISPR/Cas9 gene editing system is a new type of gene editing technology developed based on the system.CRISPR/Cas9 is a more efficient method for gene targeting than the previous methods.It has been successfully applied for gene-modified of eukaryotes since 2012,but the reports about pathogenic microorgaisms are rarely.Here,the research progress in the structure,mechanism of CRISPR/Cas9 system and its applications on pathogenic microorgaisms is reviewed.
ABSTRACT
CRISPR-based genome editing has been widely implemented in various cell types. In-silico single guide RNA (sgRNA) design is a key step for successful gene editing using CRISPR system. Continuing efforts are made to refine in-silico sgRNA design with high on-target efficacy and reduced off-target effects. In this paper, we summarize the present sgRNA design tools, and show that efficient in-silico models can be built that integrate current heterogeneous genome-editing data to derive unbiased sgRNA design rules and identify key features for improving sgRNA design. Our review shows that systematic comparisons and evaluation of on-target and off-target effects of sgRNA will allow more precise genome editing and gene therapies using the CRISPR system.
ABSTRACT
Breakthroughs of genome-editing in recent years have paved the way to develop new therapeutic strategies. These genome-editing tools mainly include Zinc-finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases. However, off-target effects are still the major issue in genome editing, and limit the application in gene therapy. Here, we summarized the cause and compared different detection methods of off-targets.
ABSTRACT
To gain more insights into the rice base editor (rBE3 and rBE4), we evaluated the mutation efficiency, off-target and inheritance of OsSERK1(D428N) and pi-ta(S918F) genes modified with rBE endonucleases. We predicted and analyzed the putative off-target sites of the sgRNA designed for OsSERK1(D428N) and pi-ta(S918F) by PCR amplification and Sanger sequencing. Then we further characterized the inheritance and stability of targeted base mutations and T-DNA segregation in the progeny of the self-fertilized T0 plants. Analysis of the DNA sequencing data of T0 plants of OsSERK1(D428N) revealed no nucleotide change at any of the four potential off-target sites. For OsSERK1(D428N) and Os08g07774 carry the same sgRNA targeting sites, base substitution at both two loci were detected at a frequency of 41.67%. The targeted base mutations could be transmitted readily to T1 progeny. Furthermore, genetic segregation caused the loss of T-DNA at a frequency between 25.0% and 40.9% in the T1 transgenic plants of OsSERK1(D428N) and pi-ta(S918F). These results demonstrated that the rBE3 and rBE4 systems could mediate specifically targeted base editing in one- or multi-site, and the targeted base editing could be stably inherited to next generation.
ABSTRACT
RNA interference mediated by short interfering RNA is widely used to study functional genes and also being developed for therapeutic applications. However, recent study demonstrated that siRNA might activate innate immune system and induce huge production of inflammatory cytokines in mammals, and also randomly inhibit expression of undesired genes. Designing highly effective siRNAs or modifying the siRNA to retain or enhance the silence efficiency and meanwhile abolish the off-target effects associated with immunostimulation then become the key techniques in application of siRNAs as safe and effective therapeutic agents.