Packaging Plasmids Research Articles

Optimizing the Procedure for Manufacturing Clinical-grade Genetically Manipulated NK cells for Adoptive Immunotherapy

BackgroundEx vivo expanded natural killer (NK) cells hold significant potential as antitumor effector cells for adoptive immunotherapy. However, producing clinical-grade genetically modified NK cells in sufficient quantities presents a considerable challenge. MethodsWe tested RPMI 1640, KBM581, SCGM, NK MACS, X-VIVO 15, and AIM-V, each supplemented with fetal bovine serum (FBS), human AB serum, human platelet lysate (HPL), or Immune Cell Serum Replacement (SR) combined with feeder cells, to produce cytotoxic NK cells. Subsequent analyses were conducted to assess cell viability, expansion folds, cytotoxicity, immunophenotype, and transcriptome profile of NK cells under certain conditions. Furthermore, transfer plasmids varying in transgene size, promoter elements, backbones and packaging plasmids with different envelopes were used to transduce NK cells and differences in transduction efficiency were compared. Nucleofection was performed every two days from Day 0 to Day 12 to determine the optimal time window for gene editing. ResultsNK cells cultured in KBM581 medium supplemented with SR exhibited the best expansion, achieving over 5000-fold increase within two weeks and exceeding 25,000-fold expansion within three weeks. Additionally, NK cells cultured in KBM581 medium with human AB serum demonstrated the highest cytolytic activities and exhibited higher expression of NKp30, 2B4, PRF1, Granzyme B, and IL2RG. Baboon envelope (BaEV) pseudotyped lentivirus outperformed BaEV-Vesicular Stomatitis Virus type-G (VSVG) hybrid envelope lentivirus, achieving robust NK cell transduction. Additionally, efficient gene knockout efficiency was achieved in NK cells on day 4 to day 6 post feeder cell activation using LONZA DN-100 program, which can strike a balance between editing efficiency and cell expansion. ConclusionsThis research presents a Good Manufacturing Practice (GMP)-compliant protocol utilizing a feeder cell expansion system for the large-scale production of highly cytotoxic Natural Killer (NK) cells. The protocol facilitates genetic modification of these cells, positioning them as promising candidates for universal therapeutic applications in immunotherapy.

ACE2-Decorated Virus-Like Particles Effectively Block SARS-CoV-2 Infection.

Over the past three years, extensive research has been dedicated to understanding and combating COVID-19. Targeting the interaction between the SARS-CoV-2 Spike protein and the ACE2 receptor has emerged as a promising therapeutic strategy against SARS-CoV-2. This study aimed to develop ACE2-coated virus-like particles (ACE2-VLPs), which can be utilized to prevent viral entry into host cells and efficiently neutralize the virus. Virus-like particles were generated through the utilization of a packaging plasmid in conjunction with a plasmid containing the ACE2 envelope sequence. Subsequently, ACE2-VLPs and ACE2-EVs were purified via ultracentrifugation. The quantification of VLPs was validated through multiple methods, including Nanosight 3000, TEM imaging, and Western blot analysis. Various packaging systems were explored to optimize the ACE2-VLP configuration for enhanced neutralization capabilities. The evaluation of neutralization effectiveness was conducted using pseudoviruses bearing different spike protein variants. Furthermore, the study assessed the neutralization potential against the Omicron BA.1 variant in Vero E6 cells. ACE2-VLPs showed a high neutralization capacity even at low doses and demonstrated superior efficacy in in vitro pseudoviral assays compared to extracellular vesicles carrying ACE2. ACE2-VLPs remained stable under various environmental temperatures and effectively blocked all tested variants of concern in vitro. Notably, they exhibited significant neutralization against Omicron BA.1 variant in Vero E6 cells. Given their superior efficacy compared to extracellular vesicles and proven success against live virus, ACE2-VLPs stand out as crucial candidates for treating SARS-CoV-2 infections. This novel therapeutic approach of coating VLPs with receptor particles provides a proof-of-concept for designing effective neutralization strategies for other viral diseases in the future.

Effects of an indole derivative on cell proliferation, transfection, and alternative splicing in production of lentiviral vectors by transient co-transfection.

Lentiviral vectors derived from human immunodeficiency virus type I are widely used to deliver functional gene copies to mammalian cells for research and gene therapies. Post-transcriptional splicing of lentiviral vector transgene in transduced host and transfected producer cells presents barriers to widespread application of lentiviral vector-based therapies. The present study examined effects of indole derivative compound IDC16 on splicing of lentiviral vector transcripts in producer cells and corresponding yield of infectious lentiviral vectors. Indole IDC16 was shown previously to modify alternative splicing in human immunodeficiency virus type I. Human embryonic kidney 293T cells were transiently transfected by 3rd generation backbone and packaging plasmids using polyethyleneimine. Reverse transcription-quantitative polymerase chain reaction of the fraction of unspliced genomes in human embryonic kidney 293T cells increased up to 31% upon the indole's treatment at 2.5 uM. Corresponding yield of infectious lentiviral vectors decreased up to 4.5-fold in a cell transduction assay. Adjusting timing and duration of IDC16 treatment indicated that the indole's disruption of early stages of transfection and cell cycle had a greater effect on exponential time course of lentiviral vector production than its reduction of post-transcriptional splicing. Decrease in transfected human embryonic kidney 293T proliferation by IDC16 became significant at 10 uM. These findings indicated contributions by early-stage transfection, cell proliferation, and post-transcriptional splicing in transient transfection of human embryonic kidney 293T cells for lentiviral vector production.

Development and utility of a SARS-CoV-2 pseudovirus assay for compound screening and antibody neutralization assays

BackgroundThe highly infectious nature of SARS-CoV-2 necessitates using bio-containment facilities to study viral pathogenesis and identify potent antivirals. However, the lack of high-level bio-containment laboratories across the world has limited research efforts into SARS-CoV-2 pathogenesis and the discovery of drug candidates. Previous research has reported that non-replicating SARS-CoV-2 Spike-pseudotyped viral particles are effective tools to screen for and identify entry inhibitors and neutralizing antibodies. MethodsTo generate SARS-CoV-2 pseudovirus, a lentiviral packaging plasmid p8.91, a luciferase expression plasmid pCSFLW, and SARS-CoV-2 Spike expression plasmids (Wild-type (D614G) or Delta strain) were co-transfected into HEK293 cells to produce a luciferase-expressing non-replicating pseudovirus which expresses SARS-CoV-2 spike protein on the surface. For relative quantitation, HEK293 cells expressing ACE2 (ACE2-HEK293) were infected with the pseudovirus, after which luciferase activity in the cells was measured as a relative luminescence unit. The ACE2-HEK293/Pseudovirus infection system was used to assess the antiviral effects of some compounds and plasma from COVID-19 patients to demonstrate the utility of this assay for drug discovery and neutralizing antibody screening. ResultsWe successfully produced lentiviral-based SARS-CoV2 pseudoviruses and ACE2-expressing HEK293 cells. The system was used to screen compounds for SARS-CoV-2 entry inhibitors and identified two compounds with potent activity against SARS-CoV-2 pseudovirus entry into cells. The assay was also employed to screen patient plasma for neutralizing antibodies against SARS-CoV-2, as a precursor to live virus screening, using successful hits. SignificanceThis assay is scalable and can perform medium-to high-throughput screening of antiviral compounds with neither severe biohazard risks nor the need for higher-level containment facilities. Now fully deployed in our resource-limited laboratory, this system can be applied to other highly infectious viruses by swapping out the envelope proteins in the plasmids used in pseudovirus production.

Single Nucleotide Polymorphisms Mutation of Tropoelastin Gene Affects Tropoelastin mRNA and Elastin Expressions in Human Aortic Smooth Muscle Cells.

We aimed to explore the effects of single nucleotide polymorphisms (SNPs) in tropoelastin gene on tropoelastin mRNA and elastin expressions in human aortic smooth muscle cells (HASMCs). Two SNP loci, rs2071307 (G/A) and rs1785598 (G/C), were selected to construct recombinant lentivirus vectors carrying wild-type and mutant tropoelastin gene. Recombinant plasmids including pWSLV-02-ELN, pWSLV-02-ELN-mut1, and pWSLV-02-ELN-mut2 were constructed, before being amplified by polymerase chain reaction (PCR) and sequenced. The prepared plasmids and the packaging plasmids (pVSV-G and psPAX2) were cotransfected into HEK293T cells to obtain recombinant lentiviruses carrying tropoelastin gene. Afterward, HASMCs were infected with recombinant lentiviruses, and the positive cells sorted by flow cytometry were amplified. Four stable HASMCs cell lines including pWSLV-02-ELN, pWSLV-02-ELN-mut1, pWSLV-02-ELN-mut2, and pWSLV-02 vector were constructed. The expressions of tropoelastin mRNA and elastin in HASMCs were detected by real-time quantitative reverse transcription-PCR and western blot, respectively. Recombinant plasmids including pWSLV-02-ELN-mut1, pWSLV-02-ELN-mut2, and pWSLV-02-ELN were successfully constructed. Recombinant lentiviruses carrying tropoelastin gene were obtained via lentivirus packaging. After infection for 24 h, 3 days and 5 days in HASMCs, tropoelastin mRNA expressions in pWSLV-02-ELN-mut1 and pWSLV-02-ELN-mut2 groups were significantly lower than that of pWSLV-02-ELN group. Besides, after infection for 24 h, 3 days, and 5 days, elastin levels in pWSLV-02-ELN-mut1 and pWSLV-02-ELN-mut2 groups were significantly lower than that in pWSLV-02-ELN group. In conclusion, SNPs mutation of tropoelastin gene affected the expression of tropoelastin mRNA and elastin, suggesting that the polymorphisms of rs2071307 and rs17855988 in tropoelastin gene might be important factors for AD development.

Two neglected but valuable genetic tools for Escherichia coli and other bacteria: In vivo cosmid packaging and inducible plasmid replication.

In physiology and synthetic biology, it can be advantageous to introduce a gene into a naive bacterial host under conditions in which all cells receive the gene and remain fully functional. This cannot be done by the usual chemical transformation and electroporation methods due to low efficiency and cell death, respectively. However, in vivo packaging of plasmids (called cosmids) that contain the 223 bp cos site of phage λ results in phage particles that contain concatemers of the cosmid that can be transduced into all cells of a culture. An historical shortcoming of in vivo packaging of cosmids was inefficient packaging and contamination of the particles containing cosmid DNA with a great excess of infectious λ phage. Manipulation of the packaging phage and the host has eliminated these shortcomings resulting in particles that contain only cosmid DNA. Plasmids have the drawback that they can be difficult to remove from cells. Plasmids with conditional replication provide a means to "cure" plasmids from cells. The prevalent conditional replication plasmids are temperature-sensitive plasmids, which are cured at high growth temperature. However, inducible replication plasmids are in some cases more useful, especially since this approach has been applied to plasmids having diverse replication and compatibility properties.

A CRISPR interference strategy for gene expression silencing in multiple myeloma cell lines

BackgroundMultiple myeloma (MM) is the second most common hematologic neoplasm which is characterized by proliferation and infiltration of plasmatic cells in the bone marrow. Currently, MM is considered incurable due to resistance to treatment. The CRISPR/Cas9 system has emerged as a powerful tool for understanding the role of different genetic alterations in the pathogenesis of hematologic malignancies in both cell lines and mouse models. Despite current advances of gene editing tools, the use of CRISPR/Cas9 technology for gene editing of MM have not so far been extended. In this work, we want to repress Rnd3 expression, an atypical Rho GTPase involved in several cellular processes, in MM cell lines using a CRISPR interference strategy.ResultsWe have designed different guide RNAs and cloning them into a lentiviral plasmid, which contains all the machinery necessary for developing the CRISPR interference strategy. We co-transfected the HEK 293T cells with this lentiviral plasmid and 3rd generation lentiviral envelope and packaging plasmids to produce lentiviral particles. The lentiviral particles were used to transduce two different multiple myeloma cell lines, RPMI 8226 and JJN3, and downregulate Rnd3 expression. Additionally, the impact of Rnd3 expression absence was analyzed by a transcriptomic analysis consisting of 3’ UTR RNA sequencing. The Rnd3 knock-down cells showed a different transcriptomic profile in comparison to control cells.ConclusionsWe have developed a CRISPR interference strategy to generate stable Rnd3 knockdown MM cell lines by lentiviral transduction. We have evaluated this strategy in two MM cell lines, and we have demonstrated that Rnd3 silencing works both at transcriptional and protein level. Therefore, we propose CRISPR interference strategy as an alternative tool to silence gene expression in MM cell lines. Furthermore, Rnd3 silencing produces changes in the cellular transcriptomic profile.

Production of Encapsidated RNA Particles as a Working Standard in Detecting Foodborne Viruses in Oysters

Monitoring foodborne viruses via nucleic acid amplification tests rely on stable RNA standards to obtain reliable testing. This study aimed to produce RNA-based standard reagents for hepatitis virus (HAV) or norovirus detections which relies on viral-like particle (VLP) technology. Using a plasmid packaging system, plasmids containing DNA encoding Qβcoat protein (CP) monomer and the VP1 gene of viruses were co-transformed into E. coli host cells. In cell lysates, expressed CP was characterized by western blot and the whole icosahedral formation of VLPs was proved by electron microscope analysis. Encapsidated RNAs were measured and assessed as a standard by a two-step reverse transcription recombinase polymerase amplification (RT-RPA). Our results showed that CP has a distinguished protein band with a molecular weight of 14.5 kDa but a few variabilities of particle size were visualized. When adjusting the pH of the lysate to lower than 6, a more intense protein band and substantial particles with homogenous particle size were observed. These VLPs were found to enclose HAV and norovirus RNA contents to 1.2×107 copies/ng and 1.9×107 copies/ng, respectively. When analyzed by RT-RPA, linear regression analysis confirmed the alternative application of RNAs enclosed in VLPs to naked RNA synthesized from in vitro transcription. Using the E. coli expression system to produce Qβ VLPs allows cost-effective production and, therefore, can be implemented in laboratories with basic equipment. These encapsidated RNAs may become an ideal “standard” for detecting foodborne viruses via a molecular test in food and clinical samples. Keywords: Molecular testing, Nanoparticles, Nucleic amplification, RNA standards, Viral-like particles

Adeno-Associated Virus Monoinfection Induces a DNA Damage Response and DNA Repair That Contributes to Viral DNA Replication.

Adeno-associated virus (AAV) belongs to the Dependoparvovirus genus of the Parvoviridae family. AAV replication relies on a helper virus, such as adenovirus (Ad). Co-infection of AAV and Ad induces a DNA damage response (DDR), although its function in AAV DNA replication remains unknown. In this study, monoinfection of AAV2 in HEK293T cells expressing a minimal set of Ad helper genes was used to investigate the role of the DDR solely induced by AAV. We found that AAV2 DNA replication, but not single stranded (ss)DNA genome accumulation and Rep expression only, induced a robust DDR in HEK293T cells. The induced DDR featured the phosphorylation of replication protein A32 (RPA32), histone variant H2AX (H2A histone family member X), and all 3 phosphatidylinositol 3-kinase-related kinases (PIKKs). We also found that the kinase ataxia telangiectasia and Rad3-related protein (ATR) plays a major role in AAV2 DNA replication and that Y family DNA repair DNA polymerases η (Pol η) and Pol κ contribute to AAV2 DNA replication both in vitro and in HEK293T cells. Knockout of Pol η and Pol κ in HEK293T cells significantly decreased wild-type AAV2 replication and recombinant AAV2 production. Thus, our study has proven that AAV2 DNA replication induces a DDR, which in turn initiates a DNA repairing process that partially contributes to the viral genome amplification in HEK293T cells. IMPORTANCE Recombinant AAV (rAAV) has emerged as one of the preferred delivery vectors for clinical gene therapy. rAAV production in HEK293 cells by transfection of a rAAV transgene plasmid, an AAV Rep and Cap expression packaging plasmid, and an Ad helper plasmid remains the popular method. Here, we demonstrated that the high fidelity Y family DNA repair DNA polymerase, Pol η, and Pol κ, plays a significant role in AAV DNA replication and rAAV production in HEK293T cells. Understanding the AAV DNA replication mechanism in HEK293T cells could provide clues to increase rAAV vector yield produced from the transfection method. We also provide evidence that the ATR-mediated DNA repair process through Pol η and Pol κ is one of the mechanisms to amplify AAV genome, which could explain AAV replication and rAAV ssDNA genome conversion in mitotic quiescent cells.

The Use of Adeno-associated virus (AAV) in Vaccine Development


 Adeno-associated virus (AAV) is a very tiny (20-26 nm) icosahedral and non-enveloped virus, and it belongs to the Parvoviridae family. AAV vectors are the most widely used option for gene therapy and delivery of therapeutic antibodies due to their relatively low immunogenicity, high safety profile, broad tropism, and their tendency to maintain long-term gene expression [1]. AVV vectors are developed by transfection of human embryonic kidney (HEK) 293 T cells with transgene, packaging and helper plasmids [2]. Several clinical studies have investigated the use of AAV vectors for gene therapy in treating of Parkinson’s disease, Alzheimer’s disease, heart disease, and prostate cancer [3]. AAV vectors have previously been used to treat muscular diseases, but in recent years, their usage as vaccine vectors to cure or prevent infectious diseases including HIV, HPV, and influenza has expanded [4].
 
 Here, we discuss the advantages and disadvantages of the use of AAV in vaccine development, and future approaches in improving the drawbacks caused by AAV-based vaccines. Numerous animal investigations have been conducted to explore vaccine vectors against various illnesses, suggesting a possibility for AAV-based vaccinations. Clinical studies on humans are, however, uncommon because, in contrast to other viral vectors, AAV induces a poor humoral and cellular immune response. Additionally, infectious vaccinations often target a large group of healthy individuals across a variety of ages, including children and teenagers. Therefore, compared to AAV-based gene therapies, vaccinations based on AAV vectors need to be more cost-effective and need more robust safety control.
 
 According to several research, AAV vector vaccines have been shown to induce a stronger or longer lasting antibody response in comparison to other vaccination approaches, such as DNA, recombinant proteins, inactivated viruses, or virus-like particles (VLPs) [5]. However, AAV vectors are thought to have a low immunogenic profile in comparison to other viral vectors. The main limitations of AAV vectors are their low transgenic capacity and widespread pre-existing immunity in humans [6]. Currently, strategies for improving AAV immunogenicity and circumventing pre-existing immunity are actively being investigated. The research undertaken so far have highlighted numerous significant benefits of AAV vectors for immunisation. Despite all the advantages, there are still a variety of challenges that limit the use of these vectors as a vaccine in humans. Thus, it is necessary to overcome these challenges in order to make AAV-based vaccines effective.

Catechol-Based Polymers with High Efficacy in Cytosolic Protein Delivery

Catechol-Based Polymers with High Efficacy in Cytosolic Protein Delivery

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine (PEI).

The current protocol describes the use of lentiviral particles for the delivery of short hairpin RNAs (shRNAs) to both human embryonic stem cells (hESCs) as well as neural progenitor cells (NPCs) derived from hESCs at high efficiency. Lentiviral particles were generated by co-transfecting HEK293T cells using entry vectors (carrying shRNAs) along with packaging plasmids (pAX and pMD2.G) using the low-cost cationic polymer polyethylenimine (PEI). Viral particles were concentrated using ultracentrifugation, which resulted in average titers above 5 x 107. Both hESCs and NPCs could be infected at high efficiencies using these lentiviral particles, as shown by puromycin selection and stable expression in hESCs, as well as transient GFP expression in NPCs. Furthermore, western blot analysis showed a significant reduction in the expression of genes targeted by shRNAs. In addition, the cells retained their pluripotency as well as differentiation potential, as evidenced by their subsequent differentiation into different lineages of CNS. The current protocol deals with the delivery of shRNAs; however, the same approach could be used for the ectopic expression of cDNAs for overexpression studies.

Generation of CAR T-cells using γ-retroviral vector.

The generation of chimeric antigen receptor (CAR) T cells requires the transfer of the CAR gene into primary T cells. Among various gene transfer strategies, gammaretroviral vectors have been widely used to generate CAR T cells for both preclinical and clinical settings. Here we describe the detailed method of generating CAR T cells utilizing gammaretroviral vectors. This approach consists of two parallel parts: (1) production of the gammaretroviral particles and (2) gammaretroviral transduction of activated T cells. The gammaretroviral particles are produced by co-transfecting the gammaretroviral vector with packaging plasmids into 293T cells. The manufactured viral particles then efficiently infect activated T cells where the CAR transgene is integrated into host genomic DNA, resulting in stable expression of the CAR molecule on the surface of T cells.

Staphylococcal phages and pathogenicity islands drive plasmid evolution

Conjugation has classically been considered the main mechanism driving plasmid transfer in nature. Yet bacteria frequently carry so-called non-transmissible plasmids, raising questions about how these plasmids spread. Interestingly, the size of many mobilisable and non-transmissible plasmids coincides with the average size of phages (~40 kb) or that of a family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs, ~11 kb). Here, we show that phages and PICIs from Staphylococcus aureus can mediate intra- and inter-species plasmid transfer via generalised transduction, potentially contributing to non-transmissible plasmid spread in nature. Further, staphylococcal PICIs enhance plasmid packaging efficiency, and phages and PICIs exert selective pressures on plasmids via the physical capacity of their capsids, explaining the bimodal size distribution observed for non-conjugative plasmids. Our results highlight that transducing agents (phages, PICIs) have important roles in bacterial plasmid evolution and, potentially, in antimicrobial resistance transmission.

A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system

Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.

Minimal impact of ZAP on lentiviral vector production and transduction efficiency

The antiviral protein ZAP binds CpG dinucleotides in viral RNA to inhibit replication. This has likely led to the CpG suppression observed in many RNA viruses, including retroviruses. Sequences added to retroviral vector genomes, such as internal promoters, transgenes, or regulatory elements, substantially increase CpG abundance. Because these CpGs could allow retroviral vector RNA to be targeted by ZAP, we analyzed whether it restricts vector production, transduction efficiency, and transgene expression. Surprisingly, even though CpG-high HIV-1 was efficiently inhibited by ZAP in HEK293T cells, depleting ZAP did not substantially increase lentiviral vector titer using several packaging and genome plasmids. ZAP overexpression also did not inhibit lentiviral vector titer. In addition, decreasing CpG abundance in a lentiviral vector genome did not increase its titer, and a gammaretroviral vector derived from murine leukemia virus was not substantially restricted by ZAP. Overall, we show that the increased CpG abundance in retroviral vectors relative to the wild-type retroviruses they are derived from does not intrinsically sensitize them to ZAP. Further understanding of how ZAP specifically targets transcripts to inhibit their expression may allow the development of CpG sequence contexts that efficiently recruit or evade this antiviral system.

Incorporation of 4-1BB Endowed the Potent CD33 Specific Chimeric Antigen Receptor T Cells with Anti-Exhaustion Capacity and Increased Central Memory Compartment

Chimeric antigen receptor (CAR) modified T cells have been an important immunotherapeutic strategy. Efficacy of CAR-T cells may be influenced by the structure of CAR, particularly the costimulator component of CAR. To improve the function of CAR-T cells, different costimulators were incorporated into CAR structures. Studies have shown that CAR-T cells containing 4-1BB costimulator have superior antileukemic efficacy and improved persistence compared with that containing CD28 costimulator. But how costimulators affect CAR-T cell function remained to be further studied. In this study, three different CARs with costimulators CD28, 4-1BB, and both CD28 and 4-1BB targeted the myeloid antigen CD33 (referred to as CD33 28z.CAR-T, CD33 BBz.CAR-T and CD33 28BBz.CAR-T, respectively) were constructed and the antileukemic functions of corresponding CD33 CAR-T cells were reported. In addition, the characteristics related to persistence and function of CAR-T cells were further analyzed.To get CAR-T cells specific for CD33, the murine anti-human CD33 scFv derived from mouse hybridoma cells (clone HIM3-4,which was established in Institute of Hematology and Blood Diseases Hospital, CAMS&PUMC) was cloned into our previously constructed pCDH-CAR plasmids, and transfected into 293T/17 packaging cells with the packaging plasmids to produce lentiviruses. Then T cells from peripheral blood of healthy donors were isolated, stimulated and infected with lentiviruses to get CD33 28z.CAR-T, CD33 BBz.CAR-T and CD33 28BBz.CAR-T, respectively.In vitro antileukemic function of CD33 CAR-T cells was detected by cytotoxic effect on target cells, CAR-T cells degranulation reflected by CD107a expression and cytokines release by ELISA. It showed that all three CD33 CAR-T cells exhibited profound proliferation and potent effector functions against AML cell lines, including specific cytotoxicity at low E:T ratios of 1:8 or even 1:20, LDH release by target cells, distinct degranulation and robust cytokine production. For in vivo effect, human CD33+ leukemia mouse model were established, and then treated with CD33 CAR-T cells, followed by observation of the body weight and overall survival of mouse. Three type CD33 CAR-T cells could all significantly prolong mouse survival without obvious weight loss.Finally, the influences of different costimulators on CAR-T cells efficacy and persistence were explored, phenotypes of three CAR-T cells were assessed by flow cytometry based on memory markers CCR7 and CD45RA and exhaustion markers PD-1, LAG-3, Tim-3 et al. Distinctions among three types CAR-T cells emerged during in vitro expansion. CD33 BBz.CAR-T cells had an increased central memory compartment, which was related to T cell persistence. While CD33 28z.CAR-T cells were effector memory T cells predominant, which were the terminally differentiated T cells. In addition, CD33 28z.CAR-T cells were more inclined to become exhausted compared to CD33 BBz.CAR-T cells, indicated by higher expression of PD-1, LAG-3 and Tim-3.In conclusion, this study reported the high specific cytotoxicity of the CAR-T cells for CD33+ AML cells. Furthermore, this study demonstrated that 4-1BB signaling domain in CARs could endowed CAR-T cells with anti-exhaustion capacity and increased central memory compartment, uncovered the reason that CAR-T cells with 4-1BB performed better in antileukemic function, and further illustrated 4-1BB CAR may be superior to incorporate into T cells to get persistent anti-tumor efficacy especially when transfused to the human body environment.Keywords: Chimeric antigen receptor, CD33, Acute myeloid leukemia, Immunotherapy, Costimulator DisclosuresNo relevant conflicts of interest to declare.

Novel CoupledCAR technology for treating colorectal cancer.

2528 Background: Chimeric antigen receptor (CAR) T cell therapy has made significant progress in the treatment of blood cancers such as leukemia, lymphoma, and myeloma. However, the therapy faces many challenges in treating solid tumors. These challenges include physical barriers, tumor microenvironment immunosuppression, tumor heterogeneity, target specificity, and limited expansion in vivo. Methods: We designed a CAR lentivirus vector that consisted of a humanized CD19-specific single-chain variable fragment (scFv), a 4-1BB costimulatory domain, and a CD3ζ signaling domain.The lentivirus was produced by transfecting HEK-293T cells with CAR lentiviral vectors and viral packaging plasmids. Patient’s CD3 T cells was cultured in X-VIVO medium containing 125U/mL 1interleukin-2 (IL-2), and transduced with CAR lentivirus at certain MOI 24h after stimulated by anti-CD3/CD28 magnetic beads. Transduction efficiency was evaluated at 7 to 9 days after CAR lentivirus transduction, and quality controls for fungi, bacteria, mycoplasma, chlamydia, and endotoxin were performed. After infusion, serial peripheral blood samples were collected, and the expansion and the cytokine release of CART cells were detected by FACS and QPCR,respectively. The evaluation of response level for patients were performed at month 1,month 3,and month 6 by PET/CT. Results: We engineered CoupledCAR T cells with lentiviral vectors encoding an anti-GCC (guanylate cyclase 2C) CAR molecule. To verify the safety and efficacy of CoupledCAR-T cells for treating solid tumors, we conducted several clinical trials for different solid tumors, including seven patients with colorectal cancer. These seven patients failed multiple rounds of chemotherapy and radiotherapy. In the clinical trial, the metastatic colorectal cancer patients were infused with autologous anti-GCC CoupledCAR-T cells range from 4.9×105/kg to 2.9×106/kg. We observed that CoupledCAR-T cells expanded significantly in the patients and infiltrated tumor tissue sites, demonstrating enhanced anti-tumor activities. PET/CT showed significant tumor shrinkage and SUV max declined, and the ongoing responses were monitored. Patient 3 achieved complete response and the best overall response rate (ORR, include complete remission, complete metabolic response, and partial response.) was 57.1% (4/7), complete remission (CR) rate was 14.3% (1/7). Conclusions: In conclusion, the clinical data demonstrated that CoupledCAR-T cells effectively expanded, infiltrated tumor tissue sites, and kill tumor cells in patients with colorectal cancer. We used immunotherapy to achieve complete remission in patients with advanced colorectal cancer for the first time. We are recruiting more colorectal cancer patients to further test the safety and efficacy of anti-GCC CoupledCAR T cells. Since our CoupledCAR technology is a platform technology, we are expanding it to treat other solid tumors using different target tumor markers.

A novel pseudovirus\u2010based mouse model of SARS-CoV-2 infection to test COVID-19 interventions

BackgroundThe spread of SARS-CoV-2, the virus that causes Coronavirus Disease 2019 (COVID-19), has been characterized as a worldwide pandemic. Currently, there are few preclinical animal models that suitably represent infection, as the main point of entry to human cells is via human angiotensin-converting enzyme 2 (ACE2) which is not present in typical preclinical mouse strains. Additionally, SARS-CoV-2 is highly virulent and unsafe for use in many research facilities. Here we describe the development of a preclinical animal model using intranasal administration of ACE2 followed by non-infectious SARS-CoV-2 pseudovirus (PsV) challenge.MethodsTo specifically generate our SARS-CoV-2 PsV, we used a lentivirus system. Following co-transfection with a packaging plasmid containing HIV Gag and Pol, luciferase-expressing lentiviruses, and a plasmid carrying the SARS-CoV-2 spike protein, SARS-CoV-2 PsVs can be isolated and purified. To better understand and maximize the infectivity of SARS-CoV-2 PsV, we generated PsV carrying spike protein variants known to have varying human ACE2 binding properties, including 19 deletion (19del) and 19del + D614G.ResultsOur system demonstrated the ability of PsVs to infect the respiratory passage of mice following intranasal hACE2 transduction. Additionally, we demonstrate in vitro and in vivo manipulability of our system using recombinant receptor-binding domain protein to prevent PsV infection.ConclusionsOur PsV system is able to model SARS-CoV-2 infections in a preclinical mouse model and can be used to test interventions or preventative treatments. We believe that this method can be extended to work in various mouse strains or to model infection with different coronaviruses. A simple in vivo system such as our model is crucial for rapidly and effectively responding to the current COVID-19 pandemic in addition to preparing for future potential coronavirus outbreaks.

Mechanistic model for production of recombinant adeno-associated virus via triple transfection of HEK293 cells

Manufacturing of recombinant adeno-associated virus (rAAV) viral vectors remains challenging, with low yields and low full:empty capsid ratios in the harvest. To elucidate the dynamics of recombinant viral production, we develop a mechanistic model for the synthesis of rAAV viral vectors by triple plasmid transfection based on the underlying biological processes derived from wild-type AAV. The model covers major steps starting from exogenous DNA delivery to the reaction cascade that forms viral proteins and DNA, which subsequently result in filled capsids, and the complex functions of the Rep protein as a regulator of the packaging plasmid gene expression and a catalyst for viral DNA packaging. We estimate kinetic parameters using dynamic data from literature and in-house triple transient transfection experiments. Model predictions of productivity changes as a result of the varied input plasmid ratio are benchmarked against transfection data from the literature. Sensitivity analysis suggests that (1) the poorly coordinated timeline of capsid synthesis and viral DNA replication results in a low ratio of full virions in harvest, and (2) repressive function of the Rep protein could be impeding capsid production at a later phase. The analyses from the mathematical model provide testable hypotheses for evaluation and reveal potential process bottlenecks that can be investigated.