Random Integration Research Articles

Energy-Efficient RIS-Enabled SISO-OFDMA Communication via Lower Bound Optimization

The pursuit of energy-efficient solutions in the context of reconfigurable intelligent surface (RIS)-assisted wireless networks has become imperative and transformative. This paper investigates the integration of RIS into an orthogonal frequency-division multiple access (OFDMA) framework for multi-user downlink communication systems. We address the challenge of jointly optimizing RIS reflection coefficients alongside OFDMA frequency and power allocations, with the aim of maximizing energy efficiency. This optimization is subject to specific quality-of-service (QoS) requirements for each user equipment (UE) and a constraint on transmission power and the RIS phase shift matrix. To address this complex optimization problem, we propose a novel practical and low-complexity approach that is based on optimizing a computationally efficient and numerically tractable lower bound on energy efficiency. The numerical results highlight the effectiveness of our approach, demonstrating a substantial increase in energy efficiency compared to scenarios without RIS, with random RIS integration, and with the scheme using the Genetic Algorithm (GA).

Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches.

Since its discovery in 2012, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system has supposed a promising panorama for developing novel and highly precise genome editing-based gene therapy (GT) alternatives, leading to overcoming the challenges associated with classical GT. Classical GT aims to deliver transgenes to the cells via their random integration in the genome or episomal persistence into the nucleus through lentivirus (LV) or adeno-associated virus (AAV), respectively. Although high transgene expression efficiency is achieved by using either LV or AAV, their nature can result in severe side effects in humans. For instance, an LV (NCT03852498)- and AAV9 (NCT05514249)-based GT clinical trials for treating X-linked adrenoleukodystrophy and Duchenne Muscular Dystrophy showed the development of myelodysplastic syndrome and patient's death, respectively. In contrast with classical GT, the CRISPR/Cas9-based genome editing requires the homologous direct repair (HDR) machinery of the cells for inserting the transgene in specific regions of the genome. This sophisticated and well-regulated process is limited in the cell cycle of mammalian cells, and in turn, the nonhomologous end-joining (NHEJ) predominates. Consequently, seeking approaches to increase HDR efficiency over NHEJ is crucial. This manuscript comprehensively reviews the current alternatives for improving the HDR for CRISPR/Cas9-based GTs.

A deep-well plate enabled automated high-throughput cell line development platform.

Cell line development (CLD) plays a crucial role in the manufacturing process development of therapeutic biologics. Most biologics are produced in Chinese hamster ovary (CHO) cell. Because of the nature of random transgene integration in CHO genome and CHO's inherent plasticity, stable CHO transfectants usually have a vast diversity in productivity, growth, and product quality. Thus, we often must resort to screening a large number of cell pools and clones to increase the probability of identifying the ideal production cell line, which is a very laborious and resource-demanding process. Here we have developed a deep-well plate (DWP) enabled high throughput (DEHT) CLD platform using 24-well DWP (24DWP), liquid handler, and other automation components. This platform has capabilities covering the key steps of CLD including cell passaging, clone imaging and expansion, and fed-batch production. We are the first to demonstrate the suitability of 24DWP for CLD by confirming minimal well-to-well and plate-to-plate variability and the absence of well-to-well cross contamination. We also demonstrated that growth, production, and product quality of 24DWP cultures were comparable to those of conventional shake flask cultures. The DEHT platform enables scientists to screen five times more cultures than the conventional CLD platform, thus significantly decreases the resources needed to identify an ideal production cell line for biologics manufacturing.

A high-fidelity, dual site-specific integration system in CHO cells by a Bxb1 recombinase.

Site-specific integration (SSI) via recombinase mediated cassette exchange (RMCE) has shown advantages over random integration methods for expression of biotherapeutics. As an extension of our previous work developing SSI host cells, we developed a dual-site SSI system having two independent integration sites at different genomic loci, each containing a unique landing pad (LP). This system was leveraged to generate and compare two RMCE hosts, one (dFRT) compatible with the Flp recombinase, the other (dBxb1) compatible with the Bxb1 recombinase. Our comparison demonstrated that the dBxb1 host was able to generate stable transfectant pools in a shorter time frame, and cells within the dBxb1 transfectant pools were more phenotypically and genotypically stable. We further improved process performance of the dBxb1 host, resulting in desired fed batch performance attributes. Clones derived from this improved host (referred as 41L-11) maintained stable expression profiles over extended generations. While the data represents a significant improvement in the efficiency of our cell line development process, the dual LP architecture also affords a high degree of flexibility for development of complex protein modalities.

Use of random integration to test equality of high dimensional covariance matrices.

Testing the equality of two covariance matrices is a fundamental problem in statistics, and especially challenging when the data are high-dimensional. Through a novel use of random integration, we can test the equality of high-dimensional covariance matrices without assuming parametric distributions for the two underlying populations, even if the dimension is much larger than the sample size. The asymptotic properties of our test for arbitrary number of covariates and sample size are studied in depth under a general multivariate model. The finite-sample performance of our test is evaluated through numerical studies. The empirical results demonstrate that our test is highly competitive with existing tests in a wide range of settings. In particular, our proposed test is distinctly powerful under different settings when there exist a few large or many small diagonal disturbances between the two covariance matrices.

Enhancing Cell Line Stability by CRISPR/Cas9-Mediated Site-Specific Integration Based on Histone Modifications.

In traditional cell line design pipelines, cost- and time-intensive long-term stability studies must be performed due to random integration of the transgene into the genome. By this, integration into epigenetically silenced regions can lead to silencing of the recombinant promoter over time. Site-specific integration into regions with active chromatin structure can overcome this problem and lead to strong and stable gene expression. Here, we describe a detailed protocol to identify integration sites with epigenetically preferable properties by chromatin immunoprecipitation sequencing and use them for stable and strong gene expression by applying CRISPR/Cas9. Furthermore, the examination of the integration sites with focus on Cas9-targeted sequencing with nanopores is described.

Construction and application of a multifunctional CHO cell platform utilizing Cre/lox and Dre/rox site-specific recombination systems.

During the development of traditional Chinese hamster ovary (CHO) cell lines, target genes randomly integrate into the genome upon entering the nucleus, resulting in unpredictable productivity of cell clones. The characterization and screening of high-yielding cell lines is a time-consuming and expensive process. Site-specific integration is recognized as an effective approach for overcoming random integration and improving production stability. We have designed a multifunctional expression cassette, called CDbox, which can be manipulated by the site-specific recombination systems Cre/lox and Dre/rox. The CDbox expression cassette was inserted at the Hipp11(H11) locus hotspot in the CHO-K1 genome using CRISPR/Cas9 technology, and a compliant CHO-CDbox cell platform was screened and obtained. The CHO-CDbox cell platform was transformed into a pool of EGFP-expressing cells using Cre/lox recombinase-mediated cassette exchange (RMCE) in only 2weeks, and this expression remained stable for at least 75 generations without the need for drug stress. Subsequently, we used the Dre/rox system to directly eliminate the EGFP gene. In addition, two practical applications of the CHO-CDbox cell platform were presented. The first was the quick construction of the Pembrolizumab antibody stable expression strain, while the second was a protocol for the integration of surface-displayed and secreted antibodies on CHO cells. The previous research on site-specific integration of CHO cells has always focused on the single functionality of insertion of target genes. This newly developed CHO cell platform is expected to offer expanded applicability for protein production and gene function studies.

Expansion of YALIcloneHR toolkit for Yarrowia lipolytica combined with Golden Gate and CRISPR technology.

Metabolic Engineering of yeast is a critical approach to improving the production capacity of cell factories. To obtain genetically stable recombinant strains, the exogenous DNA is preferred to be integrated into the genome. Previously, we developed a Golden Gate toolkit YALIcloneNHEJ, which could be used as an efficient modular cloning toolkit for the random integration of multigene pathways through the innate non-homologous end-joining repair mechanisms of Yarrowia lipolytica. We expanded the toolkit by designing additional building blocks of homologous arms and using CRISPR technology. The reconstructed toolkit was thus entitled YALIcloneHR and designed for gene-specific knockout and integration. To verify the effectiveness of the system, the gene PEX10 was selected as the target for the knockout. This system was subsequently applied for the arachidonic acid production, and the reconstructed strain can accumulate 4.8% of arachidonic acid. The toolkit will expand gene editing technology in Y. lipolytica, which would help produce other chemicals derived from acetyl-CoA in the future.

Mutant N- and K- Ras Show Distinct Effects on Growth, Signaling and Drug Response in CRISPR-Cas Edited Alleles of a Myeloma Cell Line

Myeloma is well known to have significant genetic heterogeneity among patients' tumors, affecting both progression as well as therapeutic responses. Moreover, subclonal diversity can lead to selective outgrowths that are influenced by new mutations, as well as the bone marrow microenvironment and therapeutic exposures. The ras family of genes (N -and K-) are the most common mutations (40-50% ) in myeloma. Notably, ras mutations occur among a variety of other co-existing chromosomal abnormalities. Ras mutations were found to be more commonly associated with cyclin D1 high expression (e.g., resulting from 11;14 translocations). We and others have also shown that rasmutations may not be disease-initiating but may arise during disease progression and relapse; or are subclonal events that impart selective growth advantage and emerging drug resistance. There is evidence that activating mutations (codons 12,13, 61) in K- ras may have different clinical impact than activating mutations in N- ras yet there are conflicting clinical outcomes that may be due to therapies analyzed or differences in other co-existing genetic mutations. Previous studies using transfection of genes with targeted mutations show random integration and often in multiple copies, thus, limiting interpretations. We chose to create N ras61 and K ras12 mutations by CRISPR-cas directed edits in a single endogenous ras allele in a myeloma cell line (FLAM76) that initially had two wild type alleles (and an 11;14 translocation). We then compared the impact of these edits on growth, signaling, gene expression, and drug responses. We find both the mutN ras and mutK ras edited cells have increased proliferation compared to wild type. However, we find that the N ras mutation increases MAPK signaling and downstream gene activation to a greater extent than the K rasmutation. Based on RNAseq analysis, a MAPK gene activation score was higher in mutN ras edited cells than mutK ras edited cells. Further, we see distinct gene expression patterns between mutN ras versus mutK ras edited cells (See heat map Fig) impacting distinct pathways. Notably, mutNras cells have much higher NOXA expression, increasing sensitivity to apoptosis-inducing drugs (e.g., proteasome inhibitors); and Kras induces anti-apoptotic Bcl-xL, reducing sensitivity. We have conducted a high throughput drug screen with 84 drugs, and while some drugs are active against both mutN ras and mutK ras edits (Proteasome inhibitors), we have identified drugs with differential response to K versus N ras mutations. For example, mutK ras FLAM76 cells are more resistant to proteasome inhibitors; and particularly more resistant to HSP inhibitors and HDAC inhibitors compared to wild type or mutN ras cells. In contrast, mutK ras cells are more sensitive to PikFyve and IL-12 inhibitors than mutNras or wild type cells. Gene expression analysis shows that mutKras cells result in more downregulation of apoptotic genes and higher upregulation of cell survival genes, as well as ER stress pathway genes. Our results suggest that treating myelomas containing K versus N ras activating mutations may benefit from understanding distinct pathway activations and targeting selective drug therapies. We are currently testing new pan-Ras inhibitors to determine if there are distinct responses between our N- and K- ras edited cells, and we are expanding testing to a wider panel of myeloma cell lines containing ras mutations in the context of other genetic abnormalities. Details of gene expression distinctions, pathways, and drug responses that distinguish mutN ras and mutK ras in myeloma will be presented.

Long Term Follow-up Results of Brl-201 Phase I Study, a Crispr-Based Non-Viral PD-1 Locus Specific Integrated Anti-CD19 CAR-T Cells in Treating Relapsed or Refractory Non-Hodgkin's Lymphoma

BACKGROUND ： CAR-T cell products are manufactured traditionally by using virus, which leads to the potential tumorigenesis risk and makes CAR-T cell therapy expensive and time-consuming. We developed a novel approach to generate non-viral, genome-specific integrated CAR-T cells through CRISPR/Cas9, thereby breaking through both virus usage and random integration simultaneously. Here, we update long term follow up data of trial of non-viral PD1 locus specifically integrated anti-CD19 CAR-T cells (BRL-201) in patients with relapsed/refractory (r/r) Non-Hodgkin's lymphoma (#NCT04213469). Recently, a phase I/II of BRL-201 (#NCT05741359) is ongoing with a large-scale and multi-center design to further evaluate of BRL-201 antitumor activity in lower dose level. METHODS : This phase I investigator initiated trial evaluates BRL-201 in adult patients with r/r B-NHL. Adult patients with r/r B-NHL underwent leukapheresis and a lymphodepletion chemotherapy with cyclophosphamide (500mg/m 2, D -3 to -2) and fludarabine (30mg/m 2, D -4 to -2) before BRL-201 infusion. Dose escalation are based on 3+3 escalation rule, including three cohorts: 2×10 6/kg, 4×10 6/kg, 6×10 6/kg. Besides, 3 subjects received non-standard infusion doses at 0.56~0.8 × 10 6/kg. The primary endpoint was the incidence of dose-limiting toxicities (DLT). The secondary endpoint was the proportion of patients achieving an objective response at 3 months as per investigator's assessment. In the phase I/II sponsor initiated clinical trial, we employed 3+3 rule in the escalation phase. RESULTS: Between May 3, 2020 and August 10, 2021, 25 patients with r/r B-NHL were enrolled and 21 received BRL-201 with a median age of 56 years (34-70) and a median of 4 (1-9) prior lines of therapy. Among all the treated patients, 17 patients (93.8%) were diagnosed with disease stage III or IV, and 13 patients (81.3%) were assessed with high-intermediate to high risk according to IPI or aaIPI score assessment. Two patients had undergone autologous hematopoietic stem cell transplantation (HSCT) and one patient had a history of primary refractory disease. Of 17 patients with pretreatment tumor samples PD-L1 expression detection, 4 (23.5%) had > 50% PD-L1 expression, 13 (76.5%) had ≤50% PD-L1 expression. As of May 17, 2023, the median follow- up of investigaor initiated phase I study was 29.0 months (21.5-36.2 m). All of 21 (100%) patients had an objective response to BRL-201 and 18 (85.7%) patients had a complete response (CR) as best response (Fig. 1). 7 patients achieved and maintained CR at data cut-off date, among these, 3 patients had PD-L1 expression > 50%, 1 patient was negative and 3 unknown. The median duration of response(DOR) for all 21 patients was 18.6 months (95%CI：5.1, NA ) (Fig. 2a). The median progression free survival (PFS) was 20.8 (95%CI：8.2, NA) months (Fig. 2b), while the estimated median overall survival (OS) was not reached, 12-month OS rate was 76.2% (95% CI: 60%, 96.8%) (Fig. 2c). No grade 3-4 CRS and ICANS were observed. 14 patients (66.7%) experienced grade 1-2 cytokine release syndrome (CRS) and only one patient received tocilizumab. 4 patients (19.0%) experienced grade 1-2 immune effector cell-associated neurotoxicity syndrome (ICANS). No new AEs/SAEs were observed during the last follow up. In the ongoing phase I study, 1 patient with DLBCL was treated with BRL-201 and no DLT observed during 28 days after infusion at dose of 2×10 5/kg. CONCLUSIONS : Over a median follow up of 29 months, BRL-201 was demonstrated durable response with a high median PFS of 20.8 months and 12-month OS rate of 76.2%. The median OS was not reached yet for responding patients, and the safety profile was manageable. More data will be collected to prove the value of clinical usage of BRL-201.

CRISPR-Cas9 Mediated Stable Expression of Exogenous Proteins in the CHO Cell Line through Site-Specific Integration

Chinese hamster ovary (CHO) cells are a popular choice in biopharmaceuticals because of their beneficial traits, including high-density suspension culture, safety, and exogenously produced proteins that closely resemble natural proteins. Nevertheless, a decline in the expression of exogenous proteins is noted as culture time progresses. This is a consequence of foreign gene recombination into chromosomes by random integration. The current investigation employs CRISPR-Cas9 technology to integrate foreign genes into a particular chromosomal location for sustained expression. Results demonstrate the successful integration of enhanced green fluorescent protein (EGFP) and human serum albumin (HSA) near base 434814407 on chromosome NC_048595.1 of CHO-K1 cells. Over 60 successive passages, monoclonal cell lines were produced that consistently expressed all relevant external proteins without discernible variation in expression levels. In conclusion, the CHO-K1 cell locus, NC_048595.1, proves an advantageous locus for stable exogenous protein expression. This study provides a viable approach to establishing a CHO cell line capable of enduring reliable exogenous protein expression.

Iterative gene integration mediated by 26S rDNA and non-homologous end joining for the efficient production of lycopene in Yarrowia lipolytica

Because of its potent antioxidant effects, lycopene has been used in various industries including, but not limited to, food, medical, and cosmetic industries. Yarrowia lipolytica, a non-conventional yeast species, is a promising chassis due to its natural mevalonate (MVA) pathway, abundant precursor acetyl coenzyme A content, and oleaginous properties. Several gene editing tools have been developed for Y. lipolytica along with engineering strategies for tetraterpenoid production. In this study, we engineered Y. lipolytica following multi-level strategies for efficient lycopene accumulation. We first evaluated the performance of the key lycopene biosynthetic genes crtE, crtB, and crtI, expressed via ribosomal DNA (rDNA) mediated multicopy random integration in the HMG1- and GGS1-overexpressing background strain. Further improvement in lycopene production was achieved by overexpressing the key genes for MVA synthesis via non-homologous end joining (NHEJ) mediated multi-round iterative transformation. Efficient strategies in the MVA and lipid synthesis pathways were combined to improve lycopene production with a yield of 430.5 mg/L. This strain produced 121 mg/g dry cell weight of lycopene in a 5-L fed-batch fermentation system. Our findings demonstrated iterative gene integration mediated by 26S rDNA and NHEJ for the efficient production of lycopene in Y. lipolytica. These strategies can be applied to induce Y. lipolytica to produce other tetraterpenoids.Graphical

Improved piggyBac Transformation with Capped Transposase mRNA in Pest Insects.

Creating transgenic insects is a key technology in insect genetics and molecular biology. A widely used instrument in insect transgenesis is the piggyBac transposase, resulting in essentially random genomic integrations. In contrast, site-specific recombinases allow the targeted integration of the transgene construct into a specific genomic target site. Both strategies, however, often face limitations due to low transgenesis efficiencies. We aimed to enhance transgenesis efficiencies by utilizing capped mRNA as a source of transposase or recombinase instead of a helper plasmid. A systematic comparison of transgenesis efficiencies in Aedes mosquitoes, as models for hard-to-transform insects, showed that suppling piggyBac transposase as mRNA increased the average transformation efficiency in Aedes aegypti from less than 5% with the plasmid source to about 50% with mRNA. Similar high activity was observed in Ae. albopictus with pBac mRNA. No efficiency differences between plasmid and mRNA were observed in recombination experiments. Furthermore, a hyperactive version of piggyBac transposase delivered as a plasmid did not improve the transformation efficiency in Ae. aegypti or the agricultural pest Drosophila suzukii. We believe that the use of mRNA has strong potential for enhancing piggyBac transformation efficiencies in other mosquitoes and important agricultural pests, such as tephritids.

Targeted Delivery of Chimeric Antigen Receptor into T Cells via CRISPR-Mediated Homology-Directed Repair with a Dual-AAV6 Transduction System.

CAR-T cell therapy involves genetically engineering T cells to recognize and attack tumour cells by adding a chimeric antigen receptor (CAR) to their surface. In this study, we have used dual transduction with AAV serotype 6 (AAV6) to integrate an anti-CD19 CAR into human T cells at a known genomic location. The first viral vector expresses the Cas9 endonuclease and a guide RNA (gRNA) targeting the T cell receptor alpha constant locus, while the second vector carries the DNA template for homology-mediated CAR insertion. We evaluated three gRNA candidates and determined their efficiency in generating indels. The AAV6 successfully delivered the CRISPR/Cas9 machinery in vitro, and molecular analysis of the dual transduction showed the integration of the CAR transgene into the desired location. In contrast to the random integration methods typically used to generate CAR-T cells, targeted integration into a known genomic locus can potentially lower the risk of insertional mutagenesis and provide more stable levels of CAR expression. Critically, this method also results in the knockout of the endogenous T cell receptor, allowing target cells to be derived from allogeneic donors. This raises the exciting possibility of "off-the-shelf" universal immunotherapies that would greatly simplify the production and administration of CAR-T cells.

Inducing multiple nicks promotes interhomolog homologous recombination to correct heterozygous mutations in somatic cells

CRISPR/Cas9-mediated gene editing has great potential utility for treating genetic diseases. However, its therapeutic applications are limited by unintended genomic alterations arising from DNA double-strand breaks and random integration of exogenous DNA. In this study, we propose NICER, a method for correcting heterozygous mutations that employs multiple nicks (MNs) induced by Cas9 nickase and a homologous chromosome as an endogenous repair template. Although a single nick near the mutation site rarely leads to successful gene correction, additional nicks on homologous chromosomes strongly enhance gene correction efficiency via interhomolog homologous recombination (IH-HR). This process partially depends on BRCA1 and BRCA2, suggesting the existence of several distinct pathways for MN-induced IH-HR. According to a genomic analysis, NICER rarely induces unintended genomic alterations. Furthermore, NICER restores the expression of disease-causing genes in cells derived from genetic diseases with compound heterozygous mutations. Overall, NICER provides a precise strategy for gene correction.

The caveats and setbacks of mouse genome editing tools in biomedical studies

Due to the innumerable advantages of using mice as animal models, plenty of strategies to alter mouse genetics have been developed for the purpose of studying the phenotypic outcome of gene mutations. Up until this day, there have been multiple substantial breakthroughs in gene-targeting methods, namely, the utilization of transgene insertion via random integration, gene targeting via homologous recombination, engineered nuclease-mediated targeted mutation, and the Cre-lox approach. Although having facilitated the considerable progress in biomedical research, these techniques are infamously prone to create undesired phenotypes that have been oftentimes mistaken as gene-mutation-related. Hence, being able to identify and avoid these potential artifacts is the most crucial matter in producing transgenic or targeted mutation mouse strains. In this review, we present the fundamentals in generating different mutant mice and the different ways to ensure the credibility of the target gene expression and the observed mutant phenotypes.

Arsenic affects homologous recombination and single-strand annealing but not end-joining pathways during DNA double-strand break repair.

Arsenic is a carcinogen that can cause skin, lung, and bladder cancer. While DNA double-strand breaks (DSBs) have been implicated in arsenic-induced carcinogenesis, the exact mechanism remains unclear. In this study, we performed genetic analysis to examine the impact of arsenic trioxide (As2 O3 ) on four different DSB repair pathways using the human pre-B cell line Nalm-6. Random integration analysis showed that As2 O3 does not negatively affect non-homologous end joining or polymerase theta-mediated end joining. In contrast, chromosomal DSB repair analysis revealed that As2 O3 decreases the efficiency of homologous recombination (HR) and, less prominently, single-strand annealing. Consistent with this finding, As2 O3 decreased gene-targeting efficiency, owing to a significant reduction in the frequency of HR-mediated targeted integration. To further verify the inhibitory effect of arsenic on HR, we examined cellular sensitivity to olaparib and camptothecin, which induce one-ended DSBs requiring HR for precise repair. Intriguingly, we found that As2 O3 significantly enhances sensitivity to those anticancer agents in HR-proficient cells. Our results suggest that arsenic-induced genomic instability is attributed to HR suppression, providing valuable insights into arsenic-associated carcinogenesis and therapeutic options.

Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS-STING pathway.

Viral delivery of DNA for the targeted reprogramming of human T cells can lead to random genomic integration, and electroporation is inefficient and can be toxic. Here we show that electroporation-induced toxicity in primary human T cells is mediated by the cytosolic pathway cGAS-STING (cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase-stimulator of interferon genes). We also show that an isotonic buffer, identified by screening electroporation conditions, that reduces cGAS-STING surveillance allowed for the production of chimaeric antigen receptor (CAR) T cells with up to 20-fold higher CAR T cell numbers than standard electroporation and with higher antitumour activity in vivo than lentivirally generated CAR T cells. The osmotic pressure of the electroporation buffer dampened cGAS-DNA interactions, affecting the production of the STING activator 2'3'-cGAMP. The buffer also led to superior efficiencies in the transfection of therapeutically relevant primary T cells and human haematopoietic stem cells. Our findings may facilitate the optimization of electroporation-mediated DNA delivery for the production of genome-engineered T cells.

Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy.

Manufacture of chimeric antigen receptor (CAR)-T cells usually involves the use of viral delivery systems to achieve high transgene expression. However, it can be costly and may result in random integration of the CAR into the genome, creating several disadvantages including variation in transgene expression, functional gene silencing and potential oncogenic transformation. Here, we optimized the method of nonviral, CRISPR/Cas9 genome editing using large donor DNA delivery, knocked-in an anti-tumor single chain variable fragment (scFv) into the N-terminus of CD3ε and efficiently generated fusion protein (FP) T cells. These cells displayed FP integration within the TCR/CD3 complex, lower variability in gene expression compared to CAR-T cells and good cell expansion after transfection. CD3ε FP T cells were predominantly CD8+ effector memory T cells, and exhibited anti-tumor activity in vitro and in vivo. Dual targeting FP T cells were also generated through the incorporation of scFvs into other CD3 subunits and CD28. Compared to viral-based methods, this method serves as an alternative and versatile way of generating T cells with tumor-targeting receptors for cancer immunotherapy.

Random Integration Analysis of Recombinant Adeno-Associated Virus 6 Packaged in Sf9 Insect Cells

Recently, there have been growing concerns over the integration of recombinant adeno-associated virus (rAAV) used in gene therapy. Wild-type adeno-associated virus (AAV) site specifically integrates into AAVS1 site of human genome, while rAAV randomly integrates into host chromosomes at low frequencies. This research aims to study the random integration events of rAAV6-EGFP packaged in Sf9 insect cells. Baculo-Sf9 manufacturing platform has the advantages of high-density suspension culture of Sf9 insect cells and large-scale production of rAAV vectors. In this study, we used different doses of Baculo-Sf9 produced rAAV6-EGFP to transduce HEK293T cells and A549-implanted tumors in vitro and in vivo. Using flow cytometry and fluorescence microscopy, we studied their EGFP gene expression efficiencies and EGFP fluorescence intensities. Using inverse nested PCR and DNA sequencing, random integration sites of rAAV6-EGFP genome into human chromosomes were identified. In vitro results showed that gene expression efficiencies became stable after 20 days and random integration frequencies were 0.2‒4.2%. Both in vitro and in vivo results indicated that random integration of Baculo-Sf9 rAAV6 was dose-dependent. Sequencing results showed two random integration sites, which were on human chromosomes 8 and 12. The findings suggest that we should use as low dose of rAAV vector as possible for safe gene therapy.