Vector System Research Articles

HPV16-miRNAs exert oncogenic effects through enhancers in human cervical cancer

BackgroundCervical cancer is a human papillomavirus (HPV)-related disease. HPV type 16 (HPV16), which is the predominant cause of cervical cancer, can encode miRNAs (HPV16-miRNAs). However, the role of HPV16-miRNAs in the pathogenesis of cervical cancer remains unclear.MethodsHuman cervical cancer cell lines SiHa (HPV16-positive) and C33A (HPV-negative), and cervical cancer tissues were collected to investigate the expression levels of two HPV16-miRNAs (HPV16-miR-H1 and HPV16-miR-H6). The overexpression and knockdown of HPV16-miR-H1 and HPV16-miR-H6 were performed using the lentiviral vector system and miRNA inhibitors, respectively. RNA-sequencing (RNA-seq) analysis and H3K27ac chromatin immunoprecipitation and sequencing (CHIP-seq) experiments were utilized to explore the roles of HPV16-miR-H1 and HPV16-miR-H6 facilitated by enhancers. CCK8, EdU, transwell, and wound healing assays were performed to verify the effects of HPV16-miR-H1 and HPV16-miR-H6 on cell proliferation and migration.ResultsHPV16-miR-H1 and HPV16-miR-H6 were highly expressed in both SiHa cells and tissue samples from HPV16-positive cervical cancer patients. RNA-seq analysis showed that HPV16-miR-H1 and HPV16-miR-H6 induced the upregulation of numerous tumor progression-associated genes. H3K27ac CHIP-seq experiments further revealed that HPV16-miR-H1 and HPV16-miR-H6 modulated the expression of critical genes by regulating their enhancer activity. The functional study demonstrated that HPV16-miR-H1 and HPV16-miR-H6 increased the migratory capacity of SiHa cells.ConclusionsOur data shed light on the role of HPV16-encoded miRNAs in cervical cancer, particularly emphasizing their involvement in the miRNA-enhancer-target gene system. This novel regulatory mechanism of HPV16-miRNAs provides new insights and approaches for the development of therapeutic strategies by targeting HPV16-positive cervical cancer.

A low-endotoxic Salmonella enterica Gallinarum serovar delivers infectious bronchitis virus immunogens via a dual-promoter vector system that drives protective immune responses through MHC class-I and -II activation in chickens

An effective vaccine strategy is indispensable against infectious bronchitis virus (IBV) and fowl typhoid (FT), both of which threaten the poultry industry. This study demonstrates a vector system, pJHL270, designed to express antigens in prokaryotic and eukaryotic cells. The vector system stimulates immune responses via synchronized antigen presentation to MHC class-I and -II molecules to produce balanced Th1/Th2 responses. The vaccine antigens were crafted by selecting the consensus sequence of the N-terminal domain of the spike protein (S1-NTD) and a conserved immunogenic region of the nucleocapsid protein (N321-406 aa) from IBV strains circulating in South Korea. The vaccine antigen was cloned and transformed into a live-attenuated Salmonella Gallinarum (SG) strain, JOL2854 (∆lon, ∆cpxR, ∆rfaL, ∆pagL, ∆asd). Western blot analysis confirmed concurrent antigen expression in Salmonella and eukaryotic cells. Oral immunization with the SG-based IBV vaccine construct JOL2918 induced IBV antigen and Salmonella-specific humoral and cell-mediated immune responses in chickens. PBMCs collected from immunized chickens revealed that MHC class-I and -II expression had increased 3.3-fold and 2.5-fold, respectively, confirming MHC activation via bilateral antigen expression and presentation. Immunization induced neutralizing antibodies (NAbs) and reduced the viral load by 2-fold and 2.5-fold in the trachea and lungs, respectively. The immunized chickens exhibited multifaceted humoral, mucosal, and cell-mediated responses via parallel MHC class-I and -II activation as proof of a balanced Th1/Th2 immune response. The level of NAbs, viral load, and gross and histological analyses provide clear evidence that the construct provides protection against IBV and FT.

User-Friendly Replication-Competent MAdV-1 Vector System with a Cloning Capacity of 3.3 Kilobases

Mouse adenoviruses (MAdV) play important roles in studying host–adenovirus interaction. However, easy-to-use reverse genetics systems are still lacking for MAdV. An infectious plasmid pKRMAV1 was constructed by ligating genomic DNA of wild-type MAdV-1 with a PCR product containing a plasmid backbone through Gibson assembly. A fragment was excised from pKRMAV1 by restriction digestion and used to generate intermediate plasmid pKMAV1-ER, which contained E3, fiber, E4, and E1 regions of MAdV-1. CMV promoter-controlled GFP expression cassette was inserted downstream of the pIX gene in pKMAV1-ER and then transferred to pKRMAV1 to generate adenoviral plasmid pKMAV1-IXCG. Replacement of transgene could be conveniently carried out between dual BstZ17I sites in pKMAV1-IXCG by restriction-assembly, and a series of adenoviral plasmids were generated. Recombinant viruses were rescued after transfecting linearized adenoviral plasmids to mouse NIH/3T3 cells. MAdV-1 viruses carrying GFP or firefly luciferase genes were characterized in gene transduction, plaque-forming, and replication in vitro or in vivo by observing the expression of reporter genes. The results indicated that replication-competent vectors presented relevant properties of wild-type MAdV-1 very well. By constructing viruses bearing exogenous fragments with increasing size, it was found that MAdV-1 could tolerate an insertion up to 3.3 kb. Collectively, a replication-competent MAdV-1 vector system was established, which simplified procedures for the change of transgene or modification of E1, fiber, E3, or E4 genes.

Development of pENTR-NeCo-lacZα vectors for the preparation of negative control constructs in Gateway cloning.

Gateway cloning is a useful technology for the simple and reliable preparation of various construct in many organisms. However, there is a problem regarding the negative control construct in the Gateway cloning system. In this study, we developed the pENTR-NeCo-lacZα vector system to create an empty vector that can be used as a negative control construct in Gateway cloning.

Codon Optimization-based Whole-gene Scanning Identifies Hidden Nucleotides Essential for Bombyx mori Nucleopolyhedrovirus polyhedrin Hyperexpression

During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host’s cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5ʹ proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5ʹ coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.

Abstract PO1-25-09: A new generation of breast cancer modeling has been achieved through somatic precision gene editing, offering high flexibility and efficiency

Abstract Breast cancer, along with many other cancers, is primarily caused by genetic mutations, typically involving the alteration of nucleotides (A, G, C, T) within genes (with a total of 12 possible mutations). Gene sequencing advancements have facilitated the identification of numerous gene mutations in tumor samples. However, comprehending the precise mechanisms through which these gene mutations contribute to tumor initiation, development, and their impact on tumor characteristics cannot be solely determined from these findings. Therefore, it is crucial to observe the influence of mutations on tumor initiation and development from the moment they occur. Since conducting such observations in human subjects is impractical, animal models with simulated gene mutations have become essential for studying tumor initiation and development. Mice, with a genome comprising approximately 2.7 billion nucleotides, are commonly used as animal models in cancer research due to their cost-effectiveness and high similarity to humans. About 90% of their genes share similar functions, particularly those associated with cancer development. However, achieving precise alterations to specific nucleotides among the 2.7 billion nucleotides within live mouse cells (with a total of 12 possible changes) has presented significant technical challenges. In this study, we present modifications to the CRISPR/Cas9 vector system, enabling homology-directed repair-mediated precise editing of any proto-oncogene in murine somatic mammary epithelial cells, thereby generating mammary tumor models with exceptional flexibility and efficiency. The resulting tumors exhibit significant advantages over traditional mouse models. This technological breakthrough bridges the gap between the potential of CRISPR technology and the accuracy of mouse models, facilitating the study of human tumor evolution and preclinical drug testing. Citation Format: Wen Bu, Yi li. A new generation of breast cancer modeling has been achieved through somatic precision gene editing, offering high flexibility and efficiency [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-09.

A hybrid dynamics modeling method for micro-turbine engine thrust vectoring

Thrust vector control technology is widely used in modern advanced aircrafts, space shuttles and unmanned aerial vehicles, etc. An accurate thrust vector mathematical model is of great significance to enhance the thrust vector control effect. In this paper, the P200 micro-turbine engine with an axisymmetric thrust vector nozzle placed at the rear is taken as an example, A hybrid thrust vectoring dynamics modeling method consisting of engine speed model, engine thrust model, actuator dynamics model, airflow angle model and thrust loss coefficients is proposed. The engine speed model is of Linear Parameter Varying (LPV) model with each segment is characterized by a First Oder Plus Dead Time (FOPDT) model. The engine thrust model is characterized as a cubic polynomial function of engine speed. The actuator’s dynamic model is a FOPDT model. The nozzle airflow deflection angle is proportional to the airflow deflection angle. the thrust loss coefficient is approximated by a cosine relationship with the airflow deflection angle. The experimental results show that the established model can well characterize the dynamics of the thrust vector system.

A novel lentiviral vector-based approach to generate CAR T cells targeting Aspergillus fumigatus

Abstract Invasive aspergillosis (IA) is a common and deadly mold infection in immunocompromised patients. As morbidity and mortality of IA are primarily driven by poor immune defense, adjunct immunotherapies, such as chimeric antigen receptor (CAR) T cells, are direly needed. Here, we propose a novel approach to generate Aspergillus fumigatus (AF)-CAR T cells using the scFv domain of AF-269-5 mAb and a lentiviral vector system. These cells successfully targeted mature hyphal filaments of representative clinical and reference AF isolates and elicited potent release of cytotoxic effectors and type 1 T-cell cytokines. Furthermore, AF-CAR T cells generated from peripheral blood mononuclear cells of four healthy human donors and expanded with cytokine stimulation (IL-2, IL-7+IL-15) regimens significantly suppressed mycelial growth of AF-293 after 18 h of co-culture. Moreover, cyclophosphamide-immunosuppressed NSG mice with invasive pulmonary aspergillosis that received two doses of 5 million AF-CAR T cells on days 0 and 2 after AF infection showed significantly reduced morbidity on day 4 post-infection (p<0.001) and significantly improved 7-day survival (p=0.049) compared to mice receiving non-targeting control T cells. In conclusion, we developed a novel lentiviral strategy to obtain AF-CAR T cells with high targeting efficacy, yielding significant anti-AF activity in vitro and short-term protection in vivo.

Expression and Purification of Recombinant Bowman-Birk Trypsin Inhibitor from Foxtail Millet Bran and Its Anticolorectal Cancer Effect In Vitro and In Vivo.

Trypsin inhibitors derived from plants have various pharmacological activities and promising clinical applications. In our previous study, a Bowman-Birk-type major trypsin inhibitor from foxtail millet bran (FMB-BBTI) was extracted with antiatherosclerotic activity. Currently, we found that FMB-BBTI possesses a prominent anticolorectal cancer (anti-CRC) activity. Further, a recombinant FMB-BBTI (rFMB-BBTI) was successfully expressed in a soluble manner in host strain Escherichia coli. BL21 (DE3) was induced by isopropyl-β-d-thiogalactoside (0.1 mM) at 37 °C for 3.5 h by the pET28a vector system. Fortunately, a purity greater than 93% of rFMB-BBTI with anti-CRC activity was purified by nickel-nitrilotriacetic acid affinity chromatography. Subsequently, we found that rFMB-BBTI displays a strikingly anti-CRC effect, characterized by the inhibition of cell proliferation and clone formation ability, cell cycle arrest at the G2/M phase, and induction of cell apoptosis. It is interesting that the rFMB-BBTI treatment had no obvious effect on normal colorectal cells in the same concentration range. Importantly, the anti-CRC activity of rFMB-BBTI was further confirmed in the xenografted nude mice model. Taken together, our study highlights the anti-CRC activity of rFMB-BBTI in vitro and in vivo, uncovering the clinical potential of rFMB-BBTI as a targeted agent for CRC in the future.

Gn protein expressed in plants for diagnosis of severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome virus (SFTSV) causes the highly fatal disease in humans. To facilitate diagnosis, the native form of subunit glycoprotein (Gn), a prime target for potential vaccines and therapies, was produced in Nicotiana benthamiana using a Bamboo mosaic virus-based vector system. By fusion with secretory signal tags, SSExt, derived from the extension protein, and the (SP)10 motif, the yield of the recombinant Gn (rGn) was remarkably increased to approximately 7 mg/kg infiltrated leaves. Ultimately, an rGn-based ELISA was successfully established for the detection of SFTSV-specific antibodies in serum samples from naturally infected monkeys. As validated with the reference method, the specificity and sensitivity of rGn-ELISA were 94% and 96%, respectively. In conclusion, utilizing well-suited fusion tags facilitates rGn production and purification in substantial quantities while preserving its antigenic properties. The rGn-ELISA, characterized by its commendable sensitivity and specificity could serve as a viable alternative diagnostic method for assessing SFTSV seroprevalence.Key points• SFTSV Gn, fused with secretory signal tags, was expressed by the BaMV-based vector.• The plant fusion tags increased expression levels and eased the purification of rGn.• The rGn-ELISA was established and validated; its specificity and sensitivity > 94%.

Establishment and Characterization of SV40 T-Antigen Immortalized Porcine Muscle Satellite Cell.

Muscle satellite cells (MuSCs) are crucial for muscle development and regeneration. The primary pig MuSCs (pMuSCs) is an ideal in vitro cell model for studying the pig's muscle development and differentiation. However, the long-term in vitro culture of pMuSCs results in the gradual loss of their stemness, thereby limiting their application. To address this conundrum and maintain the normal function of pMuSCs during in vitro passaging, we generated an immortalized pMuSCs (SV40 T-pMuSCs) by stably expressing SV40 T-antigen (SV40 T) using a lentiviral-based vector system. The SV40 T-pMuSCs can be stably sub-cultured for over 40 generations in vitro. An evaluation of SV40 T-pMuSCs was conducted through immunofluorescence staining, quantitative real-time PCR, EdU assay, and SA-β-gal activity. Their proliferation capacity was similar to that of primary pMuSCs at passage 1, and while their differentiation potential was slightly decreased. SiRNA-mediated interference of SV40 T-antigen expression restored the differentiation capability of SV40 T-pMuSCs. Taken together, our results provide a valuable tool for studying pig skeletal muscle development and differentiation.

Comparative performance of scFv-based anti-BCMA CAR formats for improved T cell therapy in multiple myeloma

In multiple myeloma (MM), B cell maturation antigen (BCMA)-directed CAR T cells have emerged as a novel therapy with potential for long-term disease control. Anti-BCMA CAR T cells with a CD8-based transmembrane (TM) and CD137 (41BB) as intracellular costimulatory domain are in routine clinical use. As the CAR construct architecture can differentially impact performance and efficacy, the optimal construction of a BCMA-targeting CAR remains to be elucidated. Here, we hypothesized that varying the constituents of the CAR structure known to impact performance could shed light on how to improve established anti-BCMA CAR constructs. CD8TM.41BBIC-based anti-BCMA CAR vectors with either a long linker or a short linker between the light and heavy scFv chain, CD28TM.41BBIC-based and CD28TM.CD28IC-based anti-BCMA CAR vector systems were used in primary human T cells. MM cell lines were used as target cells. The short linker anti-BCMA CAR demonstrated higher cytokine production, whereas in vitro cytotoxicity, T cell differentiation upon activation and proliferation were superior for the CD28TM.CD28IC-based CAR. While CD28TM.CD28IC-based CAR T cells killed MM cells faster, the persistence of 41BBIC-based constructs was superior in vivo. While CD28 and 41BB costimulation come with different in vitro and in vivo advantages, this did not translate into a superior outcome for either tested model. In conclusion, this study showcases the need to study the influence of different CAR architectures based on an identical scFv individually. It indicates that current scFv-based anti-BCMA CAR with clinical utility may already be at their functional optimum regarding the known structural variations of the scFv linker.

Identification of a novel RNA transcript TISPL upregulated by stressors that stimulate ATF4

Cells sense, respond, and adapt to environmental conditions that cause stress. In a previous study using HeLa cells, we isolated reporter cells responding to the endoplasmic reticulum (ER) stress inducers, thapsigargin and tunicamycin, using a highly sensitive promoter trap vector system. Splinkerette PCR and 5′ rapid amplification of cDNA ends (5′ RACE) identified a novel transcript that is upregulated by ER stress. Its endogenous expression increased approximately 10-fold in response to thapsigargin and tunicamycin within 1 h, but was down-regulated after 4 h. Because the transcript starts from an intron of a long noncoding RNA known as LINC-PINT, we designated the newly identified transcript TISPL (transcript induced by stressors from LINC-PINTlocus). TISPL was also expressed under several other stress conditions. It was particularly increased > 10-fold upon glucose starvation and 7-fold by arsenite exposure. Furthermore, in silico analyses, including a ChIP-atlas search, revealed that there is an ATF4-binding region with a c/ebp-Atf response element (CARE) downstream of the transcription start site of TISPL. Based on these results, we hypothesized that TISPL may be induced by the phospho-eIF2α and ATF4- axis of the integrated stress response pathway, which is known to be activated by the stress conditions listed above. As expected, knockout of ATF4 abolished the stress-induced upregulation of TISPL. Our results indicate that TISPL may be a useful biomarker for detecting stress conditions that activate ATF4. Our highly sensitive trap vector system proved beneficial in discovering new biomarkers.

Complexity Analysis of Chinese Text Based on the Construction Grammar Theory and Deep Learning

Due to the complexity of Chinese and the differences between Chinese and English, the application of Chinese text in the digital field has a certain complexity. Taking Chinese text in Open Relation Extraction (ORE) as the research object, the complexity of Chinese text is analyzed. An extraction system of word vectors based on construction grammar theory and Deep Learning (DL) is constructed to achieve smooth extraction of Chinese text. The work of this paper mainly includes the following aspects. To study the application of DL in the complexity analysis of Chinese text based on construction grammar, firstly, the connotation of construction grammar and its role in Chinese text analysis are explored. Secondly, from the perspective of the ORE of word vectors in language analysis, an ORE model based on word vectors is implemented. Moreover, an extraction method based on the distance of word vectors is proposed. The test results show that the F1 value of the proposed algorithm is 67% on the public WEB-500 and NYT-500 datasets, which is superior to other similar text extraction algorithms. When the recall rate is more than 30%, the accuracy of the proposed method is higher than several other latest language analysis systems. This indicates that the proposed Chinese text extraction system based on the DL algorithm and construction grammar theory has advantages in complexity analysis and can provide a new research idea for Chinese text analysis.

Design of event-triggered natural second-order observers for second-order vector systems

The problem of designing event-triggered natural second-order state observers for second-order vector systems subject to external disturbances is considered in this paper. In this work, state observer is constructed using discrete measurements at instants when an event-triggered condition is satisfied. Due to the errors that arise by using the event-triggered mechanism and the external disturbance, the method in this paper only robustly estimates the state position and the state velocity of the second-order vector observers instead of estimating them asymptotically. Instead of using the method based on a parameter-dependent Lyapunov function in investigating the stability of the dynamic error system of natural second-order observers, a convex optimization problem is established in this paper to give an event-triggered mechanism and minimized levels in evaluating the boundedness of the dynamic error system of the event-triggered natural second-order observers. An automobile suspension and two numerical examples demonstrate the effectiveness of the proposed method.

Adeno-associated viruses in gene therapy for spinal muscular atrophies: trend or triumph?

The development of gene therapy in the 21st century is largely based on application of viral vectors, which have shown their effectiveness along with a fairly high safety profile. Among the vector systems, one of the leading places was taken by adeno-associated viruses (AAV), on the basis of which drugs were created for the treatment of severe hereditary monogenic diseases, including spinal muscular atrophies (SMA). Their use, on the one hand, is justified by the flexibility of AAV as a platform for the creation of gene therapy drugs, and on the other hand, it is often perceived as a kind of trend that has significant limitations. In this review, the focus is on two main aspects: AAV as a vector for the treatment of diseases from the SMA group and possible directions of development in this area and in gene therapy in general.
 The review operates with recent data published after clinical trials and experimental studies during last decade, and also critically examines the possibilities of gene therapy using AAV, mentioning other existing approaches, incl. medical therapy for SMA.
 Attention is also paid to the situation in the field of using AAV for the treatment of other hereditary diseases and the most acute problems faced by the use of drugs created on the basis of this promising vector system.

A SAT Solver and Computer Algebra Attack on the Minimum Kochen-Specker Problem (Student Abstract)

The problem of finding the minimum three-dimensional Kochen–Specker (KS) vector system, an important problem in quantum foundations, has remained open for over 55 years. We present a new method to address this problem based on a combination of a Boolean satisfiability (SAT) solver and a computer algebra system (CAS). Our approach improved the lower bound on the size of a KS system from 22 to 24. More importantly, we provide the first computer-verifiable proof certificate of a lower bound to the KS problem with a proof size of 41.6 TiB for order 23. The efficiency is due to the powerful combination of SAT solvers and CAS-based orderly generation.

Abstract 5736: Sonochemical preparation of SP94 peptide-decorated pH-responsive biodegradable biopolyester nanoparticles for targeted therapy in hepatocellular carcinoma

Abstract Stimulation in response to nanoparticles responding to endogenous stimuli in the tumor microenvironment has been extensively studied for the release of chemotherapeutic drug. Therefore, we aimed to develop a biocompatible delivery of taxol (TAX) delivery vector system for the treatment of hepatocellular carcinoma (HCC). A basic backbone, TAX loaded poly (3-hydroxybutyrate-co-3-hydroxyvalerate) PHBV nanoparticle (PHBV-TAX-NPs), were designed and prepared via sonochemical method. The pH-responsive shell of PHBV-TAX-NPs was formed by oxidative self-polymerization of dopamine (PDA) in an alkaline environment. The HCC-targeted peptide SP94 binds to PDA-coated nanoparticles (NPs) through the Michael reaction. Moreover, the nanoparticle surface-grafted SP94 peptide serve as a specific ligand for targeting HCC. The chemical structures and properties of these nanoparticles were characterized by transmission electron microscope, dynamic light scattering-autosizer, differential scanning calorimetry, fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and thermogravimetry. As expected, excellent targeting of these SP94-PDA-PHBV-TAX-NPs was demonstrated by cellular uptake and cytotoxicity experiments in HCC cells. Compared with that of free TAX, the SP94-PDA-PHBV-TAX-NPs showed more superior antitumor efficacy in the xenograft mouse model. Notably, the SP94-PDA-PHBV-TAX-NPs were except for their potent anticancer activity, it was less toxic in vitro and in vivo compared to TAX in mice. These studies suggest the potential beneficial use of SP94-PDA-PHBV-TAX-NPs in future HCC-targeted therapy with drug-loaded nanocarriers. Citation Format: Mingfang Wu, Qi Wang, Zhengran Gong, Zixuan Liu, Haodong Zheng, Xinjie Chen, Chen Zhong. Sonochemical preparation of SP94 peptide-decorated pH-responsive biodegradable biopolyester nanoparticles for targeted therapy in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5736.

Abstract 1094: Utilizing CRISPR-Cas13 as a novel functional genomics tool to characterize PAX3-FOXO1 in alveolar rhabdomyosarcoma

Abstract Background: CRISPR-Cas13 (Cas13) is an RNA editing system that can knockdown gene transcripts with higher efficiency and specificity than RNA interference technologies. We are exploring the utility of Cas13 as a robust novel functional genomics tool to understand the biology of the PAX3-FOXO1(P3F1) gene fusion in alveolar rhabdomyosarcoma (ARMS). Our goal is to identify gene dependencies and novel druggable targets. [LB1] Aim: This study aims to functionally characterize P3F1 in paediatric cancer cell lines using Cas13. [LB2] Methods & Results: Transient transfection and viral transduction of Cas13 vector systems were used to demonstrate that Cas13 knockdown of target gene expression can be achieved in ARMS cell lines. We developed guides to specifically target P3F1 expression and tested their efficiency in HEK293T cells. Cas13 function was validated with a mCherry knockdown proof-of-concept expression in the ARMS cell lines Rh5 and Rh30. 90-95% knockdown efficiency was seen over three independent experiments. Ten P3F1 breakpoint targeting guides were designed and screened first in a HEK293T P3F1 overexpression model to select optimal high efficiency targeting guides. The highest efficiency targeting guide showed a GFP reduction of 70% in three independent experiments. High-efficiency Cas13 guides were taken forward into Rh5 cells to knockdown endogenous P3F1 expression which were validated by western blotting and RT-PCR. Western blots of Cas13 sorted Rh5 cells showed that guide 1150 was able to knockdown P3F1 protein expression by ~70%. Discussion/Conclusion: These findings demonstrate the high efficiency and specificity of the Cas13 system to silence P3F1, and other oncogenic fusion drivers. Further studies will utilize Cas13 to characterize the molecular and cellular consequences of P3F1 gene-silencing in patient-derived ARMS cell lines. Citation Format: Andrea Zhao, Lauren M. Brown, Mohamed Fareh, Paul G. Ekert. Utilizing CRISPR-Cas13 as a novel functional genomics tool to characterize PAX3-FOXO1 in alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1094.

Dose-Dependent Progression of Chorioretinal Atrophy at the Injection Site After Subretinal Injection of rAAV2/8 in Nonhuman Primates

ObjectiveProgressive retinal atrophy has been described after subretinal gene therapy utilizing the adeno-associated virus (AAV) vector platform. To elucidate whether thIS atrophy is a consequence of inherent properties of AAV, or if it is related to the surgical trauma of subretinal delivery, we analyzed data from an IND-enabling study for PDE6A gene therapy in non-human primates. DesignAnimal study (non-human primates), retrospective data analysis SubjectsForty eyes of thirty healthy non-human primates (macaca fascicularis) were included in the analysis. Two AAV dose levels (low: 1x10E11, high: 1x10E12) were compared to sham-injection (balanced saline solution; BSS). Twenty untreated eyes were not analysed. MethodsAnimals were treated with a sutureless 23G vitrectomy and single subretinal injections of AAV.PDE6A and/or BSS. The follow up period was 12 weeks. Atrophy development was followed using fundus-autofluorescence (AF), optical coherence tomography (OCT), fluorescence angiography (FA) and indocyanine green angiography (ICGA). Main Outcome MeasuresArea [mm2] of retinal pigment epithelium (RPE) atrophy on AF. Presence of outer retinal atrophy on OCT. Area [mm2] of hyperfluorescence in FA and hypofluorescence in ICGA. ResultsProgressive atrophy at the injection site developed in 54% of high dose, 27% of low dose, and 0% of sham-treated eyes. At the end of observation, the mean±SD area of atrophy in AF was 1.19±1.75 mm2, 0.25±0.50 mm2 and 0.0±0.0 mm2 respectively (sham x high dose: p=0.01). Atrophic lesions in AF (p=0.01) and FA (p=0.02) were significantly larger in high dose eyes, compared to sham-treated eyes. Rate of progression in high dose eyes was 4.1x higher compared to low dose eyes. ConclusionSubretinal injection of AAV.PDE6A induced dose-dependent, progressive retinal atrophy at the site of injection. Findings from multimodal imaging were in line with focal, transient inflammation within the retina and choroid and secondary atrophy. Atrophic changes after gene therapy with AAV-based vector systems are not primarily due to surgical trauma and increase with the dose given.